Abstract 
Title of Document:   CELLULAR PATHWAYS INVOLVED IN 
EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN 
NEURAL CREST CELLS 
 
  Shen Li, Master of Sciences 2013 
 
Directed by:      Dr. Lisa Taneyhill 
Department of Animal and Avian Sciences 
Abstract: 
Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate 
embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to 
generate diverse derivatives. As such, abnormal development of neural crest cells can lead to 
human congenital and hereditary malformations, diseases and cancers. Both internal molecular 
signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. 
How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton 
to respond to the mechanical features of their external environment during EMT is not well 
understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and 
migration, midbrain neural folds that contain premigratory neural crest cells were dissected out 
from chick embryos, explanted into chamber slides, and incubated to allow for the formation of 
migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To 
elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, 
Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular 
structures were employed. Effects of these perturbations on neural crest cell EMT and migration 
were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving 
cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory 
neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell 
bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 
treatment led to more neural crest cells coming out from these explants within a shorter time 
period compared to control. Nocodazole treatment blocked neural crest cell EMT and the 
resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. 
These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the 
target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell 
EMT and migration. Actin crosslinkers such as ?-actinin and actin-associated proteins like palladin 
also participate in pathways affected by these cytoskeletal inhibitors through their regulation of 
focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and 
migration. We are also documented the distribution of ?-actinin and palladin in migratory neural 
crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways 
regulating neural crest cell EMT and migration and the impact on various biophysical parameters 
upon perturbation of these pathways. 
 
 
 
  
 
 
 
 
Cellular pathways involved in epithelial-to-mesenchymal transitions  
in neural crest cells 
 
 
 
          By 
 
           Shen Li 
 
 
 
Thesis submitted to the Faculty of the Graduate School of the 
University of Maryland, College Park, in partial fulfillment 
of the requirements for the degree of 
Master of Science 
2013 
 
 
 
 
 
Advisory Committee: 
Dr. Lisa Anne Taneyhill, Chair 
Dr. Carol Keefer 
Dr. Arpita Upadhyaya 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Shen Li 
2013 
 
ii 
 
Dedication 
This thesis is dedicated to my grandfather who I never got to meet but miss a lot. 
 
  
 
iii 
 
 
Acknowledgements 
First and foremost I would like to thank my advisor, Dr. Lisa Taneyhill, for her 
guidance, extraordinary patience and support throughout my graduate studies. She has 
been an excellent mentor both personally and professionally. As her first international 
student, I truly appreciate her patience in correcting and shaping my academic altitude 
and techniques and her extra efforts in helping me improve my writing skills. 
Second, I would like to thank Dr. Carol Keefer, for her constant help in my graduate 
studies. She not only gave me advice frequently throughout my project but encouraged 
me a lot both in my academic life and personal life.   
Third, I would like to thank the members of the Taneyhill lab, past and present, for 
being a continued source of inspiration, support and friendship. They have all contributed 
to the lab being a productive and pleasant environment. I would especially like to thank 
Chyong-Yi Wu, Sharon Jhingory, Andrew Schiffmacher, Ranga Padmanabhan and 
Ankita Shah for their expertise, patience and assistance during my early transition into 
graduate studies. 
I would also like to thank our collaborator, Christina Ketchum in Dr. Upadhyaya?s lab, 
for her help in making those amazing movies and collecting quantitative data in my 
project. 
Lastly I would like to extend thanks to my friends and family, especially my parents, 
Chuanfeng Li and Yanbin Li, my husband, Hao Cheng for providing love and support in 
my life. I would also like to thank my friends Yangyang Wang, Summer Hu, Meng Li, 
Christina Ketchum and Qiong Hu for providing support during my graduate study. I would 
also like to thank my sweet kitty, Oktoberfest (Oktty), for her company during my thesis 
writing. 
 
 
iv 
 
 
Table of contents 
 
Dedication .............................................................................................................................................. ii 
Acknowledgements ............................................................................................................................. iii 
Table of contents ................................................................................................................................. iv 
List of figures ........................................................................................................................................ vi 
Chapter1: Literature Review ............................................................................................................... 1 
1.1 Introduction ............................................................................................................................. 1 
1.2 An overview of the neural crest ........................................................................................... 3 
1.3 EMT ......................................................................................................................................... 4 
1.4 Neural crest cell migration .................................................................................................... 8 
1.5 Neural crest cell derivatives ................................................................................................. 9 
1.6 Role of intracellular molecules during cellular movement ............................................. 10 
NM myosin II ....................................................................................................................... 11 
Rho ....................................................................................................................................... 11 
Actin ...................................................................................................................................... 12 
Microtubules ........................................................................................................................ 13 
Palladin ................................................................................................................................ 13 
?-Actinin ............................................................................................................................... 14 
Chapter 2: Materials and Methods................................................................................................... 16 
Chicken Embryos ....................................................................................................................... 16 
Neural Crest Cell Explants ........................................................................................................ 16 
Applications of inhibitors and time-lapse Imaging ................................................................. 17 
Immunohistochemisry ................................................................................................................ 19 
Video Analysis ............................................................................................................................. 20 
Chapter 3: Cellular pathways involved in neural crest cell EMT ................................................. 22 
3.1 Inhibition of non-muscle myosin II by blebbistatin irreversibly blocks EMT ................ 22 
3.2 Inhibition of ROCK by Y-27632 treatment promotes neural crest cell EMT. ............... 25 
3.3 Microtubule function is important for proper neural crest cell EMT .............................. 27 
Chapter 4: Cellular pathways involved in neural crest cell migration ......................................... 33 
4.1 Inhibition of NM myosin II by blebbistatin leads to cessation of cell migration, with 
neural crest cells migrating back towards the neural folds after washout of blebbistatin.
  ...................................................................................................................................................... 33 
4.2 Inhibition of Rho-kinase by Y-27632 accelerates neural crest cell migration. ............ 37 
4.3 Microtubule function is important in neural crest cell migration .................................... 39 
4.4 Inhibition of actin polymerization by latrunculin-A irreversibly suspends cell migration
 
v 
 
 ...................................................................................................................................................... 42 
Chapter 5:  Distribution of ?-actinin and palladin in neural crest cells ...................................... 47 
5.1 Distribution of ?-actinin and palladin in DMSO-treated (control) neural crest cell 
explants. ....................................................................................................................................... 47 
5.2 Distribution of ?-actinin and palladin in blebbistatin-treated neural crest cell explants
  ...................................................................................................................................................... 51 
5.3 Distribution of ?-actinin and palladin in Y-27632-treated neural crest cell explants .. 57 
5.4 Distribution of ?-actinin and palladin in nocodazole-treated neural crest cell explants
  ...................................................................................................................................................... 60 
5.5 Distribution of ?-actinin and palladin in latrunculin-A-treated neural crest cell explants
  ...................................................................................................................................................... 68 
Chapter 6: Discussion and future plans .......................................................................................... 72 
Reference ............................................................................................................................................ 77 
 
 
  
 
vi 
 
List of figures 
Chapter 1: Literature Review 
Figure 1. .......................................................................................................................... 4 
Figure 2. .......................................................................................................................... 5 
Figure 3. .......................................................................................................................... 7 
Figure 4. ........................................................................................................................ 10 
Chapter 2: Materials and Methods 
Figure 5. ........................................................................................................................ 17 
Figure 6. ........................................................................................................................ 18 
Figure 7. ........................................................................................................................ 19 
Figure 8. ........................................................................................................................ 21 
Figure 9. ........................................................................................................................ 21 
Chapter 3: Cellular pathways involved in neural crest cell EMT 
Figure 10. ...................................................................................................................... 22 
Figure 11. ...................................................................................................................... 24 
Figure 12. ...................................................................................................................... 26 
Figure 13. ...................................................................................................................... 31 
Figure 14. ...................................................................................................................... 31 
Chapter 4: Cellular pathways involved in neural crest cell migration 
Figure 15. ...................................................................................................................... 35 
Figure 16. ...................................................................................................................... 36 
Figure 17. ...................................................................................................................... 38 
Figure 18. ...................................................................................................................... 40 
Figure 20.  .................................................................................................................... 41 
Figure 19. ...................................................................................................................... 41 
Figure 21. ...................................................................................................................... 43 
Figure 22. ...................................................................................................................... 44 
Figure 23. ...................................................................................................................... 45 
Chapter 5:  Distribution of ?-actinin and palladin in neural crest cells 
Figure 24. ...................................................................................................................... 49 
Figure 25. ...................................................................................................................... 50 
Figure 26. ...................................................................................................................... 53 
Figure 27. ...................................................................................................................... 54 
Figure 28. ...................................................................................................................... 55 
Figure 29. ...................................................................................................................... 56 
Figure 30. ...................................................................................................................... 58 
Figure 31. ...................................................................................................................... 59 
Figure 32. ...................................................................................................................... 61 
Figure 33. ...................................................................................................................... 62 
 
vii 
 
Figure 34. ...................................................................................................................... 63 
Figure 35. ...................................................................................................................... 64 
Figure 36. ...................................................................................................................... 65 
Figure 37. ...................................................................................................................... 66 
Figure 38. ...................................................................................................................... 67 
Figure 39. ...................................................................................................................... 69 
Figure 40. ...................................................................................................................... 70 
 
Table 1. .......................................................................................................................... 32 
Table 2. .......................................................................................................................... 46 
Table 3. .......................................................................................................................... 71 
 
 
 
1 
 
Chapter 1: Literature Review 
1.1 Introduction 
Early embryonic development is a crucial time period in which organisms form a 
highly structured, functioning body from a single, fertilized egg. Any abnormal 
development in this highly regulated process can lead to severe defects or be lethal. 
Different cell types with specific functions are generated and directed to their destinations 
under strict spatio-temporal control. During this process, the neural crest plays a critical 
role due to its multi-potent, transient, and migratory properties, and is the focus of my 
thesis research.  
Neural crest cells undergo an epithelial-to-mesenchymal transition (EMT) to 
acquire motility so that they can migrate extensively to their target areas. Aggressive 
tumor cells share many characteristics with neural crest cells, contributing to the 
paradigm of tumor cell plasticity (Kasemeier-Kulesa, Teddy et al. 2008). Although we 
know that cell-cell and cell-extracellular matrix interactions must be altered to facilitate 
EMT and cell migration, the specific cellular pathways that are involved, and how these 
molecular signals function in this dynamic process, are poorly understood. In my thesis 
project, I explored the role of non-muscle myosin II (NM myosin II), Rho kinase (ROCK), 
actin and microtubules during neural crest cell EMT and migration. This project not only 
will give insight into neural crest cell EMT but will shed light on the potential pathways 
involved in tumor generation and progression. 
In this study, we incorporate time-lapse imaging techniques to accomplish our goal 
of dynamically recording neural crest cell behaviors, under normal conditions and those 
in which the above pathways have been perturbed. We use chick embryos for our 
research model because they develop externally, allow easy access for neural crest cell 
dissection, and neural crest cells have been well characterized in them. Because of the 
 
2 
 
non-transparent 3D environment of the chick embryo, it is hard to track dynamic cell 
phase changes and movements associated with EMT in vivo. In vitro time-lapse imaging, 
however, allows us to easily record neural crest cell behaviors, providing us with a 
tractable experimental model with results translatable to neural crest cell EMT and 
migration in vivo. 
  
