Gene transcription in the zebrafish embryo: regulators and networks

Gene transcription in the zebrafishembryo regulators and networksMarco Ferg Olivier Armant LixinYang Thomas Dickmeis Sepand Rastegar and Uwe Stralaquo hle

AbstractThe precise spatial and temporal control of gene expression is a key process in the development maintenance andregeneration of the vertebrate body A substantial proportion of vertebrate genomes encode genes that controlthe transcription of the genetic information into mRNA The zebrafish is particularly well suited to investigategene regulatory networks underlying the control of gene expression during development due to the external devel-opment of its transparent embryos and the increasingly sophisticated tools for genetic manipulation available forthis model systemWe review here recent data on the analysis of cis-regulatory modules transcriptional regulatorsand their integration into gene regulatory networks in the zebrafish using the developing spinal cord as example

Keywords zebrafish transcription regulator gene regulatory network cis-regulatory element genomics

INTRODUCTIONThe first step of gene expression is transcription

Differential gene transcription plays a crucial role in

the development of the vertebrate body plan Also

many homoeostatic processes and responses in the

adult organism are regulated by transcription

Thus misexpression of genes due to deregulated

transcription can lead to a variety of diseases includ-

ing cancer and type II diabetes [1ndash3] The import-

ance of differential transcriptional regulation as a

means to correctly express the information encoded

in the genome is further underscored by the fact that

a substantial proportion of genes in the genomes

code for proteins that are involved in the control

of transcription such as transcription factors (TFs)

chromatin-remodelling factors and proteins of the

basic transcription machinery [4 5] Complex gene

regulatory networks (GRNs) form the basis of

cell differentiation during development of multicel-

lular organisms and can control entire batteries

of genes coordinately In GRNs specific combin-

ations of transcriptional regulators (TRs) interact

with cis-regulatory modules (CRMs) of responsive

genes [6ndash8]

CRMs can be functionally subdivided into two

groups activating modules (promoter enhancer

locus control region [LCR]) and repressive modules

(silencer insulator) Silencers inhibit gene expression

through the interaction with repressor proteins

that in turn initiate heterochromatin formation by

recruiting histone-modifying enzyme complexes

Insulators confine the activity of an enhancer or

Marco Ferg is a post-doctoral fellow at the Institute of Toxicology and Genetics (ITG) His research interest lies in the transcriptional

regulation of genes by cis-regulatory elements

Olivier Armant is a scientist at the ITG The research he is interested in concerns early patterning mechanism in the developing

zebrafish brain

LixinYang is a project leader at the ITG His main research topic concerns the identification of gene regulatory networks in the

developing neural tube of the zebrafish

Thomas Dickmeis is a group leader at the ITG His group is interested in transcriptional regulatory mechanisms at the interface

between metabolism endocrine signalling and the circadian clock

Sepand Rastegar is a project leader at the ITG His work is focused on the identification and functional analysis of cis-regulatory

elements active in the midline of developing zebrafish embryos He is also interested in the molecular mechanisms controlling zebrafish

neurogenesis

UweStralaquo hle professor at the University of Heidelberg is interested in the molecular mechanisms controlling vertebrate development

He is director of the ITG and established the European Zebrafish Resource Centre

Corresponding author Sepand Rastegar Institute of Toxicology and Genetics Karlsruhe Institute of Technology (KIT) Postfach

3640 76021 Karlsruhe Germany Tel thorn49-721-608-22886 Fax thorn49-721-608-23354 E-mail SepandRastegarkitedu

BRIEFINGS IN FUNCTIONAL GENOMICS page 1 of 13 doi101093bfgpelt044

The Author 2013 Published by Oxford University Press All rights reserved For permissions please email journalspermissionsoupcom

Briefings in Functional Genomics Advance Access published October 22 2013

silencer to its cognate gene promoter and interaction

of CCCTC-binding factor (CTCF) with insulators

has been demonstrated to be crucial to shield distal

enhancerssilencers [9] This classical view of CTCF

being a major marker for insulator elements has been

questioned by 4C experiments showing that many

of the CTCF-mediated long range interacting sites

correlate with enhancerndashpromoter interactions

Further experiments showed that CTCF facilitates

enhancerndashpromoter interactions directly through

cohesin-mediated DNA looping [10ndash12]

Enhancers have been traditionally defined as a

class of cis-regulatory elements that are able to acti-

vate and maintain gene expression in a location- and

orientation-independent manner [13 14] They are

found in the up- and downstream regions as well as

in introns of the regulated gene However enhancers

can also overlap with coding exons [15] or the

50-untranslated region (UTR) [16] They may be

located as far as 1 megabase from the target gene

as has been shown for example for a limb-specific

enhancer of the sonic hedgehog gene [17]

Enhancers are thought to represent islands of open

chromatin that act as docking stations for TFs [18]

By a combination of distinct TFs that bind to the

same enhancer various signalling inputs can be inte-

grated and interpreted thereby leading to specific

gene expression outputs In the prevailing model

the physical interaction of enhancers with their

target promoters is facilitated by a decondensation

of the chromatin structure enabling the DNA to

loop out from the nuclear periphery to the centre

to form enhancerpromoter interactions [19ndash21]

A recent publication suggests CTCFcohesin-

mediated DNA looping is a determinant for pro-

moter choice [22] However not all loci require

looping-out for transcriptional activity [23]

