Characterization of gli2 2b genes in zebrafish hindbrain development 1

CONTENTS 1.1 Zebrafish as an emerging model for development studies 2 1.3.2 Prepatterning of neural plate along the anterioposterior and 1.3.5 Sequential onset of neuronal and glial diff

CHARACTERIZATION OF GLI2/2B GENES IN

ZEBRAFISH HINDBRAIN DEVELOPMENT

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2006

ACKNOWLEDGEMENTS

Time has passed by faster than I expected since I arrived here in July, 2001 To me the whole postgraduate study is quite challenging, and it would be impossible to get through it without the helps from many people who I should be thankful to

My foremost gratitude is to my honorific supervisors: A/P Gong Zhiyuan (Department of Biological Sciences) and A/P Vladimir Korzh (Institute of Molecular and Cell Biology), for giving me this opportunity to carry out research under their guidance Their attitude toward research and personalities as well impressed me so much that it encourages me to keep on going forward without any hesitation even in the most frustrating time

Secondly, I would like to give my heartfelt thanks to A/P Hong Yunhan and A/P Wang Shu (Department of Biological Sciences), as my degree committee members, for sharing all the ideas and insights

My research work has been done in both labs in Department of Biological Sciences and Institute of Molecular and Cell Biology Although the working styles are quite different in these two labs, the people from both labs are very energetic and also friendly

to me I have to admit that I enjoy the time that I have spent in these two big labs and I really appreciate the favors from all of you: Yan Tie, Yilian, Tong Yan, Bihui, Xinjun, Siew Hong, Xiufang, Zhiqiang, Qinwei, Huiqin, Shizhen, Wan Hai, Haiyan, Shalin, Prakash, Hu Jing, Xiaoming, Bensheng, Xukun, Shan Tao and Zeng sheng in Gong’s lab; and Sasha, Lana, Li Zhen, Steven, Catheleen, Lee Thean, Kar Lai, Sergei, Dmitri, Igor, Shangwei, William, Mike and Gao Rong from IMCB Also, I would like to thank people

in the general office and those handling the fish facility in the department and TLL/IMCB for their assistant whenever required

I am also grateful to my dearest parents for letting me go abroad to fulfill my dream although they would rather see me being together with them And I am also in debt to my beloved wife for her self-giving support and care

Finally, I would like to render my appreciation to National University of Singapore for providing me the graduate research scholarship during these years

CONTENTS

1.1 Zebrafish as an emerging model for development studies 2

1.3.2 Prepatterning of neural plate along the anterioposterior and

1.3.5 Sequential onset of neuronal and glial differentiation from neural

1.4.1.1 The cascade of Shh-Patch-Ci signaling pathway is well studied in

1.4.2 From fruit fly and zebrafish to human: evolutionary conservation and

1.4.4 Patterning of ventral neurons under the regulation of Gli through

2.1 DNA applications and genomic mapping 54

2.4.1 Zebrafish 74

3.2.2 Comparison of expression patterns of the two gli2s of zebrafish

3.3.2 gli2b expression is altered in embryos deficient in components of

3.3.3 gli2b expression in the lateral domains and MHB is not regulated by

3.4 gli2b and regulation of neural precursors 114

3.4.7 Inhibition of gli2b caused the disruption of notch1 and gfap

3.4.8 Inhibition of gli2b did not affect the segmentation of hindbrain 134

3.5.2 Inhibition of Gli2b caused abnormal development of

4.1 Zebrafish Gli2b belong to Ci/Gli zinc finger transcription factor family 153

4.4 Mutant analysis demonstrated that gli2b is regulated by an integration of

4.5 Gli2s play important roles in zebrafish development similar to that in

4.6 The interaction of Gli2b and Notch signaling in the hindbrain development 161

LIST OF FIGURES AND TABLES

Fig 1-1 Camera lucida sketches of the zebrafish embryos at selected

Fig 2-1 Mechanism of SMART cDNA systhesis (reproduced from user

Fig 2-4 pCS2+ vector map (reproduced from Dave Turner/Ralph Rupp,

tuebingen, Germany)

69

Fig 2-5 pEGFP-N2 Vector map (reproduced from http://www.clontech.com) 70

Fig 2-6 Structures of DNA and morpholino oligonucleotides R and R’ denote

continuation of the oligomer chain in the 5’ or 3’ direction, respectively (Reproduced from Corey et al., 2001)

77

Fig 3-1 Isolation and cloning of full length zebrafish gli2b cDNA

clone

90

Fig 3-2 The complete nucleotide sequence of the zebrafish gli2b

cDNA and deduced amino acid sequence

92

Fig 3-3 Alignment of the zebrafish Gli2b with zebrafish (z) and

mouse (m) Gli2 proteins

96

Fig 3-4 The phylogenetic tree of vertebrate Gli family comprising

Gli1, Gli2 and Gli3

98

Fig 3-5 gli2b genomic sequence used to design primers for gene

mapping

100

Fig 3-7 Temporal expression of gli2b as defined by RT-PCR 102

Fig 3-8 The spatial pattern of gli2b expression during zebrafish

Fig 3-9 gli2b expression in the hindbrain during late development 106

Fig 3-10 gli2b expression in the 48 hpf hindbrain is Hh dependent 109

Fig 3-13 Validation of gli2b MO for blocking translation of zebrafish

gli2b

116

Table 3-2 Phenotypes obtained after injection of gli2b morpholino

oligonucleotides in zebrafish embryos

120

Fig 3-20 Expression of early neurodifferentiation markers in the

hindbrain of Gli2b morphant

133

Fig 3-22 Gli2/Gli2b regulate nkx2.2 expressions in the hindbrain at 30

Fig 3-26 Axonal scaffold and formation of neuronal clusters in the 48

hpf Gli2b morphant

149

LIST OF COMMON ABBREVIATIONS

ace acerebellar

cyc cyclops

mib mind bomb

MO morpholino oligonucleotide

Ngn1 Neurogenin 1

RT-PCR reverse transcriptase-polymerase chain reaction

smu slow-muscle-omitted

syu sonic-you

tRNA transfer ribonucleic acid

yot you-too

Ke, Z., Lim, E.s., Korzh, V., Gong, Z Characterization of gli2b gene in zebrafish

neurogenesis 8th Biologcial Sicences Graduate Congress 2003 National University of Singapore, Singapore

