Molecular mechanisms involved in the specification of embryonic ectoderm in the zebrafish embryo

Chapter 2: Wnt signalling mediated by Tbx2b is required for cell migration Chapter 3: Zebrafish Frizzled 2 is required for dorsoventral specification in the gastrula leading to the forma

I want to express my sincere gratitude to my advisor, Vladimir Korzh, for accepting me into his laboratory If this postgraduate program would be likened to a journey through an exotic land, then I could not have found a better guide Encyclopedic in knowledge and skilled in techniques, he provided an ideal environment for me to mature into a working scientist

I want to thank members of my thesis advisory committee, Dr Thameem Dheen, Dr Suresh Jesuthasan and Prof Wan-Jin Hong for their valuable advice and guidance Dr Dheen has laid much of the foundation for the work described in this thesis Dr Suresh and Prof Hong have influenced the direction of this project more than they realized

I thank members of V.K lab.- Alexander Emelyanov, the resident guru in all things molecular who laid the foundation for Fz work and provided

me with many of the reagents used in this study; Sergei Parinov, an expert in transposon-mediated enhancer trap, an enthusiastic diver and a fabulous photographer, for encouragement and distraction when I needed it; Michael Richardson, for laying the groundwork on Fz2, and shared my appreciation of wit, understatement and sarcasm; Cathleen Teh, an expert in zebrafish

electroporation, for making the BAC-recombinant Fz construct I am also grateful to Shang-Wei Chong and Kar-Lai Poon, fellow travellers in the exotic land, for putting up with me My warmest appreciation for Lee-Thean Chu, another fellow traveller, for painstakingly proof-reading my manuscripts and this thesis

I thank Paramjeet Singh, a dear friend and mentor in my formative years, for showing me the meaning of science and how it ought to be

conducted I will never forget his advice to ‘Never let work get in the way of

1

progress!’ Many fine ideas were born over coffee or beer with him And many other silly ones were shot down by him even before I lifted a pipette, saving

me precious time He showed me that it’s not how much work I do, but how much of that work is absolutely essential to answer the question Most

importantly, is it the right question to begin with

I thank Chai Chou, fellow lunatic and masochist, for showing me the meaning of staying the course In the face of difficulties bordering on the absurd, he stoically marched on At times when the going got too tough for

me, I only have to look at him to renew my strength

I have very fond memories of my time in IGBMC (Strasbourg, France) I thank Uwe Strähle, Ferenc Müller, Patrick Blader, Thomas

Dickmeis and Nadine Fischer for making me feel at home 2000 km away I also thank Stephen Wilson, Miguel Concha and C.P Heisenberg of the

University of College, London (UCL) for their hospitality and kind tutelage in the fine art of single cell transplantation

I am also indebted to R Moon, S Vriz and U Takeda for reagents and cDNAs To Bill Chia and Xiao-Hang Yang for discussions during the initial stages of this work To Chin-Heng, Amy and everyone in the fish facility; without them there would be no work I would also like to thank many of my colleagues in Temasek Life-Science Laboratory (formally Institute of

Molecular Agrobiology) for support and encouragement This work was made possible by the generous support from the Agency for Science, Technology and Research of Singapore

Finally, to Kalwant Singh (1960-1992) He’s got a Ticket To

Heaven

This work would not be complete without the generous

contributions from Sasha, Cathleen and Mike Thank you all, from my heart

2

Chapter 2: Wnt signalling mediated by Tbx2b is required for cell migration

Chapter 3: Zebrafish Frizzled 2 is required for dorsoventral specification

in the gastrula leading to the formation of posterior structures 60

Fig 1 Phenotypes of tbx2b morphants at 24 hpf 27

Fig 2 shh expression in control and tbx2bMO injected embryo 29

Fig 3A Anti-tbx2bMO blocks transcription of tbx2b mRNA and is

distributed unevenly in the blastula 31

Fig 3B Anti-tbx2bMO impairs morphogenesis of anterior forebrain 32Fig 4 Tbx2b is required for cells to adopt a neural fate 34Fig 5 Tbx2b is required for convergence cell movement 35

Fig 6 ‘Exclusion’ phenotype can be rescued by tbx2b mRNA 38

3

Fig 7 Inhibition of Fz7 signalling leads to the ‘exclusion’ phenotype 40

Fig 9 Early function of Tbx2b and Fz7 is required for cell adhesion 46Fig 10 Cell movement during gastrulation is affected in knockdown

Fig 12 Fz2 function is required to promote neural fates 65Fig 13 Transplanted deep cells move to ventral regions 67Fig 14 Fz2 is necessary for formation of posterior structures 69

Fig 15 Regulation of shh promoter and enhancer elements in COS7

4

During gastrulation, optimal adhesion and receptivity to signalling cues are essential for cells to acquire new positions and identities via

coordinated cell movements T-box transcription factors and the Wnt

signalling pathways play important roles in these processes Here we show that Tbx2b is required cell-autonomously for cell adhesion and cell movement

in the embryonic ectoderm In chimeric embryos, cells deficient in Tbx2b are defective in cell adhesion and fail to migrate to the neural plate Using this

‘exclusion’ phenotype as a screen, we show that Tbx2b acts within the context

of Fz7 via components of the canonical Wnt pathway Independent of cell movement, Tbx2b is also required for neuronal differentiation In contrast to studies in amniotes, our screen failed to demonstrate a role for FGF signalling

in the dorsal movement of embryonic ectoderm leading to neural plate

formation Instead, our results illustrate the importance of Tbx2b-mediated Wnt signalling in this process

We have also fate-mapped a previously undefined population of deep lying vegetal cells in the zebrafish blastula, and showed that Fz2

function is essential for their migration to the ventral side of the gastrula and their subsequent incorporation into the posterior structures of the embryo This result highlights the early commitment of cell fate during the blastula period, and links the positions of cells in the gastrula to their earlier positions

in the blastula Thus, Fz2 is required for the initial specification of the

dorsoventral axis in the gastrula We further demonstrate that Fz2 is required for the specification of posterior paraxial mesoderm

5

Steven Fong, Alexander Emelyanov, Cathleen Teh and Vladimir Korzh Wnt

signaling mediated by Tbx2b regulates cell migration during formation of the neural plate (Development, submitted, joint first authorship with Emelyanov)