Promoters can be functionally subdivided into the

proximal promoter and the core promoter Proximal

promoters are located in the immediate vicinity of

the transcription start site (TSS) and contain like

enhancers or silencers recognition sites for DNA-

binding factors that are involved in transcriptional

regulation The core promoter is usually defined as

a 100ndash150 bp stretch of DNA encompassing the TSS

that is sufficient to direct initiation of transcription by

the RNA polymerase II machinery [24] Core pro-

moters can differ considerably in motif composition

(reviewed in [25ndash27]) and can be bound by a multi-

tude of promoter recognition complexes [28 29]

Not all enhancers are able to interact with every

type of core promoter [18 30 31] providing

another level of regulatory control

LCRs are like enhancers activating CRMs They

possess chromatin domain-opening activity and are

able to confer lsquocopy number-dependentrsquo expression

on a linked gene ie the number of integrated trans-

genes determines the expression level and not the

influence of the chromatin surrounding the trans-

genes (reviewed eg in [32 33])

Interestingly enhancers and silencers share im-

portant properties such as DNase I hypersensitivity

active chromatin marks and interaction with tran-

scription factors and RNA polymerase II It has

therefore been proposed that it depends on the

gene target and the developmental stage whether a

given CRM activates or represses gene transcription

[34]

CRMs contain the information required for

precise transcriptional regulation The implementa-

tion of this information is carried out by TRs

through sequence-specific interactions with CRMs

of their target genes As described above TFs activate

or repress gene transcription by binding to enhancers

or silencers respectively In addition general TFs

are involved in the basic processes of transcription

such as initiation elongation and termination of

transcription However they may be less lsquogeneralrsquo

than previously thought as recent evidence suggests

gene-specific employment of these factors [35 36]

Finally chromatin remodelling proteins alter the

state of the chromatin and thereby affect transcrip-

tion by regulating access to the CRMs

In summary GRNs can be defined as the imple-

mentation of the regulatory information encoded

in the DNA of CRMs via their interactions with

TRs Modelling a GRN related to any development

process requires knowledge of which TFs and signal-

ling molecules are involved when and where the

genes are expressed and how these factors interact

with each other and with CRMs [8] The zebrafish

is an ideal model to study the components and com-

position of GRNs Its fast and external development

and the high number of transparent embryos obtain-

able have proven very useful to address questions in

transcriptional regulation Zebrafish and other fish

species have been utilized to analyse developmental

GRNs [37 38] and promoterenhancer interactions

[31] to validate and analyse enhancer function on

a large scale [39ndash44] to address the regulatory

potential of short sequences [45] and to identify

regulatory regions driving tissue-specific expression

page 2 of 13 Ferg et al

in enhancer trap assays [46 47] Furthermore recent

progress in defining gene expression profiles on a

global scale [4] demonstrates that zebrafish is a

highly useful organism to comprehensively study

GRNs in a vertebrate organism

In this review we will illustrate how zebrafish can

be used to understand the regulatory logic of the

vertebrate genome We will summarize the current

knowledge on specific aspects of CRMs regarding

their distribution and the genomic features that

have been associated with CRMs examine the rep-

ertoire of TRs in the zebrafish genome and discuss

one example of a transcriptional regulatory network

that is well analysed in both zebrafish and mice

SEQUENCE CONSERVATIONANDHISTONEMODIFICATIONSIDENTIFYCRMSCRMs are frequently characterized by conservation

of sequence across species DNA sequences with

regulatory functions exhibit a lower mutation rate

than non-functional DNA and therefore like pro-

tein coding sequences show significant sequence

similarity across species The Fugu rubripes genome

was the first vertebrate genome to be assembled

[48] after sequencing of the human genome [49

50] Comparative studies of the human and Fugu

genomes established the presence of a large number

of highly conserved non-coding elements (HCNEs)

[51ndash54] Since then the growing number of whole-

genome sequences from various organisms and the

development of bioinformatic tools [55ndash59] have

made comparative genomics a powerful means to

identify putative CRMs [60ndash63]

The binding of TRs to CRMs provides one

mechanism of transcriptional regulation Another

level is introduced by the structure of the chromatin

which can control the accessibility of the CRMs

The fact that active regulatory CRMs are in an

open chromatin state forms the basis of large-scale

mapping of CRMs by DNAse I hypersensitivity

assays [64] DNAse I hypersensitivity provides a

comprehensive picture of the active regulatory land-

scape of the genome in a particular cell because it is a

key feature of all classes of CRMs [65 66] Specific

post-translational modifications of histones such as

methylation and acetylation at particular positions

are further landmarks of active and inactive chroma-

tin structure of CRMs For example active enhan-

cers have been shown to be marked by

monomethylation of H3K4 (H3K4me1) [67ndash69]

However this chromatin modification is not limited

only to active enhancers Several studies suggested

the existence of both active and poised classes of

enhancer elements Active enhancers in a cell are

engaged in the regulation of transcription whereas

poised enhancers are not actively regulating tran-

scription but can be rapidly converted to active

enhancers eg by external stimuli to the cell or in

the course of differentiation [70] The histone marks

associated with poised enhancers contain both the

H3K4me1 mark and trimethylation at K27 of his-

tone H3 (H3K27me3) Active enhancers are distin-

guished from poised enhancers by the presence of

acetylation of histone H3 at lysine 27 (H3K27ac)

and by the absence of H3K27me3 [70ndash72] Besides

these post-translational modifications of histones

binding of chromatin remodelling enzymes has

been shown to designate active enhancer modules

Binding of the acetyltransferase and transcriptional

co-activator p300 for example predicted with

high accuracy where active enhancers are located

in the genome in neural limb and heart tissue [69

73 74] Furthermore the chromatin remodelling

enzymes CHD7 and BRG1 appear to be present at

active enhancers [71 72]