 
3 
 
1.2 An overview of the neural crest 
The neural crest is a transient structure derived from the ectoderm. The 
development of the neural crest is strictly regulated by a series of events. The 
specification of the neural crest at the neural plate-ectoderm boundary occurs during 
gastrulation. In this process, a set of transcription factors, including Distalless-5 and 
Pax3/7, cooperatively keeps cells in the border region from becoming the neural plate or 
epidermis and induces a second set of transcription factors (FoxD3, Sox9, Id, Twist, and 
Snail) to direct the formation of the neural crest (Nikitina, Sauka?Spengler et al. 2009). 
Neural crest cells are multi-potent and thus give rise to a diverse array of derivatives, 
including sensory neurons and glia, skin pigment cells, portions of the heart, and the 
craniofacial skeleton (Nicole Le Douarin, Chaya Kalcheim 1999). Abnormal development 
of neural crest cells may lead to neurocristopathies or even cancers (Ramgolam, Lauriol 
et al. 2011) 
  
 
4 
 
1.3 EMT 
Initially contained within the dorsal neural tube, neural crest cells undergo EMT 
and depart from their site of origin, migrating extensively throughout the embryo (Fig. 1). 
Premigatory neural crest cells need to lose intercellular tight and adherens junctions and 
reorganize their cytoskeleton in order to acquire motility, which is crucial for these cells to 
exit the dorsal neural tube. Neural crest cells then migrate towards their precise 
peripheral targets, allowing for the formation of a wide variety of derivatives and, 
eventually, a functioning vertebrate embryo.   
Figure 1. Schematic illustrating 
the formation of the neural crest 
during chick neurulation. Adapted 
from (Liu 2005). 
 
5 
 
EMT is an orchestrated series of events in which cell-cell and cell-extracellular 
matrix (ECM) interactions are changed to release epithelial cells from the surrounding 
tissue. The cytoskeleton is reorganized to confer cells with the ability to move through a 
three-dimensional ECM, and a new transcriptional program is induced to maintain the 
mesenchymal phenotype (Fig. 2; (Kalluri and Weinberg 2009)). 
The structural integrity of epithelial tissues is essential for the organism to 
maintain the protective barrier against environmental hazards externally and to create 
specific and physically controlled subdomains internally. Cell-cell interactions, including 
tight junctions, cadherin-based adherens junctions, gap junctions, and desmosomes 
connected to the intermediate filament cytoskeleton, collectively maintain epithelial 
structures. Integrins involved in cell-ECM interactions also help to maintain the epithelial 
state of the cell. Tissue polarity like apical-basal differences is also defined by cell-cell 
and cell-ECM contacts (Yeaman, Grindstaff et al. 1999). Many mesenchymal cells, 
however, do not have cell-cell contacts and defined cell polarity. Instead, mesenchymal 
cells have distinct cell-ECM interactions and cytoskeleton structures. At the same time, 
Figure 2. Schematic illustrating the morphology changes of cells and involved signal pathways during 
EMT. Through EMT, polarized epithelial cells transition into motile and ECM component?secreting 
mesenchymal cells. The  figure above shows commonly used epithelial and mesenchymal cell 
markers. Adapted from (Kalluri & Weinberg, 2009).   
 
6 
 
mesenchymal cells produce signaling proteins to organize and remodel ECM, which, in 
turn, affects epithelial cells. These signaling proteins include but are not limited to growth 
factors of the epidermal (EGF), hepatocyte (HGF) and fibroblast (FGF) families, and 
transforming growth factor ? (TGF?) (Derek Radisky, 2005).  
According to their different functions, EMTs are classified as three subtypes 
(Kalluri and Weinberg 2009) (Fig. 3). Type 1 EMTs play a role in many stages of 
development, including implantation and embryo formation (Zeisberg and Neilson 2009). 
This type of EMT neither causes fibrosis nor induces invasive phenotypes. Mesenchymal 
cells generated through type 1 EMTs have the potential to undergo a 
mesenchymal-to-epithelial transition (MET) to form secondary epithelia. For example, 
during delamination of the neural crest, a population of highly motile cells migrates to and 
incorporates into many different tissues (Radisky 2005). After arrival to their destinations, 
the cells may revert to their original epithelial phenotype through MET. Type 2 EMTs are 
associated with wound healing, tissue regeneration, and organ fibrosis (Zeisberg and 
Neilson 2009). Their initiation and continued occurrence depend on inflammation- 
inducing injuries. Once the provoking injuries or infections are removed, these EMTs 
cease. Type 2 EMTs can continue to respond to ongoing inflammation and eventually 
lead to organ destruction (Ivanova, Butt et al. 2008). Type 3 EMTs are related to cancer 
progression and metastasis. During carcinoma progression, epithelial cells lose their 
polarity and detach from the basement membrane (Zeisberg, Shah et al. 2005). Cell- 
ECM interactions and signaling networks are altered, while the composition of the 
basement membrane changes. EMT then occurs to facilitate the malignant phase of 
tumor growth. Type 3 EMTs are also involved in enabling cancer cells to enter the 
circulation and exit the blood stream at a remote site, where they may form micro- and 
macro-metastases, which may involve METs and thus a reversion to an epithelial 
phenotype (Zeisberg and Neilson 2009). It should be noted that the specific signals that 
determine EMTs to form in these three settings are not clearly defined. A common set of 
 
7 
 
genetic and biochemical elements, however, appears to underlie these diverse systems. 
Neural crest cells, as a highly invasive embryonic progenitor cell population, share many 
characteristics with cancer cells, making them a good model of tumor cell plasticity and 
invasiveness. (Kasemeier-Kulesa, Teddy et al. 2008). 
 
Figure 3. Classification of EMTs according to their biological functional differences. 
(A) Type 1 EMTs associate with implantation, embryogenesis, and organ development. 
(B) Type 2 EMTs are associated with tissue regeneration and fibrosis. (C) Type 3 EMTs 
occur in the context of tumor growth and cancer progression. Adapted from (Kalluri & 
Weinberg, 2009). 
 
8 
 
1.4 Neural crest cell migration  
Negative and positive regulators collectively facilitate neural crest cell migration. 
Two main classes of these negative signaling molecules are ephrins and their Eph 
receptors and class 3 semaphorins and their neuropilin/plexin receptors (Baker and Antin 
2003) (Mellott and Burke 2008). These signaling pathways induce the collapse of cell 
protrusions to prevent neural crest cells from entering specific zones. Neuropilins 1 and 2 
(Nrp1/2) (Schwarz, Vieira et al. 2008) (Yu and Moens 2005) are expressed by cephalic 
neural crest cells and form a receptor specific for the class 3 semaphorins, allowing for 
Nrp1/2 binding. Some physical barriers like the otic placode/vesicle also prevent the entry 
of neural crest cells into specific regions in the head. Positive regulators are considered 
to fall into two categories: permissive factors, which promote general motility, and 
chemoattractants, which lead neural crest cells to specific regions. Molecular 
components of the ECM that support migration or increase motility are the examples of 
this first category. Putative chemoattractants must meet three criteria. First, the candidate 
should be expressed in neural crest cells; second, its loss should lead to cell dispersion, 
so that cells expressing the receptor for this factor develop a broader distribution than the 
control; and third, when assessed by time-lapse imaging, a localized source of the 
candidate should lead to directional movement (Theveneau and Mayor 2012). So far, the 
VEGF, PDGF and FGF family of signaling molecules ((McLennan, Teddy et al. 2010); 
(Trokovic, Jukkola et al. 2005); (Richarte, Mead et al. 2007)), the chemokine Stromal 
cell-derived factor 1 (Sdf1 or CXCL12) ((Fukui, Goto et al. 2007); (Olesnicky Killian, 
Birkholz et al. 2009)) and Cadherin-11, expressed in Xenopus neural crest cells (Kashef, 
K?hler et al. 2009), are potential neural crest cell chemoattractants. Interestingly, though, 
none of the candidates identified to date meets all three criteria described above.  
  
 
9 
 
1.5 Neural crest cell derivatives 
Neural crest cells are an evolutionarily conserved cell population that gives rise to 
various cell types including pigment cells, craniofacial cartilage and the peripheral 
nervous system (Nikitina, Sauka?Spengler et al. 2009). In general, the type of derivative 
depends upon the axial level from which the neural crest cells originate and the time of 
their emigration from the neuroepithelium. For example, cephalic neural crest cells 
generated from the midbrain and rhombomere 1 and 2 will contribute to cranial ganglia 
and stop in a relatively dorsal position, while the subpopulations forming cartilage and 
bones of the face and neck will continue further migrating ventrally and invade the 
branchial arches. The cardiac neural crest cells, arising posterior to the otic placode from 
the anterior limit of rhombomere 4 and caudalward, migrate to the developing heart (Kirby 
and Hutson 2010), and the enteric crest, arising from somites 1 to 7, colonize the gut. The 
time of migration also influences the types of derivatives that neural crest cells form. Early 
migrating cranial neural crest cells populate the branchial arches to generate bone, 
cartilage, and connective tissue (skeletal structures), whereas the later wave of neural 
crest cells stays close to the central nervous system and generates the neurons and glia 
of the cranial ganglia (Graham, Begbie et al. 2004).  
  
 
10 
 
1.6 Role of intracellular molecules during cellular movement  
Many cellular pathways have been suspected to participate in neural crest cell 
EMT and migration due to their primary roles in general cytoskeletal organization or cell 
motility. Fig. 4 shows interactions among four cytoskeletal components upon which we 
focused in our project. In addition, we performed immunohistochemistry for the actin 
crosslinking proteins ?-actinin and palladin to further explore potential cytoskeletal 
changes after inhibitor treatment. 
Figure 4. Cytoskeletal components interact with each other and facilitate cellular 
activities. The figure shows known interactions among myosin, actin, microtubule and 
ROCK. 
 
11 
 
NM myosin II: NM myosin II is a myosin family member that binds to actin and has 
actin-linking and contractile properties. By phosphorylation of its light or heavy chains, 
NM myosin II controls cell adhesion, cell migration, and tissue architecture due to its 
position downstream of convergent signaling pathways (Vicente-Manzanares, Ma et al. 
2009). Recent evidence implicates NM myosin II in neural crest cells. Berndt and 
Halloran (2008) showed that neural crest cells require blebbing or protrusive activity to 
undergo EMT in zebrafish (Berndt, Clay et al. 2008). They found that inhibition of myosin 
II or ROCK disrupted both blebbing and neural crest cell EMT. Myosin II contractility is 
also required for other forces generated in migratory cells, such as those exerted on focal 
adhesions (Mitchison and Cramer, 1996). Although the authors did not see an obvious 
effect of the myosin inhibitor blebbistatin on production of filopodia or lamellipodia, it 
remains possible that a significant effect of blebbistatin is instead on focal adhesion- 
related forces. In addition, the disassembly of apical junction complexes in calcium- 
depleted intestinal cells, which occurs through contractile F-actin ring intermediates, is 
blocked by blebbistatin treatment, indicating a requirement for NM myosin II activity in 
disassembly (Ivanov et al., 2004). 
 
Rho: Rho proteins constitute a family of small signaling GTPases that reside within the 
Ras superfamily. Rho kinase (ROCK) is involved in many basic cellular processes that 
influence cell proliferation, differentiation, motility, adhesion, survival or secretion 
(Boureux, Vignal et al. 2007). ROCK leads to stabilization of actin filaments via LIM 
kinase and cofilin, and also leads to phosphorylation and activation of NM myosin II 
(Bresnick 1999) in some cell types. Liu and Jessell (1998) showed that inhibition of Rho 
by C3 exotoxin in chick neural tube explants caused a decrease in the number of 
migratory neural crest cells without affecting neural crest cell induction (Liu and Jessell 
1998). Moreover, Rho inhibition did not affect neural crest cells that had already initiated 
migration, suggesting that Rho signaling may specifically alter EMT and not the ability of 
 
12 
 
neural crest cells to migrate. In contrast, a recent in vivo study showed that RhoA 
inhibition caused a decrease in filopodial retractions and cell velocity in migrating 
post-otic neural crest cells, suggesting that Rho signaling plays a significant role in later 
adhesion-based migration (Rupp and Kulesa 2007), at least in this subpopulation of 
neural crest cells. Shoval et al. (2012) reported that while Rho disappears from neural 
crest cell membranes upon delamination, active Rac1 becomes apparent in 
lamellipodia of mesenchymal cells (Shoval and Kalcheim 2012). Loss of Rac1 function at 
chick trunk levels inhibited neural crest cell migration but did not prevent emigration that 
is associated with N-cadherin down-regulation and the G1/S transition. Furthermore, 
inhibition of Rho stimulated premature Rac1 activity and consequent formation of 
lamellipodia, leading to neural crest cell migration (Groysman, Shoval et al. 2008). 
Recently, Clay and Halloran (2013) showed that Rho is activated in a discrete apical 
region of premigratory neural crest cells during EMT, and Rho-ROCK signaling is 
essential for apical detachment and generation of motility within the neuroepithelium 
(Clay and Halloran 2013). Previous conflicting results regarding Rho functions may be 
due to different species and/or axial levels examined. The role of ROCK pathway in chick 
cranial neural crest cell EMT and migration, however, remains unknown. 
 