Ke, Z., Emelyanov, A., Lim, E.S., Korzh, V., Gong, Z A novel member of zebrafish

gli gene, gli2b, and its expression affected by Sonic Hedgehog signaling pathway

during embryonic development Sir Edward Youde Memorial Fund Postgraduate Conference: Model Organism Research and Human Diseases 2004 Hongkong, China

SUMMARY

Three Gli proteins are known as key transcription factors that play important roles in neurodevelopment, by regulating the expression of downstream genes under Hedgehog (Hh) signaling pathway in vertebrates Zebrafish has been shown as a good model for genetic study and thus has been applied for the characterization of the components in Hh signaling cascade, including Gli family members Among zebrafish Gli family members, Gli2 is the first one identified and its dominant negative mutant

you-too was characterized However, many questions remain regarding the roles of

Gli2 in zebrafish despite the intense analysis This study demonstrated that there is a second Gli2 (Gli2b) in zebrafish which plays major roles in neurogenesis in contrast

to the previously found Gli2, which will explain the functional differences of Gli2 between zebrafish and mice reported so far

In this study, the zebrafish gli2b was cloned and mapped and its expression was

detected in the neural plate in early neurulation In contrast, during this developmental

period zebrafish gli2 was expressed mostly in the lateral mesoderm The two genes

also showed different expression patterns in the diencephalon, hindbrain and hypothalamus Combined with functional analysis, this study supports the hypothesis that subfunctionalization indeed is consistent with the maintenance of most duplicate genes in teleosts

By analysis of the expression patterns in different mutants or other genetic modified embryos, this study also demonstrates that Hh and Notch, but not Fgf

pathways are involved in regulating gli2b expression in later stages, although the early regulation of gli2b expression still remains unclear

The functions Gli2b in neurogenesis were analyzed mainly by loss-of-function approach, and several early neural markers of neural precursors as well as

differentiated neurons have been used to trace the disruption of neurogenesis after knockdown of Gli2b Also, same markers have been applied for comparative analysis

of the role of Gli2 Gli2b morphants show severe disruption in the hindbrain development, including the reduction of mitotic neural precursors and radial glial cells The segmental pattern of normal radial astrocytes (RA) is also disrupted in the

hindbrain of gli2b morphants The study demonstrates that the disruption of RA in

Gli2b morphants is due to the change in activity of proneural or neurogenic genes, rather than the disruption of the earlier patterning of hindbrain rhombomeres

By analyzing the expression of neuronal and glial markers in Gli2b morphants, this study also revealed a role of Gli2b in cell fate selection between neurogenic and gliogenic precursors as illustrated by correlation in reciprocal changes in oligodendrocytes progenitors and motor neuron progenitors along the A-P axis of Gli2b morphants

In addition, the roles of Gli2b and Gli2 in regulation of Hh downstream genes in neurogenesis have been further investigated in this study Gli2b plays a role in regulating Hh signaling in ventral hindbrain in a context dependent manner The knockdown of Gli2b combined with the dominant negative Gli2 resulted in the complete blocking of Hh signaling in hindbrain Also comparison of Gli2b functions

in WT and gli2 mutant yot-/- indicates a previously unknown role of Gli2b in

rhombomere 4

In summary, this study explored the consequence of the duplication of Gli2 in zebrafish and differences in function of Gli2 between zebrafish and mice Also, it illustrated novel developmental roles of Gli2 in neurogenesis, in particular in the development of r4 and radial astrocytes

Chapter I

INTRODUCTION

I INTRODUCTION

1.1 Zebrafish as an emerging model for developmental studies

Zebrafish (Danio rerio) emerge as one of the most promising experimental model

for developmental biologists due to its several advantages compared with other experimental models Zebrafish eggs are fertilized externally and all embryonic stages are accessible for study; this is a great advantage over the mouse, where embryonic development takes place in uterus Development of pigment, which will obscure observation from the second day, can be suppressed by incubating the embryo in 1-phenyl-2-thiourea (PTU) without apparent effect on embryonic development The other advantages include the relatively short generation time, the large brood size and rapid embryonic development Most importantly, zebrafish is amenable for large scale mutant screens using chemical mutagens (Haffter et al, 1996) and proviral insertions (Amsterdam et al, 1999) In result many mutants are already available

As a teleost species, which diverged from tetrapodea such as mice about 450 million years ago (Metscher and Ahlberg, 1999), zebrafish appears to have a partially duplicated genome (Taylor et al, 2001) and up to 30% of its genes are duplicated Taking advantage of this, important insights into both the potential function of specific protein domains in mouse orthologs can be achieved should the zebrafish co-orthologs take on distinct expression patterns and developmental roles, so called sub-functionalisation The restricted expression of zebrafish co-orthologs in distinct sub-regions of a tissue in comparison to mouse orthologs may lead to a better understanding of developmental relations in cell lineage and tissue patterning in

mammals Alternatively, the sequences of protein domains in zebrafish co-orthologs may also diverge sufficiently to reveal new functions, referred to as neo-functionalisation To facilitate the analysis of developmental events in zebrafish, various genetic approaches have been applied