CRMS IN THE ZEBRAFISHGENOMEMost genome-wide studies of the regulatory land-

scape have focused on mammalian cell line while

the dynamic changes of the histone code and TF

occupancy of CRMs on a global scale during onto-

genesis of a vertebrate are still unresolved The zeb-

rafish with its experimentally easily accessible early

stages of development has a great potential to address

these questions ChIP (chromatin immunoprecipita-

tion) on chip experiments confirmed enrichment of

the histone mark H3K4me3 found in active pro-

moters [75] also near the TSS in zebrafish [76]

Interestingly many inactive developmental regula-

tory genes are marked by the repressive mark

H3K27me3 while at the same time containing the

activating chromatin modification H3K4me3 thus

showing the characteristic signature of poised genes

that is also found in developmental regulators in

mouse embryonic stem cells [77 78] This lsquobivalentrsquo

signature was not detected before midblastula transi-

tion when the activation of the zygotic genome

occurs [79] It was suggested that such marks might

Gene regulatory networks and control of gene expression page 3 of 13

secure precise timing of the activation of develop-

mental regulators [80]

Combining H3K4me1 and H3K4me3 profiles

signatures of active enhancers (H3K4me1) and

active promoters (H3K4me3) in combination with

the readily available expression data in zebrafish

helped to computationally identify putative cis-regulatory sequences [39] The generation of gen-

omic tracks of H3K4me3 H3K4me1 and H3K27ac

histone modifications at four developmental time-

points of zebrafish embryogenesis lead to the

identification of 50 000 potential cis-regulatory

elements operating during the first 48 h of zebrafish

development a wealth of data that will help to

deduce regulatory networks on the DNA sequence

level [81]

Although many data are now available on

the location of putative CRMs involved in embry-

onic development a systematic analysis of the func-

tion of CRMs has not yet been carried out on a large

scale in zebrafish One crucial question is how con-

served the functions of the CRMs encoded in

HCNEs are Case studies illustrated several possibili-

ties For example the lateral stripe enhancer of the

neurogenin1 gene retained its function for about 395

million years of evolution as it drives expression in

the dorsal telencephalon of both mouse and zebrafish

[82] In contrast the function of CRMs in the shhgene that are conserved between mouse and zebra-

fish seems to have changed the zebrafish sonic hedge-hog (shh) enhancer ar-C directs mainly notochord

expression in zebrafish embryos and also functions

in the midline of mouse embryos However the

mouse enhancer SFPE2 which exhibits sequence

similarity with zebrafish ar-C is floor plate-specific

in the mouse and not functional in zebrafish

Another important question is why CRMs of

developmental regulators show higher sequence

conservation than CRMs of other genes why do

not all conserved sequences have an obvious

function Why are not all functional CRMs highly

conserved Moreover it is more than questionable

whether up to 90 conservation is necessary to

maintain the binding of the same TFs given the

degeneracy of the recognition sequences of many

TFs In fact functional dissection of five notochord

enhancers with evolutionarily conserved sequences

revealed two short DNA elements mediating noto-

chord expression However these elements were

embedded within the conserved sequences of

the five enhancers in an almost random fashion

suggesting that the conservation of the sequence is

only indirectly linked to binding of TFs [83] The

findings that non-coding RNAs bi-directionally

transcribed on enhancers (eRNAs) [84] significantly

increase promoterndashenhancer interactions [85] and

that activating ncRNAs (ncRNA-a) play an import-

ant role in establishing and maintaining chromatin

structure [86 87] opened up a new chapter in cis-regulation of gene expression and might help to

answer some of these questions

Taken together analysis of CRMs of the zebrafish

genome now provides a framework to understand

precisely how classes of CRMs are composed how

they interact with epigenetic marks and why some

of them are conserved across large evolutionary

distances More functional analyses will be required

and one important element of these is knowledge

about which transcriptional regulators interact with

the CRMs

CHARACTERIZATIONOF THETRANSCRIPTIONALREGULATORYREPERTOIREOFTHE ZEBRAFISH GENOMEThe availability of the entire zebrafish genome

sequence [88] and the elucidation of its gene struc-

tures by computational means to identify or predict

TSSs exonintron structure UTRs protein

domains and regulatory elements provides a rich

resource to mine genomes for genes involved in

transcriptional regulation The InterPro database

provides a resource of functional annotation on pro-

teins by classifying them into families It compiles

protein signatures from a number of databases

thereby integrating all data into a unique searchable

resource [89] A thorough search of the InterPro

protein domains led to the selection of 483 domains

associated with transcriptional regulation activities

(Figure 1A) Mining the zebrafish genome for

genes encoding at least one protein domain linked

to transcriptional regulation revealed 3302 genes

including TFs with specific DNA binding domains

genes involved in chromatin modification and factors

of the general transcriptional machinery (Figure 1B)