Actin: Actin is the most abundant protein in many eukaryotic cells and is highly 
conserved among different organisms (Schmidt and Hall 1998). The globular monomeric 
form, G-actin, can reversibly assemble to form filamentous actin (F-actin) via a process 
controlled by a large number of actin-binding proteins. The functions of actin have been 
well studied in various cellular processes such as cell motility, cell division, muscle 
contraction, and cytokinesis (Schmidt and Hall 1998). Deletion of Integrin-linked kinase 
(ILK), a major regulator that links integrins to the actin cytoskeleton, from mouse neural 
crest cells results in embryonic lethality during late gestation (Arnold, Zang et al. 2013). 
This indicates actin, as the building block of cytoskeleton, plays an essential role during 
 
13 
 
the development of neural crest cells. 
 
Microtubules: Microtubules are filamentous structures created by self-assembly of ?/? 
tubulin heterodimers (Nogales 2001; Amos 2004). Similar to F-actin microfilaments, 
microtubules are polarized by having fast-growing "+" and slow- growing "-" ends. In 
columnar epithelial cells, prominent bundles of microtubules align along the lateral 
plasma membrane. This population of microtubules orient their minus ends toward the 
cell apex and plus ends toward the cell base (Ivanov, McCall et al. 2006). The 
perijunctional space of differentiated epithelial cells is rich in microtubules (Ivanov, McCall 
et al. 2006). Microtubules appear to be involved in neural crest cell migration indirectly, 
since directional cell migration requires polarized alignment of the cytoskeleton with the 
microtubule organizing center. Francis et al. (2011) reported that knock-out mice deficient 
in the gap junction gene connexin43 exhibit developmental anomalies associated with 
abnormal neural crest and proepicardial cell migration. These migration defects are due 
to a loss of directional cell movement and are associated with abnormal actin stress fiber 
organization and a loss of polarized cell morphology (Francis, Xu et al. 2011). 
Microtubules also interact with cadherins, core transmembrane proteins in adherens 
junctions, which play a critical role during neural crest cell EMT (Coles, Taneyhill et al. 
2007; Brieher and Yap 2013).  
 
Palladin: Palladin is an actin-associated protein that plays an essential role in 
maintaining cell morphology and cytoskeletal organization in different cell types 
(Goicoechea, Arneman et al. 2008). Palladin binds directly to actin-regulating proteins 
(e.g., Vasodilator-Stimulated Phosphoprotein (VASP), profilin, CLP36, lipoma preferred 
partner (LPP), and Eps8) and actin-crosslinking proteins (e.g., ?-actinin, Lasp-1, and 
ezrin). The level of palladin expression appears to be tightly regulated by cells and is 
dramatically altered during EMT and cellular differentiation (Goicoechea, Arneman et al. 
 
14 
 
2008). Palladin and ?-actinin co-localize in focal adhesions and stress fibers (Parast and 
Otey 2000), which consist of bundles of crosslinked actin and myosin. Palladin functions 
in maintaining adherens junctions, facilitates the formation of podosomes in migrating 
cells (Goicoechea, Bednarski et al. 2008), and, along with ?-actinin in the mouse, is 
expressed in the proper spatio-temporal pattern to play a role in neural crest cell 
ontogeny (Luo, Liu et al. 2005; Tomita, Matsumura et al. 2005). The palladin knock-out 
mouse is embryonic lethal due to severe defects in cranial neural tube closure and 
herniation of the liver and intestine (Luo, Liu et al. 2005). These studies in both cultured 
cells and knock-out mice suggest that palladin's actin-organizing activity plays a central 
role in promoting cell motility. Whether palladin is an integral part of neural crest cell EMT, 
however, remains unclear. 
 
?-Actinin: ?-Actinin is an actin-crosslinking protein that functions as a platform for the 
assembly of multi-protein complexes, and is uniquely positioned as an anchor between 
the actin cytoskeleton and the cytoplasmic domains of several cell surface adhesion 
proteins. ?-Actinin is a ubiquitous protein, belonging to a highly conserved family of 
actin-binding proteins, the spectrin superfamily, which also contains the spectrins and 
dystrophin (Blanchard A, Ohanian V, et al 1989). ?-Actinin isoforms have been found and 
characterized genetically and/or biochemically from a large variety of taxa (Sj?blom, 
Salmazo et al. 2008). Through alternative splicing and gene duplication, mammalian cells 
produce at least six different protein products from four ?-actinin encoding genes, each 
found within a specific tissue type and expression profile (Sj?blom, Salmazo et al. 2008). 
Non-muscle actinin-1 is believed to play an essential role in regulating cell shape and cell 
motility (Otey et al., 1990, 1993; Gl?ck et al., 1993; Gl?ck and Ben-Ze'ev, 1994; Knudsen 
et al., 1995), while actinin-4 is associated with cell motility and cancer cell invasion 
(Honda, Yamada et al. 1998). Aside from its interactions with actin filaments, ?-actinin 
has emerged as a major platform for a number of protein-protein interactions with many 
 
15 
 
cytoskeletal and regulatory proteins. There is no direct evidence showing how ?-actinin 
functions during neural crest cell EMT.  
       In the following chapters, I detail my experiments aimed at examining the role of 
known molecular cascades regulating cell migration and/or cytoskeletal elements during 
neural crest cell EMT and migration. I have also examined the distribution of palladin and 
?-actinin pre- and post-pathway perturbation. My results have provided insight into 
specific cellular pathways regulating neural crest cell EMT and migration and the impact 
on various biophysical parameters upon pathway perturbation. 
  
 
16 
 
Chapter 2: Materials and Methods 
Chicken Embryos 
Fertilized chicken eggs (Gallus gallus) were obtained from B&E and Hy-Line North 
America, L.L.C., and incubated at 37? C in humidified incubators. The embryos were 
staged according to the number of somite pairs (somite stage, ss) and removed from the 
yolk sac, using forceps and scissors. The embryos were kept in Ringer?s solution on ice 
before dissection. 
 
Neural Crest Cell Explants  
Neural fold tissue, containing premigratory neural crest cells, from 2 to 5 ss 
embryos was dissected out using tungsten needles in PB-1 standard medium and 
explanted in 8-well chamber slides coated with a 1:100 dilution of poly-L-lysine (Sigma) in 
cell culture water, followed by fibronectin (BD Bioscience) (Coles, Taneyhill et al. 
2007)(Fig. 5). The collected tissues were incubated in cell culture media (DMEM with N2 
supplement (GIBCO-BRL), penicillin, and streptomycin) at 37? C in humidified incubators. 
Depending upon the research aims; incubation time was shortened or extended. Cell 
culture media were gradually changed to L15 (Invitrogen), a CO2 independent media, 
before imaging. 
 
 
 
17 
 
 
 
 
Applications of inhibitors and time-lapse Imaging 
To examine effects on neural crest cell EMT, chemical inhibitors ((blebbistatin 
(Sigma-Aldrich), Y-27632 (Selleckchem), latrunculin A (Sigma-Aldrich) and nocodazole 
(Sigma)) were applied after an hour of explant incubation to first allow for the adherence 
of tissues to the bottom of slides (pre-incubation) (Fig. 6). Inhibitors were then added 
before imaging. The inhibitors were removed by washout if any cell behavioral changes 
were observed in their presence. Imaging was continued for another 6 hours or until cell 
death was noted. Pre-incubation time was extended to 2 to 4 hours to permit EMT when 
effects of inhibitors on neural crest cell migration were examined (Fig. 7). Inhibitors were 
added after 2 to 3 hours of imaging to make sure that cell migration had already 
commenced. If any cell behavior changes were observed, a washout was performed and 
cells continued to be imaged. Phase-contrast images were collected using an inverted 
microscope (TE2000 PFS, Nikon, Melville, NY) with a cooled CCD camera (Coolsnap 
HQ2, Photometrics, Tucson, AZ) and a 10x 0.30 NA objective lens. Images were 
collected at a frame interval of 30 seconds. 
  
Figure 5. Neural fold explant assay. Neural fold tissues were dissected out and incubated in 
a chamber slide to allow for the formation of migratory neural crest cells for fixed and live 
imaging studies. 
 
18 
 
 
 
Figure 6. Experimental set-up for neural crest cell EMT assay. To examine 
perturbation effects on NCC EMT, inhibitors were added within an hour of incubation and 
removed if cell behaviors changed. 2mM blebbistatin and 5mM Y-27632 stocks were 
diluted to working concentrations in L15. Nocodazole stock was first diluted to 1mM 
using DMSO and further diluted to working concentrations. 
 
19 
 
 
 
 
 
 
 
 
Immunohistochemistry  
Neural crest cell explants were fixed with cold 4% paraformaldehyde overnight and 
washed twice with 0.03% Triton X-100 in 1x PBS, 20 minutes each. To fix cells after 
blebbistatin treatment, the same concentration of blebbistatin used in the experiment was 
maintained with the fixative so that the cell morphology phenotype observed in the 
presence of blebbistatin could be captured. Antigen retrieval was then performed by 
incubating cells in 10mM sodium citrate at 96 ? C for 20 minutes. After cooling to room 
Figure 7. Experimental set-up for neural crest cell migration assay. Inhibitive effects 
on NCC migration were tested by recording cell behaviors using time-lapse imaging after 
adding inhibitors. A washout was performed if cell behaviors changed. 2mM blebbistatin 
and 5mM Y-27632 stocks were diluted to working concentrations in L15. Nocodazole 
stock was first diluted to 1mM using DMSO and further diluted to working concentrations. 
10mM latrunculin-A was diluted to 100nM in L15. 
 
 
20 
 
temperature, the cells were rinsed with 1x PBS, blocked for 1 hour in 1x PBS containing 2% 
BSA and then incubated with primary antibodies (anti-palladin rabbit polyclonal antibody 
(1:800), Proteintech; anti-?-actinin mouse monoclonal antibody (1:500), Sigma) diluted in 
blocking solution overnight at 4 ? C. Cells were washed four times for 20 minutes with 
0.03% Triton X-100 in 1x PBS. Secondary antibodies (Alexafluor 488 goat anti-rabbit IgG 
(1:1000) and Alexafluor 594 goat anti-mouse IgM (1:500), both from Invitrogen) were 
diluted in blocking solution and applied. Cells were then washed and stained with 
4',6-diamidino-2-phenylindole (DAPI) to mark cell nuclei before being mounted with Aqua 
Poly/Mount. Samples were analyzed under the fluorescence microscope (Zeiss) or 
confocal microscope (Leica) at either 594 nm or 488 nm. Adobe Photoshop 9.0 was used 
to process images.  
 
Video Analysis 
Andor (South Windsor, CT) imaging software was used to make kymographs to 
calculate explant spreading. Kymographs made along a radial line across the advancing 
explant. Speed calculated from the slope of the kymograph edge as indicated in Figure 8. 
The velocity is measuring the speed of cell migration at the edge of the explants. PIV 
analysis was done using MATLAB (Mathworks, Natick MA) image analysis package 
PIVLab (http://pivlab.blogspot.com/). PIV analysis was used to calculate cell density, 
vorticity, and coherence of motion. Vorticity is an indicator of how much curl a field of 
vector displays (Fig 9). The live cell imaging and quantitative analysis was performed in 
Dr. Arpita Upadhyaya?s lab in collaboration with graduate student Christina Ketchum. 
 