Sudha Puttur Mudumana, Thomas Dickmeis, Steven Fong, Inna Friedrich, Alexander Emelyanov, Zhiyuan Gong, Uwe Strähle, Vladimir Korzh

Sleptsova-colIXa2 and determination of lineage of non-notochordal precursors of axial

structures of vertebrates (Development, in preparation)

Alexander Emelyanov, Steven Fong, Cathleen Teh, Chen Sok Lam, Vladimir

Korzh Zath3 acts as a proneural determinant in multipotential neural

precursors in hindbrain (Development, in preparation)

Michael Richardson, Steven Fong, Dmitry Bessarab, Alexander Emelyanov,

Vladimir Korzh Frizzled2 acts downstream of Wnt3a as a determinant of lateral mesoderm (in preparation, joint first authorship)

Lee Thean Chu, Steven Fong, Dmitry Bessarab, Alexander Emelyanov,

Suresh Jesuthasan, Andrej Minin and Vladimir Korzh Blastocystic cavity plays a role in establishing first cell lineages in zebrafish (Developmental Biology, in preparation)

6

Chapter 1: Introduction

7

1.1 Zebrafish as a model organism

Zebrafish (Danio rerio) has become an important model organism

in the study of vertebrate developmental biology Its main advantage is the availability of numerous genetic mutants (Development Vol 123, 1996) and the relative ease with which mutagenesis can be effected by chemical (ENU), physical (X-ray) and genetic (transposon or viral mediated insertions) means

This powerful genetics is not available in Xenopus (although Xenopus

tropicalis is currently being developed as a genetic system) and chick

Although mice and rats are valuable mammalian genetic systems (natural mutants and ‘knockouts’), the embryos of these animals are not as readily accessible and available as the hundreds of transparent, externally fertilized eggs of the zebrafish Having said this, the mutants currently available for zebrafish are by no means saturating Many genes, when mutated, will lead to very early developmental arrest or death, thus eluding screens designed around observable phenotypic alterations In addition, methods for targeted mutagenesis (similar to ‘knockout’ mice) are currently unavailable in zebrafish Thus, cloning of genes from mutants is a labour intensive candidate

gene/map-based effort To complicate matters, it is an accepted view that there was a genome duplication event early in the evolution leading to modern teleosts (Wittbrodt et al, 1998; Woods et al, 2000) As such, redundancy in gene function is not uncommon This should in no way be viewed as an

indictment against zebrafish as a model organism Its genetics and

embryological accessibility, coupled with modern molecular tools - like

dominant negative constructs and the recently developed anti-sense

morpholino oligonucleotides which work by inhibiting translation of target mRNAs (Nasevicius and Ekker, 2000) - for manipulating gene function, makes

8

for a powerful system to understand higher vertebrate biology This is

particularly evident in the study of human diseases, where many zebrafish mutants resemble human clinical disorders (Dooley and Zon, 2000)

1.2 General zebrafish development

The zebrafish embryo develops rapidly at 28°C At 2.75 hours post fertilization (hpf) or midblastula stage (512 cells), zygotic transcription begins This time point marks the midblastula transition (MBT) Cell divisions are very rapid and synchronized in the first 10 cleavages At MBT, the cell cycle begins

to lengthen and cells lose their synchrony thereafter At around this time, the first lineage restriction is observed, where the formation of the yolk syncytial layer (YSL) separates the yolk cell from the rest of the blastoderm After one

or two more cell divisions, the lineage of epithelial enveloping layer (EVL) cells becomes restricted to exclusively generate the periderm, while the deep layer cells (DEL) proceed to give rise to the embryo proper As such, the YSL

is an extra embryonic structure and can thus be seen as the equivalent of the syncytiotrophoblast of the mammalian embryo By 4.5 hpf epiboly movements signal the start of gastrulation Lineage tracing by intracellular injection of dye shows that DEL cells located near the animal pole will give rise to ectodermal fates (including the definitive epidermis) and cells located near the blastoderm margin will give rise to mesodermal and endodermal fates At 6 hpf, during the gastrula period, the dorsal organizer or shield is well defined and gastrulation

is now in full swing It is also at this stage that the future fates of respective regions of the gastrula are defined (Kimmel et al, 1990) It has been

suggested that the position of a cell in the blastula is unrelated to the position

of its descendants in the gastrula due to the extensive cell mixing that occurs

9

during the transition from blastula to gastrula (Warga and Kimmel, 1990) Subsequently, a study demonstrated that the fate of cells could be determined

as early as the 16-cell stage (Strehlow et al, 1994) However, due to the extremely large molecular weight of the labelling tracer employed in this

study, the finding was controversial It is suggested that the position of

blastomeres at the 16-cell stage may indeed predispose them to a particular region of the gastrula, and thus determine their ultimate fates, but the final contribution that any early blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed (Helde et al, 1994;

Wilson et al, 1995)

By 10 hpf, primary gastrulation is complete At this stage the three germ layers are well defined and the neural plate is formed The first somite appears at about 10.5 hpf and signals the beginning of the segmentation period The tail bud is now formed and with it, the recently proposed

secondary gastrulation begins (Kanki and Ho, 1997; Agathon et al, 2003) At

16 hpf (14 somites stage), the notochord separates from the ventral part of the neural keel and the yolk extension begins to protrude At 19 hpf (20

somites stage), the lens placode appears and neurons have growing axons

At this stage, trunk myotomes produce weak contractions By 24 hpf,

pigments, fins and a beating heart are visible By day 3, the larva breaks free from the chorion which has protected it until now, and becomes free

swimming For a thorough review and description of developmental stages, see Kimmel et al, 1995 and Westerfield, 1995

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1.3 Cell movement during gastrualtion

During gastrulation, embryonic cells undergo large scale

movements and rearrangements, leading to the formation of the germ layers

oriented around the embryonic axes In zebrafish, unlike Xenopus, the initial

cleavage planes of the early blastula are not aligned with the future

dorsoventral (D-V) axis and the time point of D-V axis specification is unclear (Helde et al, 1994) However, by early-gastrula (5.3 hpf) the shield or

organizer is formed and it defines the dorsal side of the embryo Three sets of simultaneously occurring cell movement events can be distinguished during gastrulation (Warga and Kimmel, 1990): 1) Epiboly, where radial cell

intercalations cause the thinning of the blastoderm and spread it across the yolk cell These rearrangements mix cells located deeply in the blastoderm with the more superficial ones; 2) Deep cells undergo involution to form the nascent hypoblast of the embryonic shield, and folds the blastoderm into the epiblast and hypoblast As gastrulation progresses, involuting hypoblast cells move anteriorwards relative to cells that remain in the epiblast This

movement shears the positions of cells that were neighbours before

gastrulation Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution The epiblast is equivalent to the embryonic ectoderm 3) Convergence and extension (CE) movements toward the dorsal side of the gastrula where mediolateral cell intercalations take place in both the epiblast and hypoblast By this

rearrangement, cells that were initially neighbouring one another become dispersed along the anterior-posterior axis of the embryo