Of these 2488 TR genes can be detected reliably by

deep sequencing (349 million 76 bp long paired-end

reads) of mRNA isolated from 24 h post-fertilization

(hpf) embryos This suggests that nearly 75 of the

TR gene repertoire of the zebrafish is expressed at a

significant level at 24 hpf [4] a stage of particular


importance as it represents the evolutionarily con-

served phylotypic stage of this model organism

[90] The remaining TR genes may be expressed at

very low levels below the detection limit or at later

stages or only under certain physiological conditions

Of the 3302 TR genes detected in the zebrafish

genome 2677 genes can be assigned to the TF sub-

class of TRs Precedent analysis of human and mouse

genomes suggested that 1500ndash2000 genomic loci

encode DNA-binding TFs [5] roughly 10 times

Figure 1 Zebrafish transcriptional regulators (A) InterPro protein domains specific to transcriptional regulationThe number of protein domains specific for each category of TR (transcription factor chromatin remodellingbasal transcriptional machinery) is indicated (InterPro release 19) (B) The number of genomic loci encoding geneswith at least one domain functionally linked to transcriptional control are indicated for each category (Zv9 assemblyEnsembl version 60 annotation) (C) Expression patterns of chromatin remodellers of the BTBPOZ family in24 hpf embryos bach2 and zbtb46 are expressed in the telencephalon the zbtb16 homologue is expressed in thewhole spinal cord and the posterior tuberculum in the diencephalon zbtb43 is expressed in the retina and thetectum and btbd6a is expressed in the epiphysis the telencephalon the hindbrain and the spinal cord while its para-logue btbd6b is expressed in the somites the telencephalon and other parts of the fore- and midbrain hic1 andbcl6b are expressed in the vascular system of the trunk and the head whereas the bcl6b homologue is expressedonly in the vascular system of the head


the size of the TF repertoire of the yeast Saccharomycescerevisiae (200ndash350 loci) [91] However the fraction

of the total number of genes that encode TFs does

not increase that much as only a 2-fold increase is

observed from yeast (4 TF genes) to human (8)

In zebrafish 9 of all genes encode TFs slightly

more compared with other vertebrates This might

be due to the additional genome duplication during

the evolution of teleost fishes [92] The increased

number of encoded TF genes alone cannot explain

the enormous complexity of the vertebrate body

with many distinct cell types and complex body

functions compared to unicellular organisms such

as yeast The differences seem to lie mainly in the

combinatorial action of TRs as well as in specific

post-transcriptional and post-translational modifica-

tions that will give alternative outputs in different

cellular contexts [93ndash95]

The precise spatiotemporal expression of many

TR genes is essential for normal vertebrate develop-

ment In a systematic analysis of expression of 1711

TR genes in the 24 hpf zebrafish embryo around

200 TR genes were found to be expressed in a single

tissue whereas the vast majority of TRs (nfrac14 1504)

were detected either in multiple tissues or ubiqui-

tously The highly restricted expression pattern of

these 200 TR genes makes them prime candidates

for functional studies This group of tissue-specific

genes is highly enriched for TFs (82 nfrac14 207) [4]

The central nervous system and specifically the

spinal cord and telencephalon express a majority of

the TR genes (60 of the TRs analysed by in situhybridization) A significant correlation of genes co-

expressed in different tissues was observed in a few

brain regions including the ectodermally derived

cranial sensory ganglia (otic vesicle and olfactory

bulb) and several forebrain structures as well as the

retina and the tectum [4] Interestingly retina and

tectum are functionally coupled by topographical

projections of retinal axons into the tectum

However at a global level there was no extensive

co-expression of specific TRs in multiple tissues at 24

hpf This is in contrast to the components of cell

signalling pathways which are frequently organized

into synexpression groups [96 97] Together these

results show that there is no extensive overlap be-

tween the TR expression patterns This suggests a

high flexibility in the combination of different factors

in order to generate alternative regulatory outputs

Genes encoding general TFs and chromatin

remodelling proteins are usually ubiquitously

expressed at 24 hpf with the exception of the

BTBndashPOZ family (named after Broad complex

Tramtrack and Bric a bracpoxviruses and zinc

finger) A significant proportion of these genes (38

out of the 118 mapped genes) have expression pat-

terns restricted to the somites and the central nervous

system The BTBPOZ domain is an evolutionary

conserved domain involved in proteinndashprotein inter-

actions leading to dimerization Proteins containing

both the BTBPOZ domain and C2H2 zinc finger

DNA-binding domain have been shown to promote

transcriptional repression through the recruitment of

co-repressor proteins such as histone deacetylase

(HDAC) N-CoR and SMRT [98ndash100] Six genes

of the BTBPOZ family are expressed in parts of the

nervous system of the 24 hpf zebrafish embryos

(Figure 1C) The tissue-restricted expression of these

TR genes suggests that some members of the BTB

POZmdashzinc finger family may control cellular differ-

entiation during embryogenesis possibly by the re-

cruitment of transcriptional repressors such as HDACs

Functions of ubiquitously expressed genes can still

vary dramatically from one tissue to another For

example brg1 (alias smarca4) a protein associated

with the SwiSnf-like Brg1Brm-associated factors

(BAF) chromatin remodelling complex is expressed

in a broad range of tissues during development and

in the adult zebrafish However mutation of brg1leads to specific defects in the zebrafish heart [101]