21 
 
 
  
Figure 8. Measurement of velocity. Kymographs made along a radial line across the 
advancing explant. Speed calculated from the slope of the kymograph edge 
 
Figure 9. Measurement of vorticity. Heat Map plot of vorticity of a field of cells. Negative 
values correspond to counterclockwise curl. Positive values correspond to clockwise curl. 
Figure on the right shows a histogram of vorticity. The closer a distribution is to 0, the less 
curling and more linear the movement of the cells 
 
 
22 
 
Chapter 3: Cellular pathways involved in neural crest cell EMT 
3.1 Inhibition of non-muscle myosin II by blebbistatin irreversibly blocks 
EMT 
To first elucidate standard cell behaviors characterizing neural crest cells undergoing 
EMT, we performed time-lapse imaging on control-treated (DMSO) neural folds. 
Dissected tissue was preincubated in cell culture media for 0.5-1 hour at 37?C to allow 
adherence to the fibronectin-coated chamber slide. Cell culture media was gradually 
changed to L15 (colorless CO2-independent media) before imaging. Our observations 
indicate that it took approximately 2 hours for the first few cells to break cell-cell 
adhesions and become mesenchymal, as seen by the emergence of individual cells from 
the neural folds at a velocity of 0.3743?m/min (Fig.14), which reflects the speed of cell 
migration on the edge of the explants. As time passed, more cells proceeded through 
EMT and became individual mesenchymal cells (Fig. 10 and Movie 1 in supplemental 
data). We define the neural fold tissue and migratory neural crest cells emerged from the 
neural folds together as explants. 
 
 
 
Figure 10. Cells undergo EMT and migrate out from control-treated (DMSO) neural folds. 
Successive panels show cells gradually emerging from neural fold tissue and undergoing 
migration. 
 
23 
 
To determine if NM myosin II is involved in neural crest cell EMT, we added 
blebbistatin (50?M) after attachment of neural folds. Blebbistatin binds to the ATPase 
intermediate with ADP and phosphate bound at the active site, and it slows down 
phosphate release (Kov?cs, T?th et al. 2004). Time-lapse imaging showed that neural 
crest cell EMT was suspended (movie 2 in supplemental data). During the 2.5 hours of 
incubation in blebbistatin, no cells emerged from the neural fold (Fig. 11A), and the 
velocity of cells on the edge of the neural fold decreased to 0.0182?m/min (Fig. 14), 
which is a 20-fold decrease compared to controls and is statistically significant (P < 0.05). 
Cells appeared to be moving around within the neural fold, however, indicating that cells 
retained their motility at a certain level without properly functioning NM myosin II.  
We then removed this block by washing out blebbistatin to examine its effect on EMT 
(Fig. 11B). Intriguingly, the overall motility of cells was increased to 0.3917?m/min, which 
is comparable to the controls (Fig. 14), as evidenced by an increasingly changed neural 
fold shape. Even 5 hours post-washout, not a single cell showed signs of mesenchymal 
morphology, indicating neural crest cell EMT failed to resume after the removal of 
blebbistatin. It is important to note, however, that the failure of EMT may be due to effects 
of residual blebbistatin not removed upon washout. Although the same washout 
successfully allowed for cell motility to resume in blebbistatin treatment of migrating 
neural crest cells (detailed in Chapter 4), the epithelial cells in the neural folds may be 
more sensitive to blebbistatin compared to migrating neural crest cells. Furthermore, 
washout of blebbistatin in the migration assay caused migratory crest cells to move 
towards the neural folds (see Chapter 4). It is possible that the same mechanism also is 
directing cell behavior during EMT because no neural crest cells emerged from the neural 
folds. Such explant contraction after washout was not as easily observed in our 
experimental set-up.  
 
24 
 
In zebrafish, blebbistatin treatment in the hindbrain significantly decreased the 
number of cells undergoing EMT but did not give an obvious effect on production of 
filopodia or lamellipodia (Berndt, Clay et al. 2008). Our observations also show that 
blebbistatin treatment of midbrain neural crest cells decreases or even blocks EMT. This 
confirms that neural crest cells need NM myosin II to facilitate EMT.  
Figure 11. Blebbistatin treatment irreversibly blocks neural crest cell EMT. Panel A shows snapshots from 
time-lapse imaging recording cell behavior after adding blebbistatin. During 2.5 hours, no cells had come out of the 
explant. Panel B shows cell behaviors after removal of blebbistatin (washout).  
 
25 
 
3.2 Inhibition of ROCK by Y-27632 treatment promotes neural crest cell EMT 
We tested the role of ROCK in neural crest cell EMT by employing the ROCK 
pathway inhibitor Y-27632. Y-27632 inhibits the ROCK by binding to the catalytic site 
(Ishizaki, Uehata et al. 2000). ROCK, as a downstream effector of Rho GTPases that 
regulates cytoskeletal dynamics in many aspects of cell motility (Raftopoulou and Hall 
2004; Jaffe and Hall 2005), leads to stabilization of actin filaments via LIM kinase and 
cofilin, and also leads to phosphorylation and activation of NM myosin II (Bresnick 1999) 
in some cell types. Thus inhibition of ROCK not only allows us to test the function of this 
signaling pathway in controlling cell behaviors during EMT, but also may provide a means 
to inhibit NM myosin II activity and potentially corroborate our blebbistatin results. Our 
findings, however, indicate that at least in chick cranial neural crest cells, the ROCK 
pathway is unlikely to be functioning through NM myosin II because drastically different 
cell behaviors were observed in blebbistatin-treated versus Y-27632-treated neural folds. 
Using the same experimental set-up, we noted that more neural crest cells migrated 
out in Y-27632-treated neural folds than in the controls (Fig. 12B, 4 and 6 hours). Within 
the first 2 hours, there is an apparent difference between Y-27632 treatment and control 
treatment (Fig. 12A) in that very few cells emerged from control-treated neural folds while 
a large number of cells migrated out from neural folds immersed in Y-27632. In the 
following 4 hours, many neural crest cells completed EMT and became migratory with a 
velocity of 0.4007?m/min, which is drastically greater than the velocity of cells immersed 
in blebbistatin (0.0182?m/min) and slightly higher than in controls (0.3743?m/min). 
Although there is no significant difference in the velocities between Y-27632-treated cells 
and the controls, more mesenchymal neural crest cells appeared to emerge from 
Y-27632-treated neural folds qualitatively (Fig. 12B, 4 and 6 hours, movie 3 in 
supplemental data). This suggests that Y-27632 treatment may promote neural crest cell 
EMT. 
Interestingly, our observations in midbrain neural crest cells are not in agreement 
 
26 
 
with findings reported for later stage (10ss-18ss) zebrafish hindbrain neural crest cells, 
probably because hindbrain neural crest cells migrate through a different external 
environment (through the rhombomeres), which possess a different molecular milieu than 
the midbrain. This study showed that treatment with a ROCK inhibitor led to a reduction of 
neural crest cell EMT (Berndt, Clay et al. 2008) as assessed by in situ hybridization using 
dlx2, a marker of migratory neural crest cells. This lab also showed that inhibition of 
ROCK upon C3 exotransferase disrupted neural crest cell EMT in zebrafish (Clay and 
Halloran 2013). In agreement with a more recent finding in chick trunk neural crest cells 
that disrupting RhoA signaling induces migration of post-otic neural crest cells (Rupp and 
Kulesa 2007), we showed that in midbrain neural crest cells, inhibition of the ROCK 
pathway also induced more cells to undergo EMT. 
 
  
Figure 12. Y-27632 treatment promotes neural crest cell EMT. Panel A shows neural crest 
cells undergo EMT and migrating over 6 hours. Panel B shows more cells migrating out after 
Y-27632 treatment within the same period of time. Arrows show cells that migrated out. 
 
27 
 
3.3 Microtubule function is important for proper neural crest cell EMT 
It has been reported that microtubule dynamics, along with RhoA, play an important 
role in EMT during chick gastrulation. In this example, embryos treated with nocodazole, 
a specific inhibitor of microtubules, showed a premature breakdown of the cell basement 
membrane, a crucial step in EMT (Nakaya, Sukowati et al. 2008). Microtubules also 
actively interact with cadherins, core transmembrane proteins in adhesion junctions, 
which play a critical role during EMT in neural crest cells (Coles, Taneyhill et al. 2007; 
Brieher and Yap 2013). Whether microtubules are involved in controlling neural crest cell 
EMT, however, is not known. To address this question, we employed nocodazole to 
perturb the polymerization of microtubules. Nocodazole binds to microtubule subunits 
and prevents heterodimers from repolymerizing (Saunders and Limbird 1997). A high 
concentration of nocodazole (30?M) results in the depolymerization of the whole 
microtubule lattice and affects cell junctions. Significantly lower concentrations (100nM) 
inhibit the dynamic activity of microtubules? plus ends (Nakaya, Sukowati et al. 2008) and 
can cause a decreased rate of locomotion in NRK fibroblasts cells by more than 60% but 
does not show a qualitative change in MT distribution by immunofluorescence (Liao, 
Nagasaki et al. 1995). As such, employing a range of concentrations of nocodazole in our 
study may give us different results through its differential effects. To test our hypothesis, 
we chose nocodazole concentrations of 30?M, 1?M, 100nM.  
Using the same experimental set-up, we noted that no/very few neural crest cells 
emerged from neural folds treated with high concentrations of nocodazole (Fig. 13A). 
Moreover, the velocity of the cells was 0.0208?m/min (Fig. 14), 18-fold lower than that 
observed for cells in controls and thus significantly different. Neural folds became flat, 
with cell movements occurring within them (movie 4 in supplemental data). It was hard to 
determine if the cells were still alive by the end of the treatment because cells stayed 
within the neural folds. Nevertheless, we performed a washout experiment to assess 
effects after removal of nocodazole. After washout of 30?M nocodazole (Fig. 13B), we 
 
28 
 
observed a neglible number of cells emerging from the neural folds. Many cells appeared 
to be rounding up, probably dying (based upon their morphology) and spread out with a 
velocity of 0.1713?m/min (Fig. 14). This indicates that high concentrations of nocodazole 
may irreversibly block neural crest cell EMT. It is also possible that such high 
concentration of nocodazole inhibited many cellular processes, including EMT, which 
caused most cells to die and led to the failure of neural crest cell EMT in the presence of 
nocodazole and after washout. 
In the1?M nocodazole treatment, the neural fold cells spread with a velocity of 
0.3748?m/min (Fig. 14), comparable to the controls. The boundary of the neural folds 
became blurry, indicating cells were emerging from the neural folds (movie 5 in 
supplemental data). A few cells were able to come out, but they could not maintain their 
mesenchymal morphology as they rounded up shortly after emerging from the neural fold 
(Fig. 13C and movie 5), indicating that perhaps nocodazole affected EMT by interrupting 
the maintainence of the mesenchymal cell shape. It is also possible that cells were 
rounding up because they were dying. Washout experiments showed a decreased cell 
velocity (0.1227?m/min; Fig. 14); however, some cells were able to come out as 
individual mesenchymal cells and kept migrating. Most cells rounded up and died after 4 
hours (Fig. 13D). This suggests that the block to neural crest cell EMT from 1?M 
nocodazole can be partially released, but this concentration may still be toxic to the cells 
and eventually cause cell death. The velocity of cells was greater in the presence of 1?M 
nocodazole than after washout. This suggests that lower concentrations of nocodazole 
may facilitate neural crest cell migration during EMT via some pathway that compensates 
for the perturbation of microtubules. Similar results were observed in migration assays 
with more significant data (detailed in Chapter 4). 
100nM nocodazole treatment resulted in only a few cells emerging from the neural 
folds (Fig. 13E), but the effect is reversible after removal of nocodazole (movie 6 in 
supplemental data). The velocity of cells was 0.4560?m/min (Fig. 14) in the presence of 
 