In Xenopus, all three movements are interdependent due to the

tight coupling of the ectodermal and the mesendodermal sheets (Shih et al,

11

1994) Although the zebrafish ectoderm, like that in Xenopus, is also

organized and moves as a sheet (Concha and Adams, 1998), the

mesodermal cells migrate as individuals or groups of cells Lacking the tight coupling of ectoderm to the underlying mesoderm, the three gastrulation movements in zebrafish are thus independent of each other (Warga et al, 1990) Of the three mass cell movement events, CE movement is most

extensively investigated In the zebrafish, individual cells lose their

independence and integrate their behaviour to achieve a coherent movement over the yolk (Concha and Adams, 1998) However, at the molecular level, most of these studies focused on CE within the mesoderm (reviewed in Myers

et al, 2002) Thus, the molecular mechanism governing dorsal movement of the overlying ectoderm leading to the formation of the neural plate is not well understood By 10.5 hpf (yolk-plug stage), primary gastrulation is complete

A requirement for a posterior organizer and secondary gastrulation centred around the tailbud has been proposed for the subsequent formation of the yolk extension and posterior structures of the embryo (Kanki and Ho, 1997; Agathon et al, 2003) Cells which were initially on the ventral side of the gastrula (opposite the shield) now contribute to the posterior of the animal During the gastrula stage, the D-V axis is defined by the position of the shield After the yolk-plug stage, as neurulation (the formation of the neural tube from the neural plate) proceeds, the D-V axis is now applied to the embryo proper

neural keel will eventually fold the whole sheet into a tube As this is being accomplished, the placode (the border between the neural plate and

epidermis) becomes crest like and will eventually give rise to neural crest cells When the neural plate finally folds into a tube, what was initially the margin of the neural plate now becomes the most dorsal portion of the neural tube (the roof plate) and the epidermis from both halves of the embryo meet and cover the neural tube In higher vertebrates, the neural canal is formed as the plate folds into a tube In zebrafish, the progress of neurulation is slightly different Instead of the neural plate folding in on itself, dorsal/medial

convergence of the neural plate causes a progressive thickening of the neural keel, culminating in the formation of a solid rod The neural canal is then hollowed out of this rod to give rise to the neural tube (Kimmel et al, 1994; Papan and Campos-Ortega, 1997)

1.5 Cell fate vs cell movement

It is not entirely clear if cells move according to or in search of identity Two possible scenarios have been put forward: a linear pathway model, where cell movement behaviour is a downstream consequence of cell-fate specification; and a parallel pathway model, where a cell initially

assesses its position within the gastrula and interpretation of this information leads to the parallel activation of a fate specification program and a cell

movement program (Myers et al, 2002) The zebrafish is an attractive and convenient system to study the interaction and relationship between fate specification and cell movement Up to the late gastrula stage, embryonic cells are pluripotent, and will readily adopt the fate of their new location when transplanted (Ho and Kimmel, 1993) Since techniques for cell transplantation

13

and single blastomere injection are well established, chimeric embryos can thus be generated Recently, the novel gene ‘knockdown’ technique using antisense morpholino oligonucleotides (MO) to inhibit translation of a target mRNA was demonstrated to work in zebrafish (Nasevicius and Ekker, 2000) with some limitations regarding nonspecific toxicity (Heasman, 2002) This

technique, in combination with in vivo analysis in chimeric embryos generated

by cell transplantation or single blastomere injection, enables the analysis of factors involved in cell movement and fate specification during gastrulation

1.6 The T-box genes

The spontaneous mutation, Brachyury (T), was first described in

mice (Dobrovolskaia-Zavadskaia, 1927) and the gene was finally cloned in

1990 (Herrmann et al, 1990) When opto-motor blind (omb) from Drosophila

was cloned (Pflugfelder, 1992) it became clear that these two genes formed a new family of transcription factors The highly homologous and sequence specific DNA-binding domain was termed the T-domain, and this new family was collectively referred to as the T-box genes Since then, many T-box genes were cloned from a wide variety of species (Papaioannou et al, 1998) With the completion of a few genome sequences, it is now known that there are no more than 18 T-box genes in mammals

T-box genes encode proteins ranging in size from 50 kDa to 78 kDa and are comprised of a structural and a functional domain: the T-domain, and a transcriptional activator or repressor domain The crystallographic

structure of the T-domain of the Xenopus orthologue of Brachyury (Xbra)

(Muller and Herrmann, 1997) and the human Tbx3 (Coll et al, 2002) showed that the T-domain is unique among DNA-binding domains Analyses of

14

downstream targets and binding-site selection experiments showed that

several members bind to a core consensus sequence TCACACCT, although different members preferentially bind sequences that contain two or more of these core motifs in various orientations (Sinha et al, 2000; Conlon et al, 2001) In addition, murine Tbx2 has been shown to bind a variant T-site

(Lingbeek et al, 2002) However, little is known about how T-box genes select and regulate their downstream targets (Tada and Smith, 2001) T-box proteins have been shown to function both as transcriptional activators and repressors

In all cases studied, this functionality requires sequences in the

carboxy-terminal portion of the protein Again, little is known about how the

transactivation domain selects and binds interacting partners to effect

transcriptional regulation

T-box transcription factors play important roles in vertebrate

development (Smith, 1999; Wilson and Conlon, 2002; Showell et al, 2004)