and in retinal neurogenesis [102 103] Furthermore

disrupting the dosage balance between Brg1 and the

cardiac TF Tbx5 led to impaired heart development

in the mouse indicating that normal formation of

this tissue relies on precise levels of functional BAF

complexes These results highlight the importance of

ubiquitous chromatin remodelers in the acquisition

of specific cellular fates The fact that most TRs are

expressed ubiquitously emphasizes the importance of

combinatorial action with tissue-restricted factors as

well as that of post-translational and post-transcrip-

tional regulation

Thus data on the global repertoire of TRs and on

their expression during development are now com-

plementing the CRM and epigenetic modification

data In order to be able to model transcriptional

regulation during development these data will

have to be assembled into a global GRN This will

require large-scale functional analysis by perturbing

normal networks eg by creating mutants or other

loss of function phenotypes for particular regulators

or even for CRMs Such large-scale efforts might be


built on smaller scale studies that were aimed at

understanding particular subprocesses of develop-

ment In the next paragraph we will discuss an

example of such a study that illustrates the experi-

mental advantages of the zebrafish model

GENEREGULATORYNETWORKSSPECIFYING ZEBRAFISHVENTRAL SPINALCORDNEURONSMany TFs act in specific cellular contexts thereby

addressing distinct downstream genes or GRNs

Frequently TFs are organized in pathways which

form a hierarchy of TF gene interactions The chal-

lenge is to understand these cascades and to link

the action of TFs to specific cellular outcomes The

vertebrate spinal cord offers well-studied examples

of how different cell types are specified by combin-

ations of TFs

The spinal cord is patterned along its dorsoventral

axis by antagonistic signalling interactions of the Shh

the bone morphogenetic protein and the Wnt path-

ways [104] In the ventral spinal cord the morpho-

gen Shh which is secreted from the underlying

notochord and the floor plate of the spinal cord

induces five distinct ventral neuronal subtypes via a

concentration gradient [105] Distinct concentrations

of Shh induce or repress the spatial expression of TFs

which belong predominantly to the homoeodomain

protein (HD) or basic helixndashloopndashhelix (bHLH)

families (Figure 2 [105]) Class II TFs are induced

while class I TFs are repressed by Shh (Figure 2C)

As a consequence of this Shh activity the five ventral

neural progenitor domains express each a specific

combination of TF genes (Figure 2A and B)

Further cross-repressive interactions between these

TF proteins sharply delineate the boundaries

between the ventral neuronal domains [106] These

progenitor domains will give rise to distinct popula-

tions of neuronal subtypes (Figure 2B) Close to the

ventral source of Shh V3 interneurons form fol-

lowed at a more dorsal position by motoneurons

(MNs) and then by V2 V1 and V0 interneurons

further dorsally (Figure 2B)

The Gli family of zinc finger transcription factors

mediates Hedgehog (Hh) signalling in all vertebrates

by activating or repressing the expression of down-

stream target genes [108] As is the case for mammals

zebrafish Shh is expressed in the notochord and floor

plate and specifies ventral spinal cord fates [109]

Most of the TFs expressed in the spinal cord of the

mouse that mark its distinct domains are expressed in

the zebrafish spinal cord in a similar dorsoventral

pattern (Figure 2A) It was therefore proposed that

gene regulatory networks underlying spinal cord

patterning are highly conserved during vertebrate

evolution [109]

One of the advantages of the zebrafish model

organism is the existence of a huge collection of

mutants for all major signalling pathways For

instance more than 10 zebrafish mutations have

been shown to affect different components of the

Shh signalling cascade [110] Analysis of zebrafish

mutants in Hh pathway components as well as

over-expression of Shh highlighted differences in

the requirement of Shh signalling in spinal cord

development in mouse and zebrafish embryos in

contrast to mouse Shh signalling zebrafish Hh

signalling appears to be partially dispensable for the

specification of some of the ventral neuronal sub-

types [106 111] In the total absence of Hh signalling

in maternal and zygotic smu mutants motoneuron

progenitors (pMN) are strongly reduced in numbers

and p3 progenitors are totally absent In contrast p2

p1 and p0 progenitors as well as the post-mitotic V2

V1 and V0v interneurons that develop from these

progenitor domains are present [111] These results

suggest that the activity of this signalling pathway in

zebrafish is primarily required for the specification of

the p3 progenitor domains As in mouse the expres-

sion of nkx22 and nkx29 genes in the p3 domain is

dependent on Hh signals secreted from the floor

plate and the notochord [111ndash115] The expression

of mouse Nkx22 and 29 and zebrafish nkx 22a and

29 is driven by conserved Shh responsive CRMs

which are bound by Gli factors [115ndash118] suggest-

ing that Hh directly regulates the expression of these

transcription factors In line with this hypothesis in

gli1(dtr) mutants the expression of the nkx2 genes is

greatly reduced while misexpression of Gli1 results

in ectopic induction of nkx29 Furthermore a con-

struct containing the Gli consensus binding site of

the nkx29 enhancer drives green fluorescent protein

(GFP) expression in the ventral neural tube and mu-

tation of this Gli-binding site abolishes GFP expres-

sion [115] In addition morpholino-mediated

knockdown of nkx2ab and nkx29 genes leads to an

expansion of olig2 expression which is a marker of

pMN into the p3 domain and abolishes the expres-

sion of the V3 marker sim1 (leucine zipperPAS

domain) [38] Thus zebrafish nkx2ab and nkx29


genes are required downstream of the Hh pathway

for the specification of the p3 domain and the pro-

duction of V3 interneurons and they contribute to

the correct dorsoventral positioning of the pMN

domain by repressing olig2 expression in a negative

cross-regulatory interaction similar as in the mouse

[105]