29 
 
nocodazole, higher than the cells in controls (0.3743?m/min) and the cells in 1?M 
nocodazole (0.3748?m/min), but only a few cells came out from the neural folds (Fig. 
13E). After washout, the velocity was unchanged (0.4534?m/min; Fig. 14) and still higher 
than the controls, but it appeared that more individual cells came out (Fig. 13F). These 
results imply that the partial block of EMT is removed after washout. Given that this low 
concentration of nocodazole has been observed to disrupt the plus end of microtubules, 
and that we observe a higher velocity than the control explants, this result indicates that 
the plus end of microtubules may be involved in neural crest cell EMT and that 
microtubules may play a significant role in regulating neural crest cell EMT.  
In summary, 30?M nocodazole irreversibly blocks neural crest cell EMT, and may do 
so by causing cell death; 1?M nocodazole reversibly blocks neural crest cell EMT but 
causes cell death eventually; and 100nM nocodazole partially blocks neural crest cell 
EMT but the block can be removed by washout. These results indicate that microtubules 
are involved in neural crest cell EMT and that the resumption (or lack thereof) of neural 
crest cell EMT after nocodazole treatment is dose-dependent and/or could be absent due 
to lethal effects of nocodazole at higher concentrations. 
We also examined the effects of the inhibition of actin polymerization by latrunculin-A 
treatment on neural crest cell EMT. Latrunculin A sequester monomeric actin in living 
cells (Yarmola, Somasundaram et al. 2000). Results looking at neural crest cell migration, 
however, showed that even very low concentrations of latrunculin A (100nM) irreversibly 
disrupted cell migration and led to cell death. Based on this result, we did not treat with 
latrunculin-A and document effects on EMT, since the effects of latrunculin A were very 
harsh to neural crest cells. 
As summarized in Table 1, blebbistatin treatment irreversibly blocks EMT, Y-27632 
treatment promotes EMT, the effect of nocodazole on EMT is dose-dependent, and 
latrunculin A severely impairs EMT and migration. This suggests NM myosin II, ROCK 
and microtubules are involved in neural crest cell EMT.  
 
30 
 
 
 
31 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 13. Different concentrations of nocodazole can block neural crest cell EMT 
but resumption of EMT is dose-dependent. Panels A, C, E show that neural crest cell 
EMT is blocked or partially blocked under 30?M, 1?M, 100nM nocodazole conditions 
(arrows indicate no/few cells could come out as mesenchymal cells). Panel B shows that 
no cells resume EMT and most cells appeared to be dead after the washout of 30?M 
nocodazole (arrow). Panel D shows that several cells resumed EMT (arrow), but cell 
death happened after 4 hours post-washout of 1?M nocodazole. Panel F shows the 
resumption of neural crest cell EMT post-100nM nocodazole washout (arrow).  
MEANS in ?m/min 
Control = 0.3743 
Bleb 50?M = 0.0182* 
Bleb washout= 0.3917 
Y27632 = 0.4007 
Noc 100nM =0.4560 
100nM washout =0.4534 
Noc 1?M =0.3748 
1?M washout =0.1227 
Noc 30?M =0.0208** 
30?M washout = 0.1713 
Figure 14. Velocities of cells measured on the edge of the explants in EMT 
assay. Figure on the left shows the velocity comparisons among controls and each 
condition in EMT assay. Each bar represents mean?SD. N.S; *P < 0.05, **P < 0.01. 
Table on the left gives means of the velocities on each experimental condition. 
 
32 
 
Table 1. Summary of results for neural crest cell EMT assay 
Treatment Phenotype Velocity (compared to 
control) 
DSMO-treated (control) Normal EMT  
50?M blebbistatin Blocks EMT Statistically decreases 
Bleb washout Resumes motility but not 
EMT 
Comparable 
Y-27632 Promotes EMT Comparable 
100nM nocodazole Partially blocks EMT Comparable 
100nM noco washout Resumes EMT Comparable 
1?M nocodazole Blocks EMT Comparable 
1?M noco washout Partially resumes EMT but 
cells later die 
Comparable 
30?M nocodazole Causes cell death Statistically decreases 
30?M noco washout Cells die Comparable 
 
  
 
33 
 
Chapter 4: Cellular pathways involved in neural crest cell migration 
4.1 Inhibition of NM myosin II by blebbistatin leads to cessation of cell 
migration, with neural crest cells migrating back towards the neural folds 
after washout of blebbistatin 
 To elucidate cell behaviors characterizing neural crest cell migration after EMT, we 
performed time-lapse imaging on inhibitor treated and control (DMSO-treated) neural 
crest cell explants. Dissected tissue was preincubated in cell culture media for 2 to 3 
hours at 37?C to allow adherence to the fibronectin-coated chamber slide and to permit 
EMT. Cell culture media was gradually changed to L15 before imaging. By the time we 
started imaging, there were several cells that had undergone EMT and became 
mesenchymal. In the following 2 to 10 hours of imaging, migratory mesenchymal cells 
spread out from the explants with a velocity of 0.5249?m/min (Fig. 22). We also observed 
extensive dynamics of cell-cell contacts during cell spreading and migration. At the same 
time, more cells from the interior of the explants underwent EMT and became 
mesenchymal cells (Fig. 15 and movie 7 in supplemental data). 
 We then examined the effect of blebbistatin on neural crest cell migration. We 
allowed the cells to undergo EMT and migrate out for 2 to 4 hours before adding 
blebbistatin (50?M) into media. Cell behaviors were recorded to make sure that cells 
developed to a similar stage among experiments with different inhibitor treatments. Cell 
morphology drastically changed in blebbistatin-treated explants. Cell migration was 
suspended, as the velocity of cell migration decreased to a negligible level 
(0.1133?m/min, Fig. 22). Cells exhibited blebbing behavior (Berndt, Clay et al. 2008) and 
developed rounder cell bodies, with lamellipodia disappearing and filopodia appearing 
(Fig. 16B). Within half an hour, cells accomplished morphological changes described 
above (movie 8 in supplemental data). Cells stayed alive while maintaining this 
 
34 
 
morphology with blebbistatin in the media (Fig. 16B). We expected to see resumption of 
normal motility after removal of blebbistatin. Intriguingly, cells fully resumed motility with a 
comparable migrating velocity (0.5943?m/min) to controls (Fig. 22), but they changed 
their direction and moved toward the neural folds, leading to the contraction of the explant 
(Fig. 16C). Neural crest cells quickly adopted a normal morphology, with flat cell bodies 
and lamellipodia and a reduction in filopodia. Within 5 hours after washout, cells 
appeared to be alive and had active cell-cell contacts. We also observed that a few cells 
that were relatively far away from the neural fold were unable to move back to the neural 
folds and ended up moving in random directions (in other movies recording blebbiatatin 
washout experiments, data not shown here). 
 We next examined the spatial dynamics of cell migration by measuring the vorticity 
for the movement of cells migrating out from the explants. Vorticity is an indicator of how 
much curl a field of cells displays (Vedula, Leong et al. 2012). The closer a distribution is 
to 0, the less curling and more linear the movement of the cells is. The control explants 
(Fig. 23) demonstrated a typical distribution of vorticity with the peak at zero on the x-axis.  
After washout of blebbistatin, the distribution of vorticity was much narrower, 
corresponding to lower vorticities and indicating that cells moved in a more linear manner. 
This suggests that cell migration may be directed by some yet unknown factors after the 
removal of blebbistatin. 
 The movement of neural crest cells back towards the original explanted tissue after 
removal of blebbistatin was first observed and documented in our experiments. Two 
possibilities could explain these observations. One is chemotaxis, which is the 
phenomenon whereby cells direct their movements according to certain chemicals in 
their environment. This would require a chemoattractant from the explant center or a 
chemorepellent from outside of the explant acting on the migratory neural crest cells. The 
latter one is unlikely happening because we did not include any other factor other than 
blebbistatin, which was removed by washout. It may be the case that some molecules 
 
35 
 
were induced and/or secreted from the explants after removal of blebbistatin. The 
suspected factor, however, remains unknown. The other explanation could be a change 
of cell polarity. Migratory neural crest cells changed their direction during explant 
contraction, which is one hallmark of directional migration. In order to initiate directional 
migration, cells need to break symmetry and polarize the migratory machinery. There is 
strong evidence that NM II contractility initiates symmetry-breaking by forming the 
prospective rear of the cell (Yam, Wilson et al. 2007), which allows for protrusion to occur 
at the opposite end. This might be an explanation of explant contraction. After washout of 
blebbistatin, NM myosin II recovered its function and reinitiated symmetry-breaking; 
however, the reason why cells changed the prospective rear after pausing by blebbistatin 
needs further exploration.  
 
  
Figure 15. Cell migration in control-treated (DMSO) neural crest cell 
explants. Cells that had undergone EMT migrate out of neural folds and spread 
out as time passed. Cell-cell contacts were maintained while cells were 
migrating. More cells from neural folds undergo EMT and became migratory 
mesenchymal cells. 
 
36 
 
 
  
Figure 16. Inhibition of non-muscle myosin II by blebbistatin leads to cessation of cell 
migration, and neural crest cells migrate towards neural folds after washout of 
blebbistatin. Panel A shows pre-drug cell movement: cells behaved normally like controls. 
Panel B shows cells migration was suspended and cells developed rounder cell bodies after 
blebbistatin treatment. Panel C shows cells resuming motility upon washout of blebbistatin 
and having a normal morphology but changing their migratory direction back towards the 
neural folds.  
 
37 
 
4.2 Inhibition of Rho-kinase by Y-27632 affects neural crest cell migration 
 Using the same set-up, we examined the inhibition of ROCK by Y-27632 in migratory 
neural crest cells. After adding Y-27632, we did not observe any apparent cell 
morphology changes (Fig. 17 and movie 9 in supplemental data). The quantitative data, 
however, showed that the velocity of cell migration (0.7082?m/min) was actually 
increased compared to the controls (0.5249?m/min) (Fig. 22). This result indicates 
Y-27632 treatment accelerates neural crest cell migration and is consistent with its 
function in promoting neural crest cell EMT.  
 Vorticity of cells upon Y-27632 treatment had a broader distribution, which means 
cells in Y-27632-containing media moved in a curved or non-linear path. This indicates 
that ROCK may be involved in directional migration of neural crest cells. Previous studies 
have shown in Xenopus and zebrafish that Rho regulating directional migration of neural 
crest cells through inactivation of Rac (Cascone, Audero et al. 2003). Our findings have 
now established that a similar mechanism may be occurring in chick midbrain neural 
crest cells.  
 
 
38 
 
 
  
Figure 17. Inhibition of ROCK by Y-27632 accelerates neural crest cell migration. Panel 
A shows pre-drug cell movement: cells behaved normally like the controls. Panel B shows 
that the overall cell morphology did not change upon treatment with 100?M Y-27632. 
 