Brachyury (T) has been shown to be required for cell adhesion (Yanagisawa

and Fujimoto, 1977), cell migration (Hashimoto et al, 1987), and

morphogenetic cell movements during gastrulation (Wilson et al, 1995) T and the zebrafish orthologue no tail (ntl) are known to be essential for the

specification of axial mesoderm (Smith 1999) Similar to T, Tbx16/spadetail (spt) has been shown to be required for mesodermal cell movement (Ho and Kane, 1990); and Xbra has been demonstrated to function as a switch

between cell migration and convergent extension during gastrulation (Kwan and Kirschner, 2003) In addition, a number of human disorders have been linked to mutations in T-box genes, confirming their medical importance They include Holt-Oram syndrome/TBX5, Ulnar-Mammary syndrome/TBX3, and more recently DiGeorge syndrome/TBX1, ACTH deficiency/TBX19 and cleft palate with ankyloglossia/TBX22 (Packham and Brook, 2003)

15

1.7 The Tbx2 family

Zebrafish tbx2b belongs to the Tbx2 family and was previously named tbx-c Its expression is first detected by whole-mount in situ

hybridization at 8.5 hpf in the axial mesoderm By 12 hpf it is expressed in the

eye field, ventral diencephalon and, uniquely among the Tbx2 family, the

notochord At 24 hpf, the domains of expression include the eyes, otic

vesicles, trigeminal ganglia, Rohon-Beard cells, cell of the epiphysis and pronephric ducts By this stage, the expression in the notochord has receded

to the tailbud, in the region of the chordo-neural hinge (Dheen et al, 1999)

tbx2a, another zebrafish Tbx2 gene, is highly homologous to tbx2b with

extensive overlap in their expression patterns However, tbx2a is not

expressed in the notochord and the epiphysis at any stage, but is instead expressed in the cells of neural crest, and subsequently in the branchial

arches It is very likely that these two genes are the product of a chromosomal duplication event which occurred sometime during the evolution of teleosts, about 450 million years ago (Force et al, 1999)

Tbx2b has been demonstrated to function in a complex signalling

web downstream of TGF-β signalling, ntl and floating head (flh) for the

specification of the notochord In brief, a loss-of-function experiment, via expression of a dominant negative (dn)-Tbx2b that lacked the carboxy-

over-terminal transactivation domain, resulted in embryos with reduced midline mesoderm and expanded lateral mesoderm In agreement, the opposite gain-

of-function experiment, via overexpression of the full length tbx2b, led to an

expansion of the midline mesoderm and formation of ectopic midline

structures at the expense of lateral mesodermal tissues Animal explant

experiments showed that Tbx2b acts downstream of early mesodermal

16

inducers (activin and ntl) and reveal an autoregulatory feedback loop between

ntl and tbx2b Thus, in zebrafish, two T-box factors are required for the

formation of the notochord (Dheen et al, 1999)

Tbx2 of Xenopus, chick, mouse and human have been

characterized quite extensively None of them were shown to be expressed in the notochord In chick, Tbx2, together with Tbx3,4 and 5 were shown to be involved in limb and digit identity specification (Gibson-Brown et al, 1998; Suzuki et al, 2004) Human TBX2 is known to repress the melanocyte-specific TRP-1 promoter (Carreira et al, 1998) and is itself a target for the

microphthalmia-associated transcription factor in melanocytes (Carreira et al, 2000) Murine Tbx2 is also known to repress the tumor suppressor gene p14ARF via a variant T-site in its promoter (Lingbeek et al, 2002) Although murine Tbx2 is known to contain domains for both transcriptional activation and repression (Paxton et al, 2002) no target of Tbx2 activation has been identified Recently, TBX2 was shown to be linked to the development of breast cancer (Sinclair et al, 2002), possibly via its role in the repression of p19ARF

and bypassing senescence control (Jacobs et al, 2000) TBX2 was also implicated in cell cycle control via its repression of p21WAF1cyclin-dependent

kinase inhibitor (Prince et al, 2004) The Drosophila Tbx2-related omb is

regulated by the Wingless (Wg), Decapentaplegic (Dpp) (Grimm and

Pflugfelder, 1996) and Hedgehog (Hh) (Kopp et al, 1997) signalling pathways;

Xenopus Tbx2 is known to function within the Sonic hedgehog (Shh) pathway

(Takabatake et al, 2002); and in chick, Tbx2 is known to function through both Shh and bone morphogenetic protein (BMP, vertebrate homologue of Dpp) signalling (Suzuki et al, 2004) Thus, it is of interest to determine the pathways

regulating zebrafish tbx2b

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1.8 The Wnt signalling pathway

Wnt (vertebrate Wg) signalling is involved in cell movement and fate determination (Moon et al, 1997) Based on their ability to induce a

second axis in Xenopus, Wnt proteins have been divided into two classes: the

Wnt1 class, which induces a second axis; and the Wnt5 class, which does not Molecular dissection of the two classes has shed light on their respective mechanisms and roles in development The Wnt1 class acts via β-catenin and

is shown to function in fate determination This is equivalent to the Drosophila

Wg/Armadillo pathway and is now commonly referred to as the canonical pathway (Moon et al 2002) The Wnt5 class is β-catenin-independent and is known to play essential roles in cell polarity and cell movement (Veeman et al, 2003) This has been shown to be similar to the Planar Cell Polarity (PCP)

signalling pathway in Drosophila and is now referred to as the non-canonical pathway In Xenopus, Wnt3a and Wnt8 have been implicated in the

posteriorisation of the neural plate (Bang et al, 1999; McGrew et al, 1995 and

1997) Mouse Wnt3a is involved in the specification of paraxial mesoderm (Liu

et al., 1999) In zebrafish, mutants of wnt5, wnt8 and wnt11 have been

identified (Rauch et al, 1997; Heisenberg et al, 2000; Lekven et al, 2001) Wnt5a and Wnt11 were shown to act through the non-canonical signalling pathways and affect morphogenetic cell movements (Heisenberg et al, 2000; Tada and Smith 2000; Wallingford et al, 2001); whilst Wnt8 signals through the canonical Wnt pathway to mediate mesodermal patterning by promoting ventral cell fates (Lekven et al, 2001) The two pathways share some common components: the 7-pass transmembrane Frizzled (Fz) receptors and the downstream adaptor molecule Dishevelled (Dsh)

The amino-terminal extracellular Wnt binding domain and the

18

carboxy-terminal PDZ-binding domain of the Fz protein are known to be

essential for Fz function Deletion of the PDZ-binding domain creates a

dominant negative molecule that will bind the Wnt ligand but will not transduce signal intracellularly Dsh is modular in nature and has three functional

domains The DIX domain is essential for β-catenin activity, whereas the DEP domain is involved in non-canonical signalling (Topczewski et al 2001) In