Taken together it appears that the transcription

factors and their role in the specification of different

progenitor domains and of post-mitotic interneurons

are largely conserved between mammals and zebra-

fish but that the signals required for the correct

spatial and temporal expression of these factors are

only partially maintained It has still to be determined

which other signals are required for the early expres-

sion of pMN p2 p1 and p0 progenitor domain

genes in the zebrafish [111]

Thus the patterns of expression of the TFs in the

mouse and zebrafish spinal cord are very similar and

the mechanisms underlying neuronal differentiation

appear to be conserved a conclusion also supported

by other studies [112 119 120] However expres-

sion of TFs is not necessarily always an indicator

of maintained function This is exemplified by the

GRNs controlling differentiation of two closely

related types of GABAergic interneurons the

KolmerndashAgdhur neurons KA0 and KA00 [38]

Homologues of these cells have so far not been

described in the mouse spinal cord In contrast to

other neurons in the spinal cord the cell bodies of

both KA interneuron types are positioned close to

the central canal into which they extend cilia [121]

KA0 cells are required for spontaneous free

swimming while the function of KA00 neurons is

unknown [122] The KA00 interneurons occupy a

ventral position and develop from the lateral floor

plate next to the progenitors of V3 interneurons

The KA0 cells reside at a more dorsal position and

arise from the motoneuron progenitor domain [38

123ndash125] The specification and formation of KA

interneurons involve the Notch and Hh signalling

pathways [38 123ndash125] KA00 interneurons in the

lateral floor express the TFs nkx22a nkx22bnkx29 tal2 gata2 and gata3 (Figure 3) The expres-

sion of tal2 gata2 and gata3 as well as of the GABA-

synthesizing enzyme glutamic acid decarboxylase 67(gad67) is shared between KA0 and KA00 cells

(Figure 3 [38 119]) The functional relationships

Figure 2 Schematic representation of the ventral spinal cord of mouse and zebrafish (A) TF genes expressedin the progenitor domains (p) of mouse (left) and zebrafish (right) The expression patterns of the TF genes in theprogenitors and post-mitotic interneurons of mouse and zebrafish are almost identical (B) Ventral progenitordomains and their corresponding post-mitotic neurons (C) Distinct concentrations of Shh induce the spatialexpression of class II genes or repress class I gene expression (left) Progenitor domain borders are refined andmaintained by negative cross-regulatory interactions among proteins which share common boundaries Negativecross-regulatory interaction can take place between class I and class II proteins or between proteins of the sameclass (centre) Specific combinations of homoeodomain transcription factor proteins in each progenitor domaindetermine the neuronal subtype (right) Figure modified from a previous study [107]


of these factors and their role in the specification of

the two distinct KA interneuron subtypes were

investigated in a systematic knockdown approach

using morpholinos designed against each one of

these TFs [38] Differentiation of the correct

number of KA00 cells depends on the activity of

Nkx29 that acts in cooperation with Nkx22a and

b All three of these nkx2 genes are necessary for the

expression of the zinc finger transcription factor gata2and gata3 in KA00 cells Gata2 but not Gata3 is neces-

sary for expression of the bHLH transcription factor

tal2 that acts upstream of gad67 in KA00 cells

(Figure 3) The four genes tal2 gata2 gata3 gad67 are

also expressed in KA0 cells and they depend on the

bHLH transcription factor Olig2 rather than on

Nkx2 genes in these cells Curiously knock-down

of gata2 does not affect tal2 or gad67 expression in

KA0 cells (Figure 3) suggesting different functional

connections Expression of tal2 and gad67 is rather

dependent on Gata3 in these cells Moreover tal2although like gata2 it is expressed in KA0 inter-

neurons is not required for gad67 expression in

these cells (Figure 3) Hence the functional connec-

tions between the different regulatory genes differ in

the two KA cell types although gata2 and tal2 are

also expressed in KA0 cells they are dispensable for

gad67 expression in these cells Instead olig2 and

gata3 are required for the differentiation of gad67-expressing KA0 cells (Figure 3 [38])