39 
 
4.3 Microtubule function is important for proper neural crest cell migration 
 Knowing that different concentrations of nocodazole have varying effects on 
microtubule polymerization, which affected neural crest cell EMT, we also tested the 
inhibitory effects of nocodazole in different concentrations on cell migration. 
 30?M nocodazole appeared to be toxic to cells (Fig. 18). Cell migration was 
suspended, with velocity decreasing to 0.0208?m/min (Fig. 22). Most cells also rounded 
up and died after 4 hours of drug treatment. Explant size decreased as a result of cell 
death, although a few cells appeared to survive (movie 10 in supplemental data). 
 We then tested 1?M nocodazole treatment on cells. Cell morphology did not change, 
but the velocity (0.4163?m/min) decreased a little (not significantly) compared to the 
controls (Fig. 22). Intriguingly, the velocity further decreased to 0.0534?m/min after 
removal of nocodazole by washout, which is significantly different from the controls. Eight 
hours after washout, cells failed to resume migratory capacity, and instead cell death was 
observed, although more cells appeared to survive compared to 30?M nocodazole 
treatment (Fig. 19B and movie 11 in supplemental data). This suggests that 1?M 
nocodazole is also toxic to neural crest cells. This result is similar to what we observed 
after 1?M nocodazole treatment of neural folds in the EMT assay. Cells were able to 
retain motility in the presence of 1?M nocodazole, but further lost their motility after 
removal of nocodazole. This implies that nocodazole may facilitate cell motility in some 
other way, which compensates for the loss, or partial loss of microtubule function, upon 
microtubule disruption. After removal of nocodazole, both disrupted microtubules and the 
unknown pathway(s) that nocodazole supports disappeared, leading to cessation of cell 
migration and eventually cell death. 
 Because a low concentration of nocodazole (100nM) had a reversible effect on 
neural crest cell EMT, we wanted to test if it would affect cell migration to see if the plus 
end of microtubules is involved in controlling migration. Interestingly, although no change 
in cell morphology was observed (Fig. 20A and movie 12 in supplemental), quantitative 
 
40 
 
data indicate that 100nM nocodazole accelerated the velocity to 0.7544?m/min, even 
faster than that observed in controls (0.5249?m/min) (Fig. 22). Even more surprising, the 
velocity significantly decreased to 0.2147?m/min compared to controls (P<0.01) after 
washout. This finding reveals that a relatively low concentration of nocodazole may 
promote migration. It also indicates that the polymerization of microtubules at the plus 
end may modulate neural crest cell migration. These findings are consistent with those 
observed in EMT assay. 
 Cell vorticity in 100nM nocodazole (Fig. 23) showed a broader distribution than 
controls, indicating cells moved more randomly. This result supports the notion that the 
factor(s) that maintains cell migratory direction in controls was weakened in the presence 
of nocodazole. Also, this finding suggests that the plus end of microtubules may play a 
role in directing cell migration in neural crest cells. In some cell types like HeLa and 293T 
cells, the function of the plus end of microtubule in facilitating directional cell migration 
has been reported (Zhang, Shao et al. 2013). 
  
Figure 18. 30?M nocodazole suspended cell migration and caused cell death. Panel A 
shows pre-drug cell movement: cells behaved normally like controls. Panel B shows cells 
rounded up and gradually died after 30?M nocodazole treatment. 
A 
 
41 
 
  
Figure 20. 1?M nocodazole slowed cell migration and caused cell death after washout. 
Panel A shows cells movement after adding 10?M nocodazole. Cell migration slowed down a 
little. Panel B shows that cells migration was further slowed after removal of nocodazole, with 
many cells dying after 8 hours. 
Figure 19. 100nM nocodazole did not have an apparent effect on neural crest cell 
migration. Panel A shows cells migrating normally after adding 100nM nocodazole. Panel B 
shows cells continued normal migration after washout. 
 
 
42 
 
4.4 Inhibition of actin polymerization by latrunculin A irreversibly suspends 
cell migration 
 Actin filaments are building blocks for the cytoskeleton involved in many basic 
cellular processes (Schmidt and Hall 1998). Knowing that inhibition of actin 
polymerization is detrimental in different cell types, we wanted to test what effect it would 
have on neural crest cells. Using the same experimental set-up, we noted that 100nM 
latrunculin A suspended cell migration to a significantly low level, with the velocity 
decreased to 0.1313?m/min (Fig. 22). We also observed explant contraction in some 
cases, with a velocity of 0.4352?m/min, likely due to cell death (data now shown here). 
Cell shapes were distorted, as cells rounded up within 4 hours post-drug treatment and 
appeared to be dying (Fig. 21 and movie 13 in supplemental data). After washout to 
remove latrunculin A, the cells in the explants became loose probably because of the 
inhibition of actin and the removal of media during by washout. This was not observed 
after washout of other inhibitors, indicating the importance of actin filaments in the 
cytoskeleton and the maintenance of explant contact with the fibronectin-coated chamber 
slide. Interestingly, blebbistatin and Y-27632 treatment can also interrupt actin, but in an 
indirect way through the interaction among NM myosin II, ROCK and actin filaments (as 
shown in Fig 4). Our observation indicates that proper actin activity is crucial to neural 
crest cell migration. Unlike many cell types, neural crest cells are very sensitive to 
latrunculin A.  
 As summarized in Table 2, blebbistatin reversibly blocks cell migration but cell 
migratory direction changes after removal of blebbistatin; Y-27632 accelerates cell 
migration; nocodazole has differential effects on neural crest cell migration based upon 
the concentration used; and latrunculin A causes cell death even at a very low 
concentration. This suggests NM myosin II, ROCK, microtubules and actin filaments are 
all involved in neural crest cell migration. 
 
43 
 
  
Figure 21. Inhibition of actin polymerization by latrunculin A irreversibly blocks cell 
migration. Panel A shows pre-drug cell movement: cells behaved normal like the controls. 
Panel B shows that 100nM latrunculin A suspended cell migration and caused cell death. 
Panel C shows this effect could not be released by washout and cells eventually died. 
 
44 
 
 
 
  
MEANS in ?m/min 
Control = 0.5249 
Blebbistatin = 0.1133 ** 
Bleb washout= 0.5943 
Y27632 = 0.7082  
Noc 100nM =0.7544  
100nM washout = 0.2147 * 
Noc 1?M = 0.4163 
1?M washout = 0.0534 ** 
Noc 30?M =0.0208 *** 
30?M washout=0.3577 
LatA = 0.1313 ***  
 
Figure 22. Velocities of cells measured at the edge of the explants in the 
migration assay. Figure on the left shows the velocity comparisons among controls 
and each condition in migration assay. Each bar represents mean?SD. N.S; *P < 
0.05, **P < 0.01, ***P < 0.001). Table on the left gives means of the velocities on 
each experimental condition. 
 
45 
 
 Figure 23. Vorticity of cells on explants treated in different inhibitors. The first row 
shows the distribution of vorticity in controls, after washout in blebbistatin treatment and in 
Y-27632 treatment; the second row shows the distribution of vorticity upon treatment with 
100nM nocodazole and latrunculin.  
 
46 
 
Table 2. Summary of results for neural crest cell migration assay 
Treatment Phenotype Velocity 
(compared to 
control) 
Vorticity 
DSMO-treated 
(control) 
Normal migration   
50?M blebbistatin Blocks migration Statistically  
decreases 
N/A 
Bleb washout Resumes migration 
but direction changes 
Comparable More directional 
Y-27632 Normal migration Increases Less directional 
100nM nocodazole Normal migration Comparable Less directional 
100nM noco washout Normal migration Statistically  
decreases 
N/A 
1?M nocodazole Normal migration Comparable N/A 
1?M noco washout Normal migration but 
cells die later 
Statistically  
decreases 
N/A 
30?M nocodazole Causes cell death Statistically  
decreases 
N/A 
100nM latrunculin A Causes cell death Statistically  
decreases 
Less directional 
 
  
 
47 
 
Chapter 5:  Distribution of ?-actinin and palladin in neural crest cells 
To examine the effects of chemical inhibitors on the distribution of ?-actinin and 
palladin in neural crest cell explants, we performed immunohistochemistry on cells after 
inhibitor treatment. By looking at the distribution changes of these two actin-binding 
and/or actin-associating proteins compared to control explants treated with DMSO, we 
can get additional insight into the effects these inhibitor treatments had on the actin 
cytoskeleton.  
5.1 Distribution of ?-actinin and palladin in DMSO-treated (control) neural 
crest cell explants 
We first examined ?-actinin and palladin distribution DMSO-treated explants. 
?-actinin was found in filopodial and lamellipodial structures in the cytosol, while palladin 
co-localized with ?-actinin but was also expressed in nuclei. This is the first time the 
distribution of ?-actinin and palladin has been reported in cranial neural crest cells (Fig. 
24 and Fig. 25). 
?-actinin, as a focal adhesion protein, functions as a linker between the cytoplasmic 
domain of ?-integrins and actin filaments (Critchley DR 1999). In avian trunk neural crest 
cells, ?-actinin was shown to be localized to focal contacts and along actin microfilaments 
(Delannet, Martin et al. 1994) (Testaz, Delannet et al. 1999). It was also reported that in 
chick trunk neural crest cells (from the level of mid-otocyst to the third somite pair), 
?-actinin was localized around the nucleus, in focal adhesions, and in filopodia (Anthony 
R. Mwakikunga 2011), indicative of its function to bundle actin filaments into stress fibers. 
Our findings of the distribution of ?-actinin in cranial crest cells are in agreement with 
what has been reported in trunk neural crest cells.  
Palladin, an actin associated protein identified in 2000 (Parast and Otey 2000), 
directly binds to ?-actinin and localizes to stress fibers, focal adhesions, and other 
 
48 
 
actin-based structures (Jin 2011). Although palladin is thought to be of critical importance 
in regulating cell motility (Chin and Toker 2010), there are no reports showing its location 
or function in neural crest cells. Our localization results suggest that palladin, along with 
?-actinin, may serve as a cytoskeletal scaffold and to cross-link actin. Moreover, palladin 
localization to neural crest cell nuclei indicates that it may have additional functions other 
than as an actin-crosslinker, such as nuclei-cytosol communication.  
  
 
49 
 
 
 
 
 
 
Figure 24. Distribution of ?-actinin and palladin in control (DMSO-treated) explants. 
?-actinin (A, red) and palladin (B, green) co-localize to the cytosol, lamellipodia and filopodia; 
palladin also localizes to the nucleus. DAPI (C, blue) stains the nucleus. Rectangular area is 
magnified in Fig. 25. Bar = 50?M. 
 
50 
 
  
Figure 25. Distribution of ?-actinin and palladin in control (DMSO-treated) explants 
(magnified image of Fig. 24 in rectangular area). ?-actinin (A, red) and palladin (B, green) 
co-localize to the cytosol, lamellipodia and filopodia (arrows); palladin localizes to the nucleus 
(arrowhead). DAPI (C, blue) stains the nucleus. Bar = 25?M.  
 
51 
 
5.2 Distribution of ?-actinin and palladin in blebbistatin-treated neural crest 
cell explants 
 To examine potential changes in the distribution of ?-actinin and palladin in neural 
crest cell after inhibition of NM myosin II by blebbistatin, we fixed cells after blebbistatin 
treatment and performed immunohistochemistry. To maintain the blebbistatin 
treatment-induced change in cell morphology (as seen in Fig. 16B), blebbistatin (50?M) 
was added along with the fixative. Blebbistatin-treated neural crest cells developed 
rounder and smaller cell bodies (Fig. 26), lamellipodia disappeared, and filopodia 
appeared instead (Fig. 27, arrows).  ?-Actinin was observed in the cytosol and enriched 
in filopodia, and palladin co-localized with ?-actinin in the cytosol and filopodia. Palladin 
was also noted in the nucleus and appeared to be more abundant there compared to 
controls.  
In fibroblasts, NM myosin II localizes to actin bundles contacting growing adhesions, 
forming a striated pattern with ?-actinin. In other cells, such as in neuronal growth cones, 
NM myosin II may have a more direct role controlling retrograde flow in the peripheral 
zone (Medeiros, Burnette et al. 2006) When NM myosin II is knocked out, knocked down 
by small interfering RNA or inhibited with blebbistatin, large actin bundles are not 
observed in the lamellum, whereas the lamellipodium remains intact (Ponti, Machacek et 
al. 2004). In many cells, inhibition of NM II with blebbistatin, or genetic deletion of NM II, 
increases protrusiveness (Vicente-Manzanares, Ma et al. 2009); however, we did not 
observe obvious protrusions in neural crest cells. 
 In order to examine whether ?-actinin and palladin are involved in the contraction of 
explants after washout of blebbistatin and to identify potential reasons of explant 
contraction, we performed immunostaining after washout of blebbistatin (Figs. 28, 29). 
Cell morphology was slightly different from control treated cells with slimmer cell bodies 
and directional protrusions. Interestingly, ?-actinin and palladin were not equally 
distributed in cells. Most cells had a stronger distribution of ?-actinin and palladin in 
 
52 
 
regions of the cell facing the explants (Figs. 26, 27 arrows pointing at the explant). This 
asymmetric distribution of ?-actinin and palladin may be related to the changed polarity of 
the neural crest cells. At the same time, palladin nuclear distribution strongly decreased 
after washout of blebbistatin compared to controls and blebbistatin treatment. Moreover, 
vorticity data shows cells have a more directional movement than controls after washout 
of blebbistatin. The shift in localization of palladin from the nucleus to the cytosol, and ?- 
actinin to the edge of the cell facing the explant, correlates nicely with the accompanying 
vorticity change. This was the first time that the shift in localization of palladin from the 
nucleus to the cytosol was observed. 
 