1.1.1 Forward genetic approach

A milestone of zebrafish as an exciting model for genetic studies is the generation

of a large pool of random mutations via administration of a chemical mutagen: N-Ethyl-N-nitrosourea (ENU) The pool can be feasibly maintained because the adult zebrafish is about 3 cm long so that many animals can be housed in a relatively small space The screening of mutations is facilitated by the large number of embryos (over 200) in each spawning and regular spawning throughout the year So far, several mutagenesis screens, using a variety of mutagens and screening techniques, have been accomplished at varying scales (Driever et al, 1996; Haffter and Nüsslein-Volhard, 1996; Schulte-Merker, 2000; Knapik, 2000; Golling et al, 2002; Chen et al, 2002) Today a large number of zebrafish mutants affecting early development and organogenesis are available Although large-scale of whole mount in situ hybridization can be used to facilitate the mutant screening for desired research purpose (Meng et al, 1999; Kudoh et al, 2001), mapping the mutant to its related gene

is rather laborious For ENU induced mutants, the identification of the loci by positional mapping still remains very labor-intersive and slow However, thanks to the nearly completed genome sequence released in Zebrafish Information Network (ZFIN) (http://zfin.org) (Westerfield et al, 1999a, b), and the increasing number of markers in

several genetic linkage maps (Postlethwait et al, 1998; Gates et al, 1999; Kelly et al, 2000; Talbot and Hopkins, 2000; Woods et al, 2000), the mutant cloning work now became more efficient and less time-consuming

The Radiation hybrid (RH) maps have been developed for zebrafish with markers, which include simple sequence length polymorphisms (SSLPs), cloned genes and ESTs (Kwok et al, 1998; Geisler et al, 1999; Hukriede et al, 1999, 2001) The two zebrafish RH maps, Ekker LN54 and Goodfellow T51, cover >90% of the zebrafish genome (Talbot and Hopkins, 2000)

For insertional mutagenesis using retroviruses or “trap” vectors, the mutated loci could be easier located since the zebrafish genome sequence is almost completed Several problems were encountered using insertional mutagenesis technologies The total number of mutants is relatively low Also, some mutants might be difficult to map it because of the insertion happened to be in a repetitive sequence

1.1.2 Reverse genetic approach: gain of function and loss of function

The advances of the zebrafish as a model for developmental biology are particularly apparent due to the availability of various cellular, molecular and genetic techniques Moreover, it is feasible to introduce DNA or mRNA into zebrafish embryos by microinjection, electroporation and micro-projectiles

The genetic approaches for characterizing gene activity are usually based on the

manipulation of expression of that gene in vivo and/or in vitro, which could be

realized by either over-expression (gain of function) or silence of that gene (loss of function)

For gain-of-function approach, the expression construct under a ubiquitous or tissue-specific promoter could be used for injection, resulting in the ubiquitous or tissue-specific expression of the test gene Also, more and more conditional

expressing systems including the inducible systems (hsp70) (Halloran et al, 2000;

Blechinger et al, 2002), GAL4-UAS system (Scheer and Campos-Ortega, 1999; Scheer et al, 2001), cre-loxP system (Pan et al, 2005), are designed and applied for expressing specific gene at the desired time and dose

mRNA could also be injected directly into embryos, although it could be eventually degraded in 1-2 days Some modifications can also be done on mRNA for conditional gene expression, which involve photomediated activation of caged mRNA (Ando et al, 2001) and injecting mRNA with 3’ UTR which is required for RNA

localization For example, the 3’UTR region for special localization of vasa mRNA in germ line cells, was linked with the bacterial toxin gene kid to study the role of the

germ cells in somatic development, which resulted in discovery of the unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males (Slanchev et al, 2005)

Besides gain-of-function analyses by transgenic approaches, loss-of-function analyses are also important for gene characterization The well established gene knock-out technology in mice is still unavailable in zebrafish because of the lack of

ES cell culture Successful production of a stable embryonic stem cells line from another fish model, medaka, was reported (Hong et al, 1998) Recently, Ma et al, (2001) also reported successful production of germline zebrafish chimeras from short

term embryonic cell culture (Ma et al, 2001)

Development of knockdown technology based on morpholino oligonucleotides (MOs) provided a possibility to inactivate specific genes through blocking the desired protein translation or mRNA splicing in zebrafish (Nasevicius and Ekker, 2000; Ekker and Larson, 2001;Malicki et al, 2002) It has been shown to effectively and specifically induce phenotypes similar to that of mutants (Nasevicius and Ekker, 2000) The advantages of using morpholinos to knockdown target gene are discussed

in detail in Chapter II Short interfering RNA (siRNA) might be another good candidate for loss-of-function analysis It is used successfully in mice and frogs, but some groups reported the non-specific knockdown by siRNA in zebrafish (Zhao et al, 2001; Oates et al, 2000)

Besides the small molecule induced gene knockdown, the identification of desired genetic mutants could be used for the study of that gene function TILLING (Targeting Induced Local Lesions in Genomes), has been developed by combining the ENU-induced large-scale point mutation approach with the high throughput sequencing technology (McCallum et al, 2000; Wienholds et al, 2002) This is a method for finding mutations in known genes with identified sequences And it could

be used to identify some mutants that had no severe phenotypes therefore could not be studied by forward genetic approaches