Drosophila, it is known that the DEP domain is important for the differential

recruitment of Dsh to the plasma membrane by Fz upon binding of Wg ligand, and provides signalling specificity in the PCP and Wingless signalling

pathways (Axelrod et al,1998) The situation with the PDZ domain is more ambiguous It has been shown to affect either (Axelrod et al, 1998), or both (Sokol, 1996), the β-catenin and CE pathaway The data suggest that the PDZ domain may serve a regulatory role, rather than as an obligate

transduction module (Wharton, 2003)

Fz receptors play a key role in diverting the Wnt signals into

specific pathways In vitro studies have demonstrated that Wnts may interact

with any Fz receptor, however the binding affinity varies among different

ligand-receptor pairs (Hsieh et al, 1999; Rulifson et al, 2000) Recently a Fz4

‘knockout’ mouse was generated (Wang et al, 2001) which implicated Fz4 in the maintenance of the nervous system during adulthood In Xenopus, Fz3

interacts with XWnt1 and is required for the formation of the neural crest

(Deardorff et al, 2001) Xenopus Fz7 appears to operate via the canonical

β-catenin pathway for fate specification (Sumanas et al, 2000), a non-canonical pathway essential for convergent extension (Djiane et al, 2002), and a Dsh-independent pathway that controls cell-sorting behaviour in the mesoderm (Winklbauer et al 2001) It was also demonstrated that rat Fz2 is capable of interacting with XWnt5A in the Wnt/Ca2+ pathway (Slusarski et al, 1997b) In

19

addition, it has been suggested that zebrafish Fz2 may function as a receptor for Wnt5 and may play a role in the regulation of gastrulation through the non-canonical pathway (Sumanas et al, 2001)

The signalling cascade downstream of Dsh is complex The

proteins Axin and GSK3β form a multiprotein complex with Adenomatous Polyposis Coli (APC) Axin regulates multiple signalling pathways by serving

as a scaffold protein, controlling diverse cellular functions in proliferation, fate determination, and suppression of tumorigenesis (Luo and Lin, 2004)

Recently, it was demonstrated that Axin also plays an important role in a JNK signalling pathway by utilizing discriminatory domains for its distinct roles in the both the β-catenin pathway and the JNK pathway (Zhang et al 2000) GSK3β is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling In the absence of Wnt signalling, GSK3β is recruited to the APC complex via interaction with Axin, where it hyperphosphorylates β-catenin, marking it for ubiquitylation and destruction, thereby inhibiting the canonical pathway Activation of Wnt signalling leads to the inhibition of GSK3β activity and an accumulation of stabilized cytoplasmic (signalling) β-catenin, which becomes available to bind the TCF/LEF family of transcription factors leading

to target gene expression (Nelson and Nusse, 2004)

β-catenin, in addition to its role in Wnt signalling, is also an integral component of the adhesion complex by linking cadherins through α-catenin to the actin cytoskeleton Phosphorylation-dependent release of β-catenin from the cadherin complex not only regulates the integrity and function of the adhesion complex, but may also be an alternative mechanism for activating β-catenin signalling For an in-depth review of the potential interaction between Wnt, β-catenin and cadherins, see the review by Nelson and Nusse (2004) In

20

contrast, the non-canonical pathway was shown to act through the DEP and PDZ domains of Dsh but not APC, and activates the JNK/SAPK signalling cascade instead (reviewed in Moon, 1997 and 2002) However, in view of the role of Axin in JNK signalling (Zhang et al, 2000), this may need to be

reviewed Below is a figure summarizing the essential components of the human Wnt signalling pathway and their interactions with each other (courtesy

of Biocarta, http://www.biocarta.com/pathfiles/h_wntPathway.asp)

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1.9 Neural induction

As mentioned earlier, formation of the neural plate during

gastrulation involves concurrent cell fate specification and cell movement events The embryonic ectoderm has the potential to acquire either epidermal

or neural fates Spemann and Mangold showed that the vertebrate organizer produces signals to induce the neural fate (Spemann et al, 1924; Spemann, 1938) These neural-inducing signals were initially thought to play instructive roles Subsequent experiments showed that they function viatranscriptional repression of BMP gene expression and clearance of BMP ligands by

secreted inhibitors Thus, neural induction depends on the suppression of BMP signalling by organizer-derived inhibitors, and in the absence of cell-cell signalling ectodermal cells will adopt a neural fate This is referred to as the

‘default model’ of neural induction (reviewed in Munoz-Sanjuan et al, 2002)

In Xenopus, BMP-inhibiting factors include chordin, follistatin,

noggin, cerberus and xnr3 (reviewed in Harland, 1994; Harland, 2000) Both the Fibroblast Growth Factor (FGF) and Wnt signalling pathways have been implicated in this process In chick, gain-of-function experiments (by

transplantation of neural plate tissue or implantation of beads containing proteins of interest into the non-neural epiblast) suggest that the early events

of neural induction are regulated by FGF signalling, which could be blocked

by continued Wnt signalling (reviewed in Wilson et al, 2001) In Xenopus,

although FGF was shown to be a direct neural inducer (Lamb et al, 1995) and

a truncated FGF receptor was shown to block neural induction by

endogenous Xenopus inducers (Launay et al, 1996), gain-of-function

experiments (by injection of mRNAs) also demonstrated that neural induction depends on Wnt/β-catenin inhibition of BMP (Baker et al, 1999; Wessely et al,

22

2001) In the zebrafish, Wnt/β-catenin-dependent bozozok (boz) is sufficient

to suppress expression of bmp (Fekany-Lee et al 2000) While a secondary

axis consisting of ectodermal derivatives and lateral mesoderm was induced

by FGF8 (Furthauer et al 1997), in vivo blockade of FGF signalling in

Xenopus and zebrafish caused notochord deficiency but failed to disrupt

neural development (Schulte-Merker et al, 1995) As such, there seem to be conflicting requirements for FGF and/or Wnt signalling in neural induction between amniotes and anamniotes This difference in molecular mechanism needs to be further clarified