 
53 
 
 
  
Figure 26. Distribution of ?-actinin and palladin in blebbistatin-treated explants. 
?-actinin (A, red) and palladin (B, green) co-localize to the cytosol and filopodia; palladin 
localizes to the nucleus. DAPI (C, blue) stains the nucleus. Rectangular area is 
magnified in Fig. 27. Bar = 50?M 
 
54 
 
 
Figure 27. Distribution of ?-actinin and palladin in blebbistatin-treated explants 
(magnified image of Fig. 26 in rectangular area). ?-actinin (A, red) and palladin (B, green) 
co-localize to the cytosol and filopodia (arrows); palladin localizes to the nucleus (arrowhead). 
DAPI (C, blue) stains the nucleus. Bar = 25?M.  
 
55 
 
 
  
Figure 28. Distribution of ?-actinin and palladin in blebbistatin-treated explants after 
washout to remove blebbistatin. ?-Actinin (A, red) and palladin (B, green) co-localize to the 
cytosol but show an asymmetric distribution in cells. Expression of palladin in nuclei is much 
reduced compared to blebbistatin-treated explants. Cells also developed apparent polarized 
cell bodies. DAPI (C, blue) stains the nucleus. Rectangular area is magnified in Fig. 29. Bar = 
50?M. 
 
56 
 
  
Figure 29. Distribution of ?-actinin and palladin in blebbistatin-treated explants after 
washout to remove blebbistatin (magnified image of Fig. 28 in rectangular area). 
?-Actinin (A, red) and palladin (B, green) co-localize to the cytosol but show an asymmetric 
distribution in cells (arrows); palladin localization to the nucleus is reduced. Cells developed 
polarized cell bodies. DAPI (blue) stains the nucleus. Bar = 10?M. 
 
 
57 
 
5.3 Distribution of ?-actinin and palladin in Y-27632-treated neural crest cell 
explants 
To examine the change in the distribution of ?-actinin and palladin in neural crest cell 
explants after inhibition of ROCK, and subsequently the Rho pathway, by Y-27632, we 
fixed cells after Y-27632 inhibition and performed immunohistochemistry. Compared to 
control-treated explants, Y-27632 treated cells did not have as flat of a cell shape and had 
more cell-cell contacts (Fig. 30) by filopodia and lamellipodia. ?-actinin and palladin are 
also uniformly co-localized in the cytosol, and palladin showed reduced distribution in 
nuclei, less than controls (Fig. 31).  
Rho GTPases are important regulators of both actin dynamics and cell-substratum 
adhesions in migratory cells, and thus are critical proteins involved in neural crest cells. 
The two subgroups of the Rho GTPases, Rho and Rac, with their effects on actin and 
adhesion dynamics, have been well studied (Bishop and Hall 2000; Wennerberg and Der 
2004). Rho induces the formation of stress fibers and focal adhesions, while Rac induces 
lamellipodial extensions and the focal complexes at the leading edge of a migratory cell 
(McHardy, Warabi et al. 2005). In Swiss 3T3 fibroblasts, injection of Y-27632 induced 
rapid disassembly of the focal adhesions and active formation of lamellipodia and 
associated focal complexes, which were due to the activation of Rac (Rottner et al., 
1999).  It has been reported that Y-27632 treatment alters neural crest morphology by 
reducing stress fibers and focal adhesions (Groysman, Shoval et al. 2008). Also, in 
neurons, Y-27632 (10 ?M) can trigger neuronal polarity formation at very early time 
points (Bito, Furuyashiki et al. 2000).  
 
 
58 
 
  
Figure 30. Distribution of ?-actinin and palladin in Y-27632-treated explants. ?-actinin (A, 
red) and palladin (B, green) co-localize to the cytosol; DAPI (C, blue) stains the nucleus. 
Rectangular area is magnified in Fig. 31. Bar = 50?M. 
 
 
59 
 
 
  
Figure 31. Distribution of ?-actinin and palladin in Y-27632-treated explants (magnified 
image of Fig. 30 in rectangular area). ?-actinin (A, red) and palladin (B, green) co-localize to 
the cytosol. DAPI (C, blue) stains the nucleus. Bar = 10?M. 
 
 
60 
 
5.4 Distribution of ?-actinin and palladin in nocodazole-treated neural crest 
cell explants 
To examine the changes in the distribution of ?-actinin and palladin in neural crest 
cell after inhibition of the polymerization of microtubules by nocodazole in different 
concentrations, we fixed cells after nocodazole treatment and performed 
immunohistochemistry. Compared to control treated explants, different concentrations of 
nocodazole had different effects on cell morphology as well as the distribution of ?-actinin 
and palladin (Figs. 32-38). 
With 30?M nocodazole treatment, cells developed two drastically different 
morphologies (Fig. 32). As shown in Fig. 33, cells that appeared to survive in 30?M 
nocodazole developed very flat cell shapes, with ?-actinin enriched in protrusions. From 
the time-lapse imaging data, we find that some neural crest cells that directly attach to the 
fibronectin bottom can survive this treatment. Most cells, like those shown in Fig. 34, 
however, have retracted cell bodies, with ?-actinin and palladin condensed in several 
dot-like areas. These cells were dead shortly after 30?M nocodazole treatment. 
With 1?M nocodazole treatment, neural crest cells adopted a flatter morphology 
(Figs. 35, 36) and seemed to have more filopodia compared to controls. ?- ?ctinin and 
palladin co-localized in the cytosol, and ?-actinin was enriched in filopodia. Palladin also 
appeared to localize more abundantly in nuclei compared to control cells. 
100nM nocodazole treatment increased cell motilities in time-lapse imaging 
experiments, whereas the cell morphology and distribution of ?-actinin and palladin was 
quite similar to the controls. ?lpha-actinin is slightly more enriched in protrusions in the 
experimental cells than in controls (Figs. 37 and 38). The large red dots in the ?-actinin 
staining are precipitates. 
It has been shown that perturbation of microtubule depolymerization 
by nocodazole slows the movement of undifferentiated neural crest cells and causes 
retraction of processes led by the immature neurons, implicating microtubules in the 
 
61 
 
morphogenesis of neural crest cells into neurons (Haendel, Bollinger et al. 1996). Other 
than this finding, however, nothing is known about the function of microtubules in neural 
crest cells. Here, we report that nocodazole can alter cell morphology as well as the 
distribution of ?-actinin and palladin. 
 
 
Figure 32. Distribution of ?-actinin and palladin in 30uM nocodazole-treated explants. 
?-Actinin (A, red) and palladin (B, green) co-localize to the cytosol, ?-actinin is more enriched 
in filopoida, and palladin localizes to the nucleus. DAPI (C, blue) stains the nucleus. 
Rectangular areas are magnified in Fig. 33 and Fig. 34. Bar = 50?M. 
 
 
62 
 
  
Figure 33. Distribution of ?-actinin and palladin in 30uM nocodazole-treated 
explants (magnified image of Fig. 32 in white rectangular area). Cells surviving in 
30uM nocodazole developed a flatter morphology. ?-Actinin (A, red) and palladin (B, green) 
co-localize to the cytosol, ?-actinin is enriched in filopodia (arrows), and palladin localizes to 
the nucleus (arrowheads). DAPI (C, blue) stains the nucleus. Bar = 25?M. 
 
 
63 
 
 
  
Figure 34. Distribution of ?-actinin and palladin in 30uM nocodazole-treated 
explants (magnified image of Fig. 32 in yellow rectangular area). Dying cells are 
smaller, with ?-actinin (A, red) and palladin (B, green) co-localizing and condensing into 
several dot-shape area in the cytosol (arrows). Palladin localizes to the nucleus in these cells 
(arrowheads).  DAPI (C, blue) stains the nucleus. Bar = 10?M. 
 
 
64 
 
 
Figure 35. Distribution of ?-actinin and palladin in 1uM nocodazole-treated 
explants. ?-Actinin (A, red) and palladin (B, green) co-localize to the cytosol and filopodia, 
and palladin localizes to the nucleus. DAPI (C, blue) stains the nucleus. Rectangular area is 
magnified in Fig. 36. Bar = 50?M. 
 
 
65 
 
 
 
Figure 36. Distribution of ?-actinin and palladin in 1uM nocodazole-treated 
explants (magnified image of Fig. 35 in rectangular area). ?-actinin (A, red) and 
palladin (B, green) co-localize to the cytosol and filopodia, ?-actinin is enriched in filopodia 
(arrows), and palladin localizes to the nucleus (arrowheads). DAPI (C, blue) stains the nucleus. 
Bar = 25?M. 
 
 
66 
 
 
Figure 37. Distribution of ?-actinin and palladin in 100nM nocodazole-treated explants. 
?-Actinin (A, red) and palladin (B, green) co-localize to the cytosol, and palladin localizes to the 
nucleus. DAPI (C, blue) stains the nucleus. Rectangular area is magnified in Fig. 38. Bar = 
50?M.  
 
 
67 
 
 
  
Figure 38. Distribution of ?-actinin and palladin in 100nM nocodazole-treated explants 
(magnified image of Fig. 37 in rectangular area). ?-Actinin (A, red) and palladin (B, green) 
co-localize to the cytosol, ?-actinin is enriched in protrusions (arrows), and palladin localizes to 
the nucleus (arrowheads), but is reduced compared to the controls. DAPI (C, blue) stains the 
nucleus. The large red dots in the ?-actinin staining are precipitates. Bar = 25?M. 
 
68 
 
5.5 Distribution of ?-actinin and palladin in latrunculin A-treated neural crest 
cell explants 
To examine the changes in the distribution of in neural crest cell after inhibition of the 
polymerization of actin filaments by latrunculin A, we fixed cells after latrunculin A 
inhibition and performed immunohistochemistry (Fig. 39). Because latrunculin A had a 
severe effect on cells and caused cell death, the cytoskeleton was likely partially distorted 
due to a reduction and/or loss of polymerization of actin. Immunostaining showed that 
cells looked smaller than controls due to retraction of the cell body. ?-Actinin and palladin 
were localized to the cytosol. Palladin was also found in nucleus but appeared reduced in 
experimental cells compared to controls. 
As the building blocks of cytoskeleton, disruption of actin filaments immediately leads 
to the arrest of neural crest cell movement and causes cell death (Haendel, Bollinger et al. 
1996). We observed the same results with very low concentration of latrunculin A. We 
also first report the morphological changes of cranial neural crest cells treated with 
latrunculin A by analyzing the distribution of ?-actinin and palladin.  
As summarized in Table 3, ?-actinin and palladin co-localize in the cytosol and in 
filopodia, while palladin localizes to nucleus. The nuclear localization of palladin shifted 
from the nucleus to the cytosol after washout of blebbistatin. At the same time, cells 
appeared to have asymmetric distribution of ?-actinin and palladin in the cytosol after 
washout of blebbistatin. Y-27632 treatment resulted in uniform distribution of ?-actinin 
and palladin in the cytosol. 
 
69 
 
 
  
Figure 39. Distribution of ?-actinin and palladin in 100nM latrunculin A-treated explants. 
Latrunculin A caused substantial cell death. ?-Actinin (A, red) and palladin (B, green) are still 
co-localized to the cytosol. DAPI (C, blue) stains the nucleus. Rectangular area is magnified in 
Fig. 40. Bar = 50?M. 
 