1.1.3 Transgenic approach

The transgenic technology has been used in fish since mid-1980s (Zhu et al, 1985) And it became widely used after the focus of transgenesis was shifted from

applied research to basic research Besides the transgenic approach used for regulating

gene expression in vivo as mentioned earlier, the other well established transgenic

technique is using tissue-specific promoters and the living color reporter genes, such

as GFP and RFP, for developmental analysis Without the requirement to sacrificing embryos and fishes using the tradional reporter genes such as lacZ (β-galactosidase) and luciferase, expression of GFP reporter allows researchers to observe dynamic

patterns of gene expression in live embryos and fishes

The living color transgenic fish in developmental analysis is widely used in different genetic approaches For gene study, a transient or stable transgenic

expression by injecting the construct with studied gene promoter and gfp gene

provides dynamic information of this gene expression during fish development By modifying the promoter researchers could understand how the regulatory elements are distributed within genome and which enhancers are used to regulate gene expression

in different tissues Using the strategy by injecting different potential regulatory elements with promoter-reporter fragment, an enhancer responsible for regulating

netrin1 expression in the floor plate was identified, which was independent of Sonic

Hedgehog (Shh) signaling but required Nodal signaling and FoxA2 for its activity (Rastegar et al, 2002)

Also, transgenic fish can be used for analyzing organogenesis if the transgenic

fish shows GFP/RFP in specific organ For example, the patterns of gata1 mRNA

expression detected by RNA in situ hybridization, was faithfully represented by GFP

expression patterns in gata1-gfp zebrafish transgenic line, which is under the

promoter of gata1 encoding an erythroid-specific transcription factor (Long et al,

1997) Two color transgenic line with GFP in pancreas and RFP in liver, which is

under specific promoter of insulin and lfabp respectively, was applied for studying

liver and pancreas development as well as the transdifferentiation between these two endoderm-derived organs (unpublished data of Gong’s lab) In addition, the transgenic fishes with expression of fluorescent protein in specific organs can be used for the large scale screening of mutants (Rubinstein, 2003)

Various approaches have been developed for fish transgenesis The simplest way

is by direct transgenesis, which is to introduce a single gene by microinjection, electroporation or transfection into early stage fish embryos that are able to transmit the transgene through the germline This method, which is highly inefficient, relies on the random integration of purified DNA into chromosome

To overcome low efficiency of transgenesis, two other techniques were developed The first one is using pseudotyped retroviruses that have a broad host range (Burns et

al, 1993) A common mouse retrovirus was developed that had a replacement of its

normal species/tissue-specific env protein gene by the G-protein of vesicular

stomatitis virus These modified retroviruses can infect fish and human cells and integrate one DNA copy into a chromosome of the host cell This method has been successfully used to perform retrovirus-mediated insertional mutagenesis in zebrafish (Amsterdam et al, 1999; Golling et al, 2002) However, this technique has several drawbacks that prevent its wide use in transgenic studies: (1) Pseudotyped retroviruses are difficult to prepare at the high titer required for efficient integration

(2) This technique is mostly used for mutagenesis rather than expression of transgenes because high-titer retroviruses do not express transgenes (Linney et al, 1999) (3) Pseudotyped retroviruses do not integrate randomly and (4) they are capable of infecting lab workers who conduct the experiments and thus the safety issues must be taken into account (Smith et al, 1996)

The second technique is based on DNA-based transposons This technique takes advantage of random spread of repetitive sequence in genome and selectable transposons In humans, the LINE and SINE families among transposons comprise the largest family of retro-elements, which correspond approximately to 33% of the genome In zebrafish, the same family of SINEs has been named DANA and

mermaid The transposons can be used as delivery vehicle when supplied with

transposases either from a plasmid carrying the transposase gene or from an mRNA co-delivered with the transposon Several transposon systems have been developed

Firstly, Tc1/mariner transposons from C elegans were tested in zebrafish but the integration rate was the same in presence and absence of transposase Tdr1 and Tdr2 have been found in zebrafish that belong to Tc1/mariner-like elements However,

these transposons are transposition defective due to gaps, stop codons, and frame-shift mutations in the putative transposase-coding sequences

One “artificial” transposase was reactivated from the non-functional salmonid

transposase gene using the site-specific mutagenesis and was named as Sleeping

Beauty It showed high efficiency for the genome integration of target genes In

addition, a non-Tc1/mariner type element, Tol2, was identified in an albino medaka

after it was transposed into and inactivated a pigment gene It shows activity in both mammalian cells and medaka (Koga et al, 2001; Koga et al, 2003) This technique is also successfully applied in zebrafish (Kawakami et al, 1998 and 2000), and was recently used for enhancer trap screen to identify regulatory regions of developmental

zebrafish genes in vivo (Parinov et al, 2004)

1.1.4 Duplication of teleost genome: pros and cons of duplicated genes

As a member of teleosts, which diverged from tetrapodea such as mouse about

450 million years ago (Metscher et al., 1999), zebrafish appears to have a duplicated genome (Taylor et al., 2001) However, it does not mean that every gene in tetrapodea has both orthologs retained in zebrafish As it was hypothesized that the duplicated genes would eventually lose one copy unless they either have developed novel functions or redistributed the functions of their ancestor gene Therefore, many genes but not all which are unique in mammals retain two or more copies in teleost fish during evolution (Amores et al 1998; Volff and Schartl 2003)

One principle of maintaining the functional gene duplicated is through the neofunctionalization of a duplicate, characterized by newly acquired positively selected protein activities (Force et al., 1999) Due to the presence of one more copy, the mutation of one gene may result in gaining novel function without affecting its original function coming from their ancestor gene

The other principle of that is through the subfunctionalization, which could arise

as a consequence of the differential decay of specific regulatory or coding sequences

in each gene copy and the subsequent need for the presence of both duplicates with

complementary activities to achieve the function(s) of the original unique gene (Force

et al., 1999) In fact, empirical data show that duplicate pairs are retained at a higher rate than the classic models of duplicate gene evolution predict (Graf and Kobel, 1991; Hughes and Hughes, 1993)