These data suggest that the expression state of TFs

per se is not sufficient to determine the cell fate or

differentiation status of cells and that one would

need to know the complete expression repertoire

of TRs to predict the fate of a cell Also the post-

transcriptional and post-translational modifications of

the TRs might play a role as for instance the phos-

phorylation state of the Olig2 protein determines the

choice between the MN and the oligodendrocyte

cell fate [126 127] Even this information might

not be enough as the epigenetic history of the cell

might also play a role by determining which CRMs

are accessible to the TR combination present in it

CONCLUSIONSANDPERSPECTIVESOver the last 20 years zebrafish has become a valu-

able model organism to address questions of gene

regulation and regulatory networks This model has

been shown to be advantageous in reverse and

forward genetic screens in reporter assays to study

enhancerpromoter function as well as in ChIP

studies targeting histone modifications and TRs

thereby delivering the resources for an in-depth

analysis of GRNs The above described analysis of

KolmerndashAgdhur interneuron differentiation demon-

strated that although TFs can be expressed in two

specific cell types they do not necessarily have the

same regulatory function in both cell types It

simultaneously underlines the major advantage of

zebrafish the potential to easily and rapidly address

complex questions where the questions were actually

phrasedmdashin the organism The examples of cell

specification in a single organ which have been

presented here also demonstrate what would be

required to investigate the mechanisms underlying

TR on a global scale throughout development

A systematic knock out of TRs by the transcription

activator-like effector (TALE) [128] or CRISPR

CAS9 [129] systems would help to understand

their integration in GRNs TALEs could also be

utilized to identify and characterize CRMs

Depending on the fused effector nucleases can

excise CRMs while repressors and activators could

temporarily change the activity of the targeted

CRM(s) [130] Experiments like this could help to

Figure 3 Scheme outlining the regulatory inter-actions in KA00 and KA0 interneurons In KA0 cells(right) tal2 and gata2 are expressed but not requiredfor gad67 expression In contrast these two genes arenecessary for gad67 expression in KA00 cells In KA00

cells on the other hand gata3 is expressed but notfunctionally linked to gad67 The expression of TF genesin different cells does not necessarily reflect conservedfunction even in a case as described here where thesame downstream gene (gad67) is expressed in bothcell types


resolve how multiple CRM modules work together

to define the expression domains of a gene and

would demonstrate if a CRM identified in reporter

assays is actually required for accurate expression in a

given context The ENCODE project demonstrated

that functional enhancers can be identified by bind-

ing of TRs at distinct loci in multiple cell lines

A similarly systematic ChIP-seq study in zebrafish

might shed light on the changes of CRM TR occu-

pancy over time in development Provided the

availability of good antibodies TF-binding sites

could be discovered rapidly in fish In summary

the time is ready for a zebrafish ENCODE project

to systematically mine the embryo of this animal

system for regulatory interactions and to combine

these with expression states and systematic functional

studies of TRs

Key Points

CRMs are DNA sequences that regulate gene expression TRs modulate gene expression on the protein level by

interactionwith CRMs Gene regulatory networks are formed by a multi-layered

interaction of TRs with CRMs resulting in the precise regulationof gene expression

The midline composed of notochord and floor plate is anorganizing centre for the specification of neuronal fate in thespinal cord controlling complex gene regulatory networks

FUNDINGThe work in our laboratory was supported by

the EU IP ZF-Health FP7-Health-2009-242048

NeuroXsys Health-F4-2009 No 223262 the

Interreg network for synthetic biology in the

Upper Rhine valley (NSB-Upper Rhine) the

BMBF funded network Erasys Bio BMBF KZ

0315716 and the BioInterfaces programme of the

Helmholtz association

References1 Ghiasvand NM Rudolph DD Mashayekhi M et al

Deletion of a remote enhancer near ATOH7 disrupts retinalneurogenesis causing NCRNA disease Nat Neurosci 201114578ndash86

2 Lee TI Young RA Transcriptional regulation and itsmisregulation in disease Cell 20131521237ndash51

3 Lettice LA Horikoshi T Heaney SJ et al Disruption ofa long-range cis-acting regulator for Shh causes preaxialpolydactyly Proc Natl Acad Sci USA 2002997548ndash53

4 Armant O Marz M Schmidt R et al Genome-widewhole mount in situ analysis of transcriptional regulatorsin zebrafish embryos Dev Biol 2013380(2)351ndash62

5 Vaquerizas JM Kummerfeld SK Teichmann SA et alA census of human transcription factors function expres-sion and evolution Nat Rev Genet 200910252ndash63

6 Davidson EH Emerging properties of animal gene regula-tory networks Nature 2010468911ndash20

7 Ettensohn CA Encoding anatomy Developmental generegulatory networks and morphogenesis Genesis 201351383ndash409

8 Peter IS Davidson EH Evolution of gene regulatorynetworks controlling body plan development Cell 2011144970ndash85

9 Bell AC West AG Felsenfeld G The protein CTCF isrequired for the enhancer blocking activity of vertebrateinsulators Cell 199998387ndash96

10 Handoko L Xu H Li G et al CTCF-mediated functionalchromatin interactome in pluripotent cells NatGenet 201143630ndash8

11 Parelho V Hadjur S Spivakov M et al Cohesins function-ally associate with CTCF on mammalian chromosomearms Cell 2008132422ndash33

12 Rubio ED Reiss DJ Welcsh PL et al CTCF physicallylinks cohesin to chromatin Proc Natl Acad Sci USA 20081058309ndash14

13 Banerji J Olson L Schaffner W A lymphocyte-specific cel-lular enhancer is located downstream of the joining region inimmunoglobulin heavy chain genes Cell 198333729ndash40

14 Banerji J Rusconi S Schaffner W Expression of a beta-globin gene is enhanced by remote SV40 DNA sequencesCell 198127299ndash308

15 Tumpel S Cambronero F Sims C et al A regulatorymodule embedded in the coding region of Hoxa2 controlsexpression in rhombomere 2 Proc Natl Acad Sci USA 200810520077ndash82

16 McLellan AS Kealey T Langlands K An E box in the exon1 promoter regulates insulin-like growth factor-I expressionin differentiating muscle cells AmJPhysiol Cell Physiol 2006291C300ndash7