1.10 BMP and axis specification

It has also been demonstrated that modulations in BMP levels affect fate specification along the D-V axis without affecting those along the anterior-posterior (A-P) axis (Barth et al, 1999) In lower vertebrates, induction

of the D-V axis may occur as early as the cleavage stage This is marked by the enrichment of maternal determinants such as β-catenin in the prospective embryonic dorsal pole (Jesuthasan and Strähle, 1996; Aanstad and Whitaker, 1999; Ober and Schulte-Merker, 1999), and this process is dependent on maternal Wnt signalling (Miller et al, 1999) Induction of the A-P axis occurs at the onset of gastrulation and involves a combination of several pathways, including the Wnt signalling pathway (Christian and Moon, 1993; Glinka et al, 1998; Piccolo et al, 1999; Bradley et al, 2000; reviewed in Gamse and Sive, 2000) Wnts are capable of inducing a secondary axis if expressed vegetally prior to midblastula transition (MBT) However, Wnts will promote ventral and posterior fates if expressed after MBT (Christian and Moon, 1993)

23

1.11 Summary

In this thesis, we use the zebrafish as a model to study the

molecular mechanisms behind the specification of embryonic ectoderm Specifically, we attempt to isolate the role of Tbx2b during the formation of the neural plate, a prominent event during gastrulation, where known signalling casdades like Wnt, FGF and BMP are involved in cell fate specification and large scale morphogenetic cell movement within the ectodermal layer The transparancy of the zebrafish embryo, couple with established techniques of embryology and availability of mutants and molecular tools, allow us to

demonstrate that Tbx2b plays a role in cell adhesion and cell movement during this critical period of development In addition, we show that a hitherto

unmapped group of deep vegetal cells of the blastoderm that expresses Fz2

migrates to the ventral region of the gastrula, and thus establish a link

between the animal-vegetal position of the blastula and the dorso-ventral axis

of the gastrula

24

Chapter 2: Wnt signalling mediated by Tbx2b is required for cell migration during formation of the neural plate

25

2.1: Results

The previous study on tbx2b (Dheen et al, 1999) was performed

with dn-Tbx2b lacking the C-terminal transactivation domain Another

approach with dominant negative constructs, one without the N-terminal domain, was initially employed for this study but was found to be non-specific and pleiotropic (data not shown) and was thus abandoned MO-mediated loss-of-function approach (targeted gene ‘knockdown’) was reputed to be

T-more specific (Nasevicius and Ekker, 2000) and two anti-tbx2bMO were designed for this study The first (tbx2bMOv1) straddles the start codon of

tbx2b, with the sequence 5’-GGA AAG GGT GGT AAG CCA TCA CAG T-3’

(start codon underlined); and the second (tbx2bMOv2) targets a region 5’

upstream of the the first , with the sequence 5’-GGT AAG CCA TCA CAG TCC CTG TAA A-3’ The two MOs were injected into 1-cell stage embryos

(pan-embryonic injection) to establish their efficacy tbx2bMOv2 was found to

be more effective in producing a phenotype than tbx2bMOv1 and was

therefore used for all experiments in this study (henceforth referred to as

tbx2bMO).

2.1.1 Phenotype of Tbx2b morphants

Pan-embryonic ‘knockdown’ of Tbx2b with tbx2bMO delayed the

onset and progression of gastrulation by up to 2 hours and produced

phenotypes at 24 hpf in a dose-dependent manner (Table 1, Fig 1) The phenotypes generated were arbitrarily classified into 3 groups (Gp) Group 1 morphants were severely affected, with development retarded by about 4h and anterior-posterior (A-P) axis shortened Eyes were either not present or

26

Figure 1 Phenotypes of tbx2b morphants at 24 hpf a, WT

embryo at 24 hpf b, Severely affected embryos Development was

retarded by about 4h compared to WT Morphants had severely

shortened A-P axis Eyes were not present or small Notochord

was absent and somites fused Embryos were non-motile c,

Embryos were less retarded than Gp1and A-P axis less truncated

Notochord was malformed in some cases Somites were U-shapedbut seldom fused Eyes were small and closely spaced Embryos

were motile d, Mildly affected embryos with slightly retarded

development A-P axis was shorter and embryos were curled Eyeswere still smaller than WT Embryos were motile

small Notochord was absent and somites were fused and embryos were not motile (Fig 1b) In Group 2, morphants were less retarded than Group1 and the A-P axis was less truncated Notochord was malformed in some embryos Somites were U-shaped, but seldom fused and eyes were small and closely spaced (Fig 1c) Group 3 morphants were slightly retarded, but motile The A-

P axis was short and curled, and eyes were small (Fig 1d) In summary,

similar to boz-/- embryos (Fekany-Lee et al, 2000) the tbx2b morphants have

short A-P axis, absent or malformed notochord, fused somites, small and/or cyclopic eyes, and smaller brain

Table 1 tbx2bMO produced phenotypes in a dosage dependent manner.

tbx2bMOv1 produced identical phenotypes at higher doses

Injections of a sense MO up to 10 pmol did not produce any phenotypes To

establish the specificity of tbx2bMO, the midline axial mesoderm of the

morphants were analyzed and compared to embryos obtained from the

overexpression of dn-Tbx2b Similar to dn-tbx2b injected embryos, the

morphants had malformed notochord and floor plate, as shown with the

marker sonic hedgehog (shh) (Fig 2a-d) In the most severely affected Group

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Figure 2, shh expression in control and tbx2bMO injected embryo.

a, lateral anterior view of prechordal plate in ctrl embryo shh is

expressed in the forebrain/midbrain boundary and the tegmentum (tg)

b, Gp1 embryos showed attenuated expression of shh in the

tegmentum (asterisks) c, Ctrl at 19hpf showed expression of shh in

floorplate of the trunk only but not in the notochord (asterisks) d, Gp2

embryos with only isolated clumps of shh expressing floorplate cells

remaining (arrow head) but shh expression persisted in the notochord

(asterisks) e, Dorsal view of Gp1 embryo showed the almost complete

loss of shh in the trunk region of the embryo The boxed section is

enlarged in f, showing the shh positive cells as remnants of notochord

(arrow)

29

1 morphants, only isolated cells from the remnants of the notochord were

present This is reminiscent of the mutant flh (Fig 2e-f), and is therefore in

agreement with the conclusions derived from the previous study (Dheen et

al,1999) In addition, when co-injected with tbx2bMO pan-embryonically, translation of tbx2b-myc containing the target site of tbx2bMO was blocked in

a dose-dependent manner (Fig 3Aa), suggesting that the phenotype

observed with tbx2bMO was indeed specific to the ‘knockdown’ of tbx2b.