It has been argued against the zebrafish as a vertebrate model that the additional gene duplication in zebrafish will cause the erroneous redundant roles thus affecting the understanding of the species-specific role of the studied genes However, an advantage of zebrafish as a model for neurogenesis might actually come from the

“drawback” of the genomic difference compared with mammals The restricted expression of zebrafish co-orthologs in distinct sub-regions of a tissue in comparison

to mouse orthologs may lead to a better understanding of developmental relations in cell lineage and tissue patterning in mammals It has been shown that many duplicated genes, instead of acquiring novel functions, redistribute their ancestral subfunctions (Hughes, 1994, 1999; Force et al., 1999; Hughes, 1999; Stoltzfus, 1999)

For a diversity of studies, polyploidy in zebrafish and other model fish species might also be advantageous For example, it should be possible to identify regulatory elements in each of the zebrafish duplicates by comparing orthologous sequences in zebrafish and pufferfish A given human gene often has many expression domains, and if these expression domains have been subdivided between the fish duplicates (Force et al 1999), then by comparing the zebrafish and pufferfish sequences it might

be possible to identify the regulatory elements associated with expression domains in zebrafish These data might then be used to associate regulatory elements with

expression domains in humans Furthermore, sequence-level studies on species that experienced genome duplication may help us to determine whether our own genome

is the product of an ancient genome duplication event because they indicate what the

evolutionary products of genome duplication look like (Wolfe, 2001)

1.2 Early stages of embryonic development of the Zebrafish

As mentioned earlier, zebrafish embryo develops into larva in a short period of time It takes only two days from the first cleavage of zygote to the moving larva just hatching out As a prerequisite, the primary nervous system of zebrafish embryo develops early Therefore, it is required to define the staging series in developmental studies for comparable analysis from different researchers to become possible As for zebrafish, its embryogenesis can be broadly divided into seven consecutive periods based on Kimmel et al, 1995: zygote period (0-¾ hour); cleavage period (¾-2¼ hours); blastula period (2¼-5¼ hours); gastrula period (5¼-10 hours); segmentation period (10-24 hours), pharyngula period (24-48 hours) and hatching period (48-72 hours) at the end of which swimming early larva hatch

Starting from the fertilized egg, zebrafish embryo makes its first cleavage at about ¾ hours post fertilization (hpf) The chorion swells and surrounds the newly fertilized egg and the animal-pole cytoplasm separates from the vegetal-pole yolk to form a blastodisc (Fig 1-1A) (Kimmel et al, 1995) Early cleavages are meroblastic and all blastomeres remain connected by cytoplasmic bridges until the 16-cell stage, while the yolk is not cleaved

At 128-cell stage (2¼ hpf), the blastodisc begins to look like a ball and this stage

marks the beginning of blastula period (Kimmel et al, 1995) During this period blastomeres pile up at the animal pole (Fig 1-1B-D) until about 4 hpf, at which point the blastodisc begins to thin and moves over the surface of the yolk cell (Fig 1-1 B-E)

The important processes during this period include midblastula transition (MBT), formation of the yolk syncytial layer (YSL) and the beginning of gastrulation in the form of epiboly Initially the cells of the blastoderm divide synchronously, however, with the onset of MBT the divisions become metasynchronous and cell cycle lengthens The blastomeres at the margin of the blastoderm have a unique fate They lie against the yolk and remain cytoplasmically connected to it throughout cleavage (Kimmel et al, 1995)

Following the blastulation stage, the embryos initiates morphogenetic cell movement and generate three primary germ layers: ectoderm, mesoderm and endoderm This movement starts at 50% epiboly in the zebrafish embryo Epiboly is characterized by thinning and spreading of both the YSL and the blastodisc over the yolk cell Eventually the yolk cell is completely engulfed by cells at the end of gastrula During the early stages of epiboly, the blastodisc thins considerably from a high mound of cells to a cupshape of multi-layers of cells Epiboly continues with the morphogenetic cell movements of involution, convergence and extension, producing the primary germ layers, ectoderm, mesoderm and endoderm and the embryonic axis

At 50% epiboly (Fig 1-1F), the blastoderm has advanced over half of the yolk cell (Kimmel et al, 1995) Cells at the leading edge, or margin, of the blastula involute,

moving toward the yolk and then back toward the animal pole under the overlying cells (Fig 1-1G) This creates two layers of cells that contribute to formation of embryo proper The upper layer is epiblast or embryonic ectoderm and the lower layer

is known as hypoblast or mesendoderm Epiblast cells at the margin

continue to involute throughout the gastrulation period Involuted cells produce mesoderm and endoderm whereas cells that remain in the epiblast form epidermis and central nervous system (CNS)

As involution begins, cells in both germ layers start to converge toward a single point, producing a thickening at the margin This thickening, apparent at 60% epiboly,

is known as the embryonic shield (equivalent to Spemann’s organizer in amphibians) and marks the dorsal side of the embryo Formation of the embryonic shield indicates the beginning of rapid convergence movements and defines the dorsal side of the

embryo expressing goosecoid (one of the earliest markers of shield), which involute

to form the axial hypoblast (Stachel et al, 1993) About midway through gastrulation, the axial hypoblast becomes clearly distinct from the paraxial hypoblast, which flanks

it on either side Anterior paraxial hypoblast will generate muscles to move the eyes, jaws and gills More posteriorly, much of the paraxial hypoblast is present as the segmental plate that will form the somites The dorsal epiblast begins to thicken towards the end of gastrulation marking the beginning of development of neural plate

An organ- and tissue-level fate map generated by injecting single cells with lineage-tracer dyes at the onset of gastrulation defines where the differentiated cells and cell types are formed (Fig 1-2) (Warga and Kimmel, 1990)