 
70 
 
 
 
 
 
 
 
Figure 40. Distribution of ?-actinin and palladin in 100nM latrunculin A-treated explants 
(magnified image of Fig. 39 in rectangular area). Latrunculin caused cell retraction and 
eventual death. ?-actinin (A, red) and palladin (B, green) are still co-localized to the cytosol. 
DAPI (C, blue) stains the nucleus. Bar = 25?M. 
 
71 
 
 
Table 3. Immunohistochemistry results and relationship with vorticity 
Treatment Phenotype Distribution of 
?-actinin 
Distribution of 
palladin 
Vorticity 
DSMO-treated 
(control) 
Normal 
migration 
Cytosol, 
lamellipodia and 
filopodia 
Nucleus, cytosol, 
lamellipodia and 
filopodia 
 
50?M 
blebbistatin 
Blocks 
migration 
Cytosol, filopodia Nuclear localization 
much higher than 
controls, cytosol 
and filopodia 
N/A 
Bleb washout Resumes 
migration but 
direction 
changes 
Asymmetric in 
cytosol with more 
localization on the 
side toward 
neural fold 
Very little nuclear 
localization, 
co-localization with 
?-actinin 
More 
directional 
Y-27632 Normal 
migration 
Uniform 
distribution in 
cytosol 
Less nuclear 
localization than 
controls, uniform 
distribution in 
cytosol 
Less 
directional 
100nM 
nocodazole 
Normal 
migration 
Cytosol, enriched 
in filopodia 
Uniform distribution 
in cells 
Less 
directional 
1?M 
nocodazole 
Normal 
migration 
Cytosol, enriched 
in filopodia 
More nuclear 
localization than 
controls, cytosol   
N/A 
30?M 
nocodazole 
Causes cell 
death 
Cytosol , enriched 
in filopodia (prior 
to death ) 
Nucleus and 
Cytosol 
N/A 
100nM 
latrunculin-A 
Causes cell 
death 
Cytosol (prior to 
death) 
Nucleus and 
cytosol 
Less 
directional 
 
72 
 
Chapter 6: Discussion and future plans 
The neural crest is referred as the fourth germ layer because neural crest cells are 
able to give rise to variety of cell types, tissues and organs; it has fascinated 
developmental biologists for over a hundred years. Proper development of vertebrate 
embryos requires neural crest cells to undergo EMT and migrate to targets in a strictly 
regulated spatio-temporal manner. The specific cellular pathways regulating the 
processes, however, are poorly understood. Neural crest cells also share many similar 
properties with cancer cells as they are both migratory and plastic. Thus, deciphering the 
mechanisms controlling neural crest cell EMT and migration will shed light on the 
potential pathways that could be targeted for cancer therapies. Through time-lapse 
imaging, we were able to dynamically record neural crest cell behaviors. Here we show 
NM myosin II, ROCK, actin and microtubules to be critical factors involved in neural crest 
cell EMT and migration. We also performed immunohistochemistry of ?-actinin and 
palladin, two actin-linkers, on the explants treated with inhibitors of above cytoskeleton 
components to test our hypothesis of the potential roles of these factors on neural crest 
cell migration.  
Previous research in zebrafish embryos showed inhibition of NM myosin II by 
blebbistatin disrupted cell blebbing, which is required for hindbrain neural crest cells to 
undergo EMT in vivo (Berndt, Clay et al. 2008). Here we show in chick cranial neural 
crest cell explants, blebbistatin irreversibly blocks neural crest cell EMT in vitro. A 
previous study showed that the ROCK pathway regulates NM myosin II by 
phosphorylation and activation of NM myosin II (Bresnick 1999) in some cell types. Our 
findings, however, indicate that ROCK and NM myosin II likely are two distinct pathways 
in chick neural crest cells. We measured the velocity of cells on the edge of the explants 
to examine the spreading speed of the explants in order to characterize cell motility. In the 
 
73 
 
EMT assay, velocity drastically decreased to a negligible level (0.0182?m/min), 20-fold 
lower than the controls (0.3743?m/min). While cells were able to resume their motility 
after washout to a comparable level to the controls, they failed to resume EMT with no 
cells managing to come out from the neural fold after 5 hours. This finding is in 
agreement with what was found in hindbrain neural crest cell in zebrafish. In the neural 
crest cell migration assay, we showed that blebbistatin also blocks cell migration with a 
velocity decreased to 0.1133?m/min, 4.6-fold lower than the controls (0.5249?m/min); 
however, cell migration can be fully resumed to similar velocity (0.5943?m/min) as the 
controls after removal of blebbistatin. Intriguingly, cells changed their original direction to 
the neural folds causing the contraction of the explants. We noted that cells developed 
rounder cell bodies and increased number of filopodia in the presence of blebbistatin and 
resumed their normal morphology after removal of the drug. In the EMT assay, after 
removal of blebbistatin, epithelial cells became motile again but lost their ability to break 
the boundary of the neural folds and develop to individual mesenchymal cells. This 
indicates that NM myosin II may facilitate the loss of cell-cell adhesions at the boundary 
of the neural folds to promote neural crest cell EMT. In the migration assay, as mentioned 
in Chapter 4, chemotaxis and/or changes in cell polarity could be potential reasons for 
explant contraction after removal of blebbistatin. Our immunohistochemistry results, 
provide evidence for the theory of polarity changes through asymmetric distribution of 
?-actinin and palladin. The vorticity data showed that during explant contraction, cells 
appeared to be more linear or non-curled than cells in controls, which suggests alteration 
of NM myosin II by blebbistatin treatment and washout caused a change in the signals 
that direct neural crest cell migration. Collectively, NM myosin II has different functions 
during neural crest cell EMT and migration.  
Functions of the Rho pathway during neural crest cell EMT and migration have been 
long discussed in both chick and zebrafish embryos. Although Rho has been implicated 
in neural crest cell EMT and migration, different research models have yielded conflicting 
 
74 
 
conclusions. Liu and Jessell first reported RhoB was expressed in the chick dorsal neural 
tube and migrating neural crest cells. This group also showed that inhibition of Rho by C3 
exotoxin prevented the delamination of neural crest cells from trunk neural tube explants 
but had little effect on later migration of neural crest cells (Liu and Jessell 1998). 
Research on later stage zebrafish hindbrain neural crest cells also showed that the use of 
a ROCK inhibitor led to a reduction of EMT (Berndt, Clay et al. 2008). This group also 
recently showed inhibition of ROCK upon C3 exotransferase disrupted neural crest cell 
EMT in zebrafish (Clay and Halloran 2013). We found that the Rho inhibitor, Y-27632, 
promotes EMT, with more mesenchymal cells emerging from neural folds compared to 
the controls within the same time period. Consistent with our findings in the EMT assay, 
Y-27632 treatment also led to an increase the velocity of cells migrating out from the 
neural folds. This is the first time that an inhibitory effect of the Rho pathway has been 
documented in cranial neural crest cells. Our findings are in agreement with what has 
been observed in chick hindbrain neural crest cells. This study showed that alterations of 
RhoA in vivo induced more cells to become migratory(Rupp and Kulesa 2007). Another in 
vitro research on late stage trunk neural crest cells also reported C3 and Y-27632 both 
enhanced cell emigration without affecting cell proliferation (Groysman, Shoval et al. 
2008). The conflicting findings may be caused by different species and/or axial levels. In 
addition, we also found in the vorticity test that Y-27632 treatment slightly altered cell 
behaviors toward a more curled direction. It has been reported in Xenopus and zebrafish 
that, through inactivation of Rac, RhoA is involved in regulation of neural crest cell 
directional migration (Matthews, Marchant et al. 2008). Our findings provided more data 
regarding the function of RhoGtpase in regulating cell directional migration.  
Only one other report in chick trunk neural crest explants has examined microtubule 
function in neural crest cells. This study revealed that perturbation of microtubule 
depolymerization by nocodazole slowed the movement of undifferentiated neural crest 
cells and caused retraction of processes extended by the immature neurons, implicating 
 
75 
 
microtubules in the morphogenesis of neural crest cells into neurons (Haendel, Bollinger 
et al. 1996).  In our EMT and migration assay using different concentrations of 
nocodazole, we found that the effect of nocodozole on both neural crest cell EMT and 
migration was dose-dependent. Similar dose-dependent effects were observed with 
endothelial cells. In this study, micromolar concentrations of nocodazole irreversibly 
changed the barrier function and caused cell death, whereas nocodazole in 100 to 200 
nM concentrations reversibly increased the permeability of the endothelial monolayer 
(Smurova, Birukova et al. 2008). 30?M nocodazole caused irreversible blocks on neural 
crest cell EMT and migration leading to most cell death. This block may due to the toxicity 
of high concentration of nocodazole on cells that perturbs many cellular process including 
EMT and migration. 1?M nocodazole blocks neural crest cell EMT by affecting the cell 
capacity to maintain mesenchymal cell shape, but cells were able to move with a 
comparable velocity as the controls. Washout in the EMT assay led to a decrease in 
velocity, but the mesenchymal cell type was resumed, indicating a partial resumption of 
EMT even though cells eventually died after a few hours. Interestingly, the velocity 
decrease after washout was also observed in the migration assay and was significant. 
Although no apparent change in cell morphology was observed in presence of 1?M 
nocodozaole, cells eventually died 8 hours after washout. This suggests nocodazole 
potentially has an unknown function facilitating cell motility other than through 
microtubule depolymerization. This function compensates for the lost of microtubule 
function inhibited by nocodazole, leading to mild changes in cell behavior compared to 
after washout, in which the compensated function and microtubule function both 
disappear, causing a cessation of cell migration. 100nM also affects neural crest cell EMT, 
with few cells emerging from the explants that had an accelerated velocity compared to 
control and 1?M nocodazole treatment. This implicates the plus end of microtubule in 
neural crest cell EMT. The velocity did not change after washout in the EMT assay but 
significantly decreased in the migration assay, which is consistent with the findings in the 
 
76 
 
1?M nocodazole treatment on migration assay. Note that the velocity of cells in the 
migration assay in the presence of 100nM nocodazole is faster than the control and 
significantly decreased after washout, which further supports our hypothesis with regards 
to an additional function in facilitating cell motility provided by nocodazole. Vorticity data 
on 100nM nocodazole treatment showed a broader distribution, suggesting that the plus 
end of microtubules plays a role in cell migration potentially in modulating the 
directionality of cell movement.  
 A previous study has shown that microfilament disruption by cytochalasin 
immediately arrests the movement of neural crest cells and causes them to round-up 
(Haendel, Bollinger et al. 1996). An indirect study of actin filaments on ILK, a linker 
between integrins and actin cytoskeleton, showed that knock out of ILK in mouse neural 
crest cells led to embryonic lethality (Arnold, Zang et al. 2013). In agreement with 
previous findings, our experiment with 100nM latrunculin A caused drastic changes in cell 
morphology and cell death in migratory neural crest cells. This harsh effect could not be 
removed after washout of latrunculin-A. This suggests that actin filaments, as the building 
blocks of cytoskeleton, are involved in neural crest cell migration. 
Although the in vitro experiment attempts to mimic the in vivo events during EMT and 
migration, this might not be the case in vivo. It might be different or contradictory to what 
we observed since cells receive cues from external environment including ephrins and 
semaphorins (that help define their migratory pathways) while they are migrating. The 
intrinsic molecular factors in charge of cell behaviors during EMT and migration may be 
different, since they may be regulated by external signals in vivo. With that, our findings 
still demonstrate, for the first time, the changes of cell behaviors, velocity, and vorticity 
after different inhibitor treatments during EMT and migration in chick cranial neural crest 
cells.  
Further exploration could focus on validating these in vitro findings in vivo by 
employing knock-down techniques to eliminate specific pathway components. Several 
 
77 
 
intriguing findings also require further attention. Deciphering the underlying mechanism of 
cell movement after washout of blebbistatin will help understand pathways controlling 
directional cell migration. At the same time, the unknown pathway that nocodazole 
regulates with respect to facilitating cell motility also is worth exploring.  
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