The tbx2b expression is first detectable by whole mount in situ

hybridization (WISH) in the axial mesoderm at 8 hpf However, RT-PCR

showed that the transcripts are present as early as 50% epiboly (5.5 hpf, Fig 3Ab) To date, developmental functions have not been demonstrated for any gene before the onset of expression as detected by WISH However, this early presence of the transcript presented a problem, in that the expression of

tbx2b could not be accurately determined We did the next best thing we

could, by measuring the levels of transcript in the dorsal and ventral half of 5.5 hpf embryos (when the shield could be seen with confidence) with real-time

PCR The result indicated that tbx2b is present at a higher level in the ventral gastrula, compared to control EF1α transcript (Fig 3Ac) Could there be an early function for tbx2b at such a low level, and what might that function be?

Cross sections of the morphants at the level of the eyes (Fig 3B) showed that the neural tube was severely malformed: the ventricle in the anterior forebrain was not expanded, the eyes were not invaginated to form proper retinal cups, and the lenses were either absent or very small (Fig 3Bb and 3Bd) This hinted at a role for Tbx2b in neural development However, the malformation

of embryonic structures presented a problem Traditional methodologies such

as analyses with molecular markers became ambiguous When a marker failed to stain by WISH in a structure that was either absent or defective, as

30

50% Shield 75% 90% 14-hpf24hpf

c

Figure 3A Anti-tbx2bMO blocks translation of tbx2b

mRNA tbx2b is distributed unevenly in the gastrula a,

Recombinant tbx2b-myc mRNA containing the target

sequence for tbx2bMO in the 5’UTR, start codon and part

of the T-domain of tbx2b was injected into 1-cell stage

embryos alone, or with varying concentrations of

tbx2bMO Western blot with anti-myc antibody shows that

translation of tbx2b-myc was blocked in the presence of

tbx2bMO in a dose dependent manner b, RT-PCR of

tbx2b transcript using primers specific to its 5’-UTR

showed that it was present as early as 50% epiboly c,

Real-time PCR showed that tbx2b transcript was found at

a higher level in the ventral half of 5.5 hpf gastrula

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Figure 3B Anti-tbx2bMO impairs morphogenesis of anterior

forebrain a-d, tbx2b is required for proper development of the

eyes and forebrain a, Dorsal view of control 24hpf embryo with

expression of pax2.1 in the optic stalk (os) and midbrain-hindbrain

boundary (mhb) b, Plastic cross section of control 24hpf embryo

forebrain (counter-stained with toluidine blue) at the level of the

lens c, Dorsal view of tbx2bMO injected 24hpf embryo shows the

absence of pax2.1 expression in the optic stalk (asterisk) d, Cross

section of the embryo shows severe disorganisation of the

forebrain and eyes

32

highlighted by the marker pax2.1 in the eyes (Fig 3Bc), it became difficult to

establish the relationship of cause and effect To circumvent this, the

behaviour of Tbx2b-deficient cells in chimeric embryos were analyzed instead The chimeras were generated by two complimentary approaches - cell

transplantation and single-blastomere injection - to target ectodermal

derivatives

2.1.2 Chimeric embryos

Fluorescein-dextran labelled cells of the animal blastoderm were transplanted from 4 hpf wild type (WT) donor embryos into the same location

in WT hosts of the same stage By 24 hpf these labelled cells were found in

the brain, eyes, and epidermis (Fig 4a-c) In contrast, labelled tbx2bMO

injected cells appeared only in the epidermis (Fig 4d-f), supporting the

suggestion that Tbx2b may be required during the formation of the neural plate To confirm this, a single central blastomere of 16-cell stage embryos was injected (1/16 injection, Fig 4g-i) with fluorescein, either with or without

tbx2bMO, and the position of the labelled cells later determined At 3.25 hpf,

both control and tbx2bMO injected embryos showed labelled clones in the

ectoderm (Fig 4g), suggesting that right up to this stage there were no

discernible behavioural differences between control and tbx2bMO injected

cells At 24 hpf, all control embryos had labelled cells in the eyes, epidermis and central nervous system (CNS, Fig 4h) In addition, about 30% of them showed labelled cells in the somites of the posterior trunk, but no labelled cells were found in the axial mesoderm (ie the notochord and the floor plate) This is in agreement with the findings of earlier fate mapping exercises (Helde

et al, 1994; Strehlow et al, 1994) In contrast, tbx2bMO injected embryos had

33

3.25h

Dorsal view,

16-cell stage

Side view,3.25h

Figure 4 Tbx2b is required for cells to adopt a neural fate a-f, Deep

cells from embryos co-injected with lysine-fixable fluorescein-dextran

(70kD)/tbx2bMO at 30% epiboly were transplanted into same stage WT

host a, Distribution of transplanted control cells in vivo, and b, after

photoconversion c, Cross section shows transplanted cells in the

neuroretina and brain d, Distribution of transplanted tbx2b 'knockdown' cells in vivo, and e, after staining f, Cross section shows presence of

transplanted cells only in the epidermis (arrowheads) g-i, Single cell

injection of 16-cell stage embryos with tbx2bMO restricts its decendents

to an epidermal fate g, One central blastomere of 16-cell stage embryos

was injected with fluorescein either with or without tbx2bMO, and

embryos developed to 24hpf Both control and tbx2bMO injected

embryos showed a similar distribution of clonal population of labelled

cells in vivo at 3.25h h, Dorsal view of control 24hpf embryo shows

labelled cells in all derivatives of the neuroectoderm (eyes, CNS and

epidermis) i, In tbx2bMO injected embryo labelled cells were present

only in epidermis

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Figure 5 Tbx2b is required for convergence cell movement a-b,

WISH with the pan-neural marker sox19 shows a delay in

convergence of the neural plate and absence of some domains of

expression (arrow) in tbx2bMO injected embryo (b) compared to

control (a) c-f, WISH with bmp2 (c, d) and fz7 (e, f) show similar

expression patterns between control and tbx2b morphant at 5 hpf g-i,

Transplanted fluorescein labelled tbx2b deficient cells continue to

express sox19 in 5 hpf WT host.

labelled cells only in the epidermis (Fig 4i) The clonal decendants from the single labelled blastomere are of similar lineage to those cells that were

transplanted, ie ectodermal Thus, two separate approaches that labelled and tracked ectodermal cells support the observation that in the absence of Tbx2b labelled ectodermal cells do not contribute to the neural plate