Eventually the yolk cell is completely engulfed by cells at the end of gastrula By the end of gastrulation at 10 hpf when tail bud forms, the epiboly is complete and the three germ layers are defined and both mesoderm and ectoderm become patterned along the anterior-posterior and dorsoventral axis (Fig 1-1F-H) (Kimmel et al, 1990; Kimmel et al, 1995)

After 10 hpf, the embryo shows a morphogenetic movements involving development of somites, appearance of rudiments of primary organs and elongation of embryo with more prominent tail bud (Fig 1-1F-H) In parallel, the dramatic change

of morphology takes place during neurulation

Fig 1-1 Camera lucida sketches of the zebrafish embryos at selected stages

(reprint from Kimmel et al, 1995)

1.3 General introduction to neural development

1.3.1 The induction of neural ectoderm

Whether the ectoderm will form epidermis or choose neural fate as default has been debated for decades The previous view is that induction of neural ectoderm is achieved by the activity of molecules produced by discrete groups of cells, which could induce formation of a secondary axis including neural tissue derived from the host when transplanted to the ventral side of an embryo (Spemann and Mangold, 1924) This group of cells is generally known as the Spemann organizer, and is found within the dorsal blastopore lip of amphibians, the shield of zebrafish, and primitive node of chick and mouse (Spemann and Mangold 1924; Oppenheimer, 1936; Saude et

al, 2000; Shih et al, 1996; Waddington, 1932, Beddington 1994) The secondary neural tube in lineage tracing experiments performed on twinned amphibian embryos was generated from the part of the host ectoderm that normally form epidermis (Gimlich and Cooke, 1983) This raised an assumption of the “epidermis default” model that is the ventral ectoderm cells, which did not receive signals from organizers, would chose the epidermal fate by default

This assumption has been considered true until 1989 when several research groups demonstrated that animal caps or whole embryos subjected to a prolonged dissociation during gastrula stages form neural tissue (Godsave and Slack, 1989; Grunz and Tacke 1989; Sato and Sargent, 1989), indicating that a neural fate could be

the default fate of ectoderm in the absence of signals from the organizer The Dpp (the

Drosophila homolog of BMP2/4)/BMP proteins, which belong to the TGFβ ligand

family in Drosophila or vertebrates, function as epidermal inducer and are inhibited

by some BMP antagonist secreted from organizer tissue Several BMP antagonists

were isolated from Xenopus organizer tissue, including Noggin, Follistatin, Chordin,

Cerberus and Xnr3 (Lamb et al,1993; Hemmati-Brivanlou et al, 1994; Sasai et al, 1995; Bouwmeester et al, 1996; Hansen et al, 1997) While addition of BMPs to dissociated ectodermal cells induces an epidermal fate and blocks them from adopting

a neural fate, disruption of BMP signaling by a dominant-negative BMP receptor caused the inhibition of epidermal differentiation and the expansion of the neural plate

In zebrafish, an expansion of the neural plate at the expense of non-neural ectoderm

was also observed in the homozygous mutants swirl, snailhouse and somitabun that

have defects in genes endoding zebrafish Bmp2, Bmp7 and the intracellular component of the BMP signaling transduction pathway Smad5 (reviewed by

Weinstein and Hemmati-Brivanlou, 1999) In zebrafish, chordino mutant carrying mutated chordin homolog, showed ventralized phenotype, consistent with its function

as an antagonist of the ventralizing BMP signals (Wagner et al, 2002)

Although the BMP-inhibitor model was attractive in its simplicity, several apparently inconsistent results emerged near the end of the 1990s The genetic elimination of the mouse node in its entirety failed to block neural differentiation, indicating that neural induction begins prior to the formation of the organizer region and thus must be initiated by signals derived from other cell types (Klingensmith et al, 1999) In addition, members of other families of signaling molecules, notably the fibroblast growth factors (FGFs) have now been proposed as early-acting factors that

initiate neural induction (Streit et al, 2000; Wilson et al, 2000) So the suppression of BMP signaling may maintain rather than initiate the process of neural differentiation

It has been proposed that cooperation of FGF with inhibition of Wnt signaling can

repress the expression of BMP genes at a transcriptional level (Wilson et al, 2001), in

contrast to the organizer-derived factors such as Noggin and Chordin, which act extracellularly to block BMP activity Therefore, these new findings might serve to expand rather than overrun the BMP-inhibition hypothesis of neural induction

1.3.2 Prepatterning of neural plate along the anterioposterior and dorsoventral axis

The neural induction during gastrulation results in the formation of neuroectoderm, or neural plate After that, neural plate eventually develops the central nervous system (CNS), through the process called neurulation The newly induced neural tissue is thought to have the anterior, or forebrain, character Later, the organizer produces signals, which may include Wnts, FGFs, and retinoic acid that induce more posterior identity in the CNS

The anteroposterior (A-P) patterning, as described in Xenopus, starts from the

beginning of gastrulation Transplantation experiments revealed that the source of posteriorizing activity in zebrafish is outside of the organizer, at the lateral and ventral embryonic margin Lateral marginal cells, consisting of prospective nonaxial mesoderm and endoderm, induced host cells to develop hindbrain characteristics whereas shield cells were unable to cause this effect (Woo and Fraser, 1997) Koshida and others (1998) found that ventral marginal cells inhibited expression of anterior

neural fates and induced expression of posterior neural fates Fate mapping in zebrafish reveals that cells that contribute to major subdivisions of the nervous system

at the onset of gastrulation occupy overlapping positions and will segregate by the end

of epiboly A refined map by Woo and Fraser (1995) is shown in Fig.1-2, clearly indicating that the distinct nervous system regionalization slowly emerging at gastrulation from 6 hpf to 10 hpf

Fig 1-2 6 and 10 hpf neural fate maps, modified from Woo and Fraser (1995)

(A) Animal pole view of shield stage embryos (B) Dorsal View of 10 hpf embryo The color code for the regional fates is as follows: red=telencephalon; black= diencephalons (excluding the retina); green=retina; orange=midbrain; dark blue=hindbrain; light blue=spinal cord; purple=somite; yellow=neural crest, ear placodes and epidermis

Wnt signaling is involved in anteroposterior patterning of neural plate bozozok

that encodes a homeodomain protein might be important for neural anteroposterior patterning by preventing dorsal marginal cells from expressing a posteriorizing gene,

wnt8 Analysis of zebrafish maternal-zygotic headless mutants has suggested that

caudalizing factors, in particularWnts, operate in the context of basal repression of caudal genes provided byTcf3 homologs (Kim et al, 2000; Dorsky et al, 2003)

Canonical Wnt signalingfacilitates the expression of downstream target genes through

ß-catenin, which associates with Lef/Tcf proteins that bind to DNA regulatory elements of target genes (Barker et al, 2000)

Retinoic acid was also involved in anterioposterior patterning of neural plate

Applying the constitutively active Retinoic Acid Receptors (RAR) in Xenopus

embryos resulted in a decrease in the anterior neural tissue, whereas the dominant-negative receptors resulted in an expansion of the anterior neural structures These results suggested that xRAR activation plays a role in promoting and activating the expression of posterior neural markers (posterior hindbrain and spinal cord), while causing the correct spatial restriction of anterior markers, although it is not required for the establishment of the anterior genetic program (Blumberg et al, 1997)

Another patterning center of the nervous system is the anterior neural ridge (ANR) Removal of the front row of cells from the nascent neural plate, but not more posterior rows, caused loss of expression of telencephalic markers (Houart et al, 1998) Transplantation of ANR cells to more posterior regions induced telencephalic characteristics in the diencephalon Thus, ANR cells appear to be the source of signal patterning for nearby neural tissues and promoting telencephalon development ANR patterning occurs as neural tissue is induced and anteroposterior polarity is established

A good candidate for involvement with ANR function might be masterblind (mbl), a mutation of the scaffolding protein Axin1, which is also a negative regulator

of Wnt signaling (Heisenberg et al, 2001) The mutant embryos are phenotypically

similar to wild type embryos in which ANR cells have been removed (Houart et al, 1998) The epiphysis, which occupies dorsal diencephalon, is expanded anteriorly in

mbl mutant embryos suggesting that mbl may function as part of an anterior signaling

system that restricts development of more posterior neural fates (Masai et al, 1997) and yet later on it was shown that primary activity of the ANR is due to secreted antagonist of Wnt signaling Tlc (Houart et al, 2002) By the end of gastrulation the isthmic organizer at the midbrain-hindbrainboundary (MHB) becomes a source of additional Wnts and FGFsand the prechordal plate and ANR become sourcesof Wnt antagonists (Eroshkin et al, 2002; Hashimoto et al,2000; Houart et al, 2002; Kim et al, 2002; Shinya et al,2000) These additional sources of caudalizing factors and theirantagonists are thought to further modulate the shape of the caudalizing activity gradient in the anterior neural plate

Patterning of the neural tube along its dorsoventral axis is initiated during the neural plate stages Initially, the mediolateral coordinates of the neural plate, corresponding later to the ventro-dorsal coordinates of the neural tube, are established

by secretion of signaling molecules from non-neural tissue surrounding the neural plate (Tanabe et al, 1996) Nonneural ectoderm, adjacent to prospective dorsal neuroectoderm, produces Bmps, which promote formation of neural crest and dorsal neural tube fates Mouse Wnt1 and Wnt3a are expressed in the roof plate as soon as the neural tube closes and double mutant embryos deficient in these genes have fewer DI1-DI3 interneurons at E10.5 as showed by the expression of proneural genes, such

as Math1 and Ngn1, in precursor cells These results indicate that Wnt signaling

might be another good candidate for dorsal neural patterning

An important organizing centre of the ventral neural tube is the axial mesoderm which is comprised of notochord and prechordal plate mesoderm and which underlies the midline of the neural plate A second organizing centre, the floor plate, subsequently is established in the ventral neural tube itself as a consequence of the action of signals derived from the axial mesoderm Shh is one of several vertebrate

homologues of the Drosophila hedgehog gene product, expressed sequentially in the

axial mesoderm and then in the floor plate Shh has been shown to induce medial/ventral cell fates such as floor plate and motor neurons but repress dorsal cell fates in vertebrate embryos Expression of many genes involved in establishing different cell fates along dorsoventral axis is regulated by Shh through the downstream transcription factors, Gli proteins A detail description of the Shh/Gli signaling pathway will be given in section 1.4

1.3.3 The neurulation process in zebrafish and other vertebrates

Neurulation results in the formation of neural tube The primary neurulation has been carefully described in frog, chick, mice, and rabbit (Davidson and Keller,1999; Morriss-Kay et al, 1994; Peeters et al, 1998; Smith and Schoenwolf, 1991) Despite of slight difference in these animals, the basic steps which are specific to primary neurulation are conserved These include columnarization of the ectoderm to form the neural plate, thickening of the edges of the neural plate to form the neural folds, convergent extension of the neural plate that assists bending to form the neural groove and also elongates the neural tube, and closure of the groove to form the neural tube

(Colas and Schoenwolf, 2001)