In both transplantation and single-blastomere injection

experiments, there were reduced numbers of Tbx2b deficient labelled cells This could arise from either an increase in cell death or a decrease in cell

proliferation Staining of tbx2b morphants with acridine-orange and terminal

deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay showed no significant increase in apoptosis, implying that the reduction in cell count was a result of impairment in cell proliferation This is supported by reports implicating human TBX2 in cell cycle control and oncogenesis (Jacobs et al, 2000; Prince et al, 2004)

2.1.3 Tbx2b is required for the patterning of the neural plate

The early pan-neural transcription factor Sox19 (Vriz et al, 1995) is expressed in the neural plate at 10 hpf (Fig 5a) In tbx2b morphants it was

expressed in a broader area, with a pronounced gap in expression around the

midline (100%, n=37, Fig 5b, arrow) This suggests that the dorsal

convergence gastrulation cell movement was delayed in the morphants bmp2 and fz7 were expressed in the morphants at 5 hpf in a manner similar to

control embryos despite a delay in epibolic movement (Fig 5c-f) In addition, transplanted

donor cells from tbx2b morphants expressed sox19 in WT hosts at 5 hpf (Fig

5g-i) These results suggest that fate specification is not affected by the loss

36

of Tbx2b function

When control 1/16 injected embryos were stained for both

fluorescein and sox19 at 10 hpf, control labelled cells were found in the neural plate and epidermis (Fig 6a) In contrast, tbx2bMO-injected labelled cells

were excluded from the neural plate at 10 hpf (Fig 6b) and by 24 hpf these embryos had labelled cells only in the epidermis This suggests that the lack

of labelled cells in the 24 hpf morphants observed in previous experiments was due to the absence of labelled cells in the neural plate by 10 hpf 1/16

injection of tbx2b mRNA did not lead to the accumulation of labelled cells

within the neural plate, instead labelled cells with normal morphology were found in the midline region at 10 hpf and in the notochord at 24 hpf in about

10% of the injected embryos (n=30, Fig 6c-d) Although this result is

consistent with the role of Tbx2b in the formation of axial mesodermal

structures (Dheen et al, 1999) it is nevertheless interesting and surprising since the clonal population derived from the initial injected cell was essentially

ectodermal in origin Thus, overexpression of tbx2b caused a re-specification

of fate across germ layers

The ‘exclusion’ phenotype was significantly reduced by co-injection

of a tbx2b mRNA lacking the target site for tbx2bMO in a dose dependent

manner (Fig 6e) This was accompanied by the appearance of

overexpression phenotype of tbx2b in some embryos where labelled cells

were found in the midline and the notochord at 10 and 24 hpf, respectively The appearance of the overexpression phenotype in this rescue experiment suggests that the dose of mRNA used (1 ng) is optimal; higher doses will not likely lead to a greater degree of reduction in the ‘exclusion’ phenotype These

results support the specificity of tbx2bMO and demonstrate the requirement of

Tbx2b in the patterning of the neural plate

37

Figure 6 ‘Exclusion’ phenotype can be rescued by tbx2b mRNA.

Compared to control (a) 1/16 injection of tbx2bMO led to 'exclusion'

phenotype (b) at 10 hpf Neural plate highlighted with sox19 (blue) and

fluorescein labelled cells in brown In contrast, 1/16 injection of tbx2b

mRNA (1.0 ng) led to (c) labelled cells with normal morphology in the

midline (arrow) at 10 hpf and, (d) the notochord (arrows) at 24 hpf,

consistent with the role of tbx2b in axial mesodermal specification e,

Exclusion phenotype at 10 hpf from tbx2bMO (0.05 pmol) was rescued with

tbx2b mRNA lacking the target site in a dosage dependent manner In the

presence of tbx2bMO, 1.0 ng mRNA led to labelled cells in the midline

similar to (a) and (b) while significantly reducing the number of embryos with

‘exclusion’ phenotype 0.5 ng mRNA was less effective in reducing the

exclusion phenotype but did not lead to labelled cells in the midline

38

2.1.4 Inhibition of Wnt pathway can produce the ‘exclusion’ phenotype

The ‘exclusion’ phenotype provided an assay to screen for

signalling pathways that Tbx2b might be operating in (Table 2) Consistent

with the role of BMP suppression in neural induction, 1/16 injection of bmp2

mRNA led to exclusion in all experimental embryos This is in contrast to injection of dn-BMPR, which blocked BMP signalling (Neave et al, 1997) Thus, the physiological relevance of this phenotype in the context of neural induction is validated 1/16 injections of PKA (a negative regulator of the Hh pathway, Fig 7a), dn-FGFR3 and XFD (Fig 7b-c, which blocked FGF

signalling) (Hammerschmidt et al, 1996; Schulte-Merker et al, 1995) failed to recapitulate the exclusion phenotype and thus ruled out the involvement of the Hedgehog and FGF pathways upstream of Tbx2b In the case of XFD, closer

inspection of labelled cells within the sox19 positive neural plate showed that

they are small and morphologically undifferentiated (Fig 7d), a phenotype not observed in PKA and dn-BMPR injected embryos, suggesting that FGF

signalling may be required for the differentiation of neural cells subsequently

In contrast, inhibition of Wnt signalling by overexpression of a dn-Fz7 that lacked the C-terminal intracellular PDZ-binding domain led to the ‘exclusion’ phenotype (Fig 7e)

Detailed analyses showed that a similar effect could be obtained

with anti-fz7MO (fz7MO, Fig 7f), but not with MOs against the closely related

fz7a (Fig 7g) and the more distantly related fz2 fz7 is expressed in the

ectoderm during gastrulation (Fig 8a); thus, it is present in the dorsal

ectodermal cells at the time when they acquire neural fates In addition,

ectopic overexpression (by injecting DNA construct directly into the cell of cell stage embryos) of a dn-Fz7-EGFP/BAC (with the EGFP inserted into

1-39

Figure 7 Inhibition of Fz7 signalling leads to the ‘exclusion’

phenotype Flat mounts of 10 hpf embryos, double stained for

fluorescein (brown) and sox19 (blue), after single cell injection at

16-cell stage with (a) PKA, (b) dn-FGFR3, (c) XFD (enlarged in d),

(e) dn-Fz7, (f) fz7MO, (g) fz7aMO and (h) XdsΔDEP.