In zebrafish, the formation of neural tube is slightly different The neural tube of zebrafish or teleost is thought to form by thickening of the neural plate ectoderm to form the neural keel, then its transformation into the neural rod and secondary cavitation of that to form the lumen Initially, the neural keel was thought to be a mass

of mesenchymal cells, and neurulation therefore equivalent to the secondary process that occurs in the tailbud of most animal groups (Reichenbach et al, 1990) However, more careful examination using several model systems, including the zebrafish, showed that this is not correct (Geldmacher-Voss et al, 2003; Kingsbury, 1932; Miyayama and Fujimoto, 1977; Strahle and Blader, 1994) Instead, the neural tube of fish does form by a medial migration of neural plate cells that is just like the medial movements of primary neurulation, while the cells along the midline cling tightly together, making a nearly invisible seam rather than a long open ditch (Lowery et al, 2004) Therefore, the primary neurulation is conserved in all of the studied vertebrate models including zebrafish

1.3.4 Transcription factors in regionalization of neural tube

In parallel with the definition of extrinsic signaling molecules, advances in the biochemical characterization of transcriptional regulatory mechanisms in mammalian cells, combined with cloning genes responsible for homeotic transformations of body

parts in Drosophila melanogaster in the late 1980s, helped to identify many

transcription factors on cell fate determination in CNS Many of them contain homeodomains and play pivotal roles at several successive steps in neural induction,

regional patterning and cell fate determination Recent studies have provided evidence that a group of homeodomain proteins expressed by ventral progenitor cells act as intermediary factors in the interpretation of graded Shh signaling (Pierani et al, 1999; Briscoe and Ericson, 1999) They can be divided into two classes on the basis of their pattern of expression and mode of regulation by Shh (Briscoe et al, 2000) The expression of each class I protein is repressed at a distinct Shh threshold concentration and, as a consequence, their ventral boundaries of expression delineate progenitor domains Conversely, the expression of each class II protein requires Shh signalling and is achieved at a distinct Shh threshold concentration So their dorsal boundaries delineate progenitor domains

The combinatorial expression profile of these two classes of homeodomain proteins defines several cardinal progenitor cell domains within the ventral neural tube This feat is achieved through selective cross-repressive interactions between the complementary pairs of class I and class II homeodomain proteins that abut the same progenitor domain boundary Such interactions seem to have three main roles First, they establish the initial dorsoventral domains of expression of class I and class II proteins Second, they ensure the existence of sharp boundaries between progenitor domains Third, they help to relieve progenitor cells of a requirement for ongoing Shh signalling, consolidating progenitor domain identity

How the neural patterning role of Shh is integrated with other more general regulators of neurogenesis also remains unclear In vertebrates, as in insects, neurogenesis is regulated by signalling pathways that involve Notch and basic

helix–loop–helix (bHLH) proteins (Chan et al, 1999) Notch ligands, and many bHLH proteins, are expressed within discrete domains along the dorsoventral axis of the ventral spinal cord (Matise et al, 1997; Shawber et al, 1996; Sommer et al, 1996), and

in some regions of the CNS bHLH factors have been suggested to influence neuronal subtype identity (Fode et al, 2000) It will therefore be important to determine whether individual Notch ligands and bHLH proteins with distinct patterns of expression in the spinal cord have equivalent functions in neuronal specification

1.3.5 Sequential onset of neuronal and glial differentiation from neural progenitors

The most fundamental decision that a neural cell makes is to choose either a neuronal or a glial cell fate since glial origins and diversity mirror that of the neurons they accompany For instance, longitudinal glia of the CNS are derived from longitudinal glioblasts that have a neuroectodermal origin Other CNS and brain glia are derived from the same neuroblasts that give rise to neurons (Nelson and Laughon, 1994; Jones et al, 1995) The extrinsic signaling molecules and cell-intrinsic factors instruct multipotent progenitor cells, thereby restricting their potential to become specialized neurons, oligodendroctytes or astrocytes In the developing spinal cord, Shh and BMPs establish the dorso-ventral gradient that is required for the correct spatial and temporal expression of transcription factors and other genes that regulate the emergence of neuron and glia populations from the neuroepithelium (Mekki-Dauriac et al, 2002; Patten and Placzek, 2002)

Accumulating evidence suggests that vertebrate neurogenic bHLH transcription

factors promote neuronal fate while simultaneously repressing glial fate, thus fulfilling the criterion for a molecular switch between neuronal and glial fates It was

shown that ectopic expression of Neurogenin 1 (Ngn1) repressed astroglial fate in cultured neural stem cells (Sun et al, 2001) and over-expression of Neurod in mouse

retina completely blocked the generation of Muller glial cells (Morrow et al, 1999)

Conversely, neuronal markers from many brain regions of mash1-/-math3-/- and

Mash1-/-Ngn2-/- animals were lost while GFAP and S100b, two astroglia markers,

were up-regulated ectopically and precociously (Tomita et al, 2000; Nieto, 2001)

Similarly, in mash1 and math3 double mutant retina, bipolar neurons were replaced by

Muller glial cells (Tomita et al, 2000) These studies are consistent with the idea that neurogenic bHLH factors regulate the neuron-glia decision in vertebrate species Notch is another important molecule that has been implicated in the neuron-glia decision Ectopic Notch activation promotes Muller glial generation in mouse retina (Furukawa, 2000), and Notch1 and Notch3 instructively commit adult rat CNS stem cells into the astroglial lineage (Tanigaki et al, 2001) Given that Notch activates the Hes family of transcription repressors which in turn inhibits the transcription of neurogenic bHLH factors (reviewed by Kageyama and Nakanishi, 1997), and that the glia-promoting activity of Notch can be mimicked by Hes in some settings (Furukawa

et al, 2000), it is highly likely that Notch promotes glial fate at least partially via repressing repressors of glial development, i.e the neurogenic bHLH factors It should be noted that the function of Notch is highly context-dependent In many systems, activation of Notch keeps progenitor cells in an undifferentiated state instead