17 Lettice LA Heaney SJ Purdie LA et al A long-range Shhenhancer regulates expression in the developing limb andfin and is associated with preaxial polydactyly Hum MolGenet 2003121725ndash35

18 Li Q Barkess G Qian H Chromatin looping and theprobability of transcription Trends Genet 200622197ndash202

19 Carter D Chakalova L Osborne CS et al Long-rangechromatin regulatory interactions in vivo Nat Genet 200232623ndash6

20 Chambeyron S Bickmore WA Chromatin decondensationand nuclear reorganization of the HoxB locus upon induc-tion of transcription Genes Dev 2004181119ndash30

21 Su W Porter S Kustu S et al DNA-looping and enhanceractivity association between DNA-bound NtrC activatorand RNA polymerase at the bacterial glnA promoterProc Natl Acad Sci USA 1990875504ndash8

22 Guo Y Monahan K Wu H etal CTCFcohesin-mediatedDNA looping is required for protocadherin alpha promoterchoice Proc Natl Acad Sci USA 201210921081ndash6

23 Morey C Da Silva NR Perry P et al Nuclear reorganisa-tion and chromatin decondensation are conserved but


distinct mechanisms linked to Hox gene activationDevelopment 2007134909ndash19

24 Butler JE Kadonaga JT The RNA polymerase II corepromoter a key component in the regulation of geneexpression Genes Dev 2002162583ndash92

25 Kadonaga JT Perspectives on the RNA polymerase II corepromoter Wiley Interdiscip Rev Dev Biol 2012140ndash51

26 Juven-Gershon T Kadonaga JT Regulation of geneexpression via the core promoter and the basal transcrip-tional machinery Dev Biol 2010339225ndash9

27 Thomas MC Chiang CM The general transcriptionmachinery and general cofactors Crit Rev Biochem Mol Biol200641105ndash78

28 Muller F Tora L The multicoloured world of promoterrecognition complexes EMBOJ 2004232ndash8

29 Goodrich JA Tjian R Unexpected roles for core promoterrecognition factors in cell-type-specific transcription andgene regulation Nat Rev Genet 201011549ndash58

30 Espinosa JM Verdun RE Emerson BM p53 functionsthrough stress- and promoter-specific recruitment oftranscription initiation components before and after DNAdamage Mol Cell 2003121015ndash27

31 Gehrig J Reischl M Kalmar E et al Automated high-throughput mapping of promoter-enhancer interactions inzebrafish embryos NatMethods 20096911ndash6

32 Maston GA Evans SK Green MR Transcriptional regula-tory elements in the human genome Annu Rev GenomicsHumGenet 2006729ndash59

33 Raab JR Kamakaka RT Insulators and promoters closerthan we think Nat RevGenet 201011439ndash46

34 Kolovos P Knoch TA Grosveld FG et al Enhancers andsilencers an integrated and simple model for their functionEpigenetics Chromatin 201251

35 Ferg M Sanges R Gehrig J et al The TATA-bindingprotein regulates maternal mRNA degradation and differ-ential zygotic transcription in zebrafish EMBO J 2007263945ndash56

36 Guo S Yamaguchi Y Schilbach S et al A regulator oftranscriptional elongation controls vertebrate neuronaldevelopment Nature 2000408366ndash9

37 Morley RH Lachani K Keefe D et al A gene regulatorynetwork directed by zebrafish No tail accounts for its rolesin mesoderm formation Proc Natl Acad Sci USA 20091063829ndash34

38 Yang L Rastegar S Strahle U Regulatory interactionsspecifying Kolmer-Agduhr interneurons Development20101372713ndash22

39 Aday AW Zhu LJ Lakshmanan A etal Identification of cisregulatory features in the embryonic zebrafish genomethrough large-scale profiling of H3K4me1 and H3K4me3binding sites Dev Biol 2011357450ndash62

40 Ariza-Cosano A Visel A Pennacchio LA et al Differencesin enhancer activity in mouse and zebrafish reporter assaysare often associated with changes in gene expression BMCGenomics 201213713

41 Chatterjee S Bourque G Lufkin T Conserved and non-conserved enhancers direct tissue specific transcription inancient germ layer specific developmental control genesBMCDev Biol 20111163

42 Yip KY Cheng C Bhardwaj N et al Classification ofhuman genomic regions based on experimentally

determined binding sites of more than 100 transcription-related factors Genome Biol 201213R48

43 Tamplin OJ Cox BJ Rossant J Integrated microarray andChIP analysis identifies multiple Foxa2 dependent targetgenes in the notochord Dev Biol 2011360415ndash25

44 Ritter DI Dong Z Guo S et al Transcriptional enhancersin protein-coding exons of vertebrate developmental genesPLoSOne 20127e35202

45 Smith RP Riesenfeld SJ Holloway AK et al A compactin vivo screen of all 6-mers reveals drivers of tissue-specificexpression and guides synthetic regulatory element designGenome Biol 201314R72

46 Ellingsen S Laplante MA Konig M et al Large-scaleenhancer detection in the zebrafish genome Development20051323799ndash811

47 Levesque MP Krauss J Koehler C et al New tools forthe identification of developmentally regulated enhancerregions in embryonic and adult zebrafish Zebrafish 20131021ndash9

48 Aparicio S Chapman J Stupka E et al Whole-genomeshotgun assembly and analysis of the genome of Fugurubripes Science 20022971301ndash10

49 Lander ES Linton LM Birren B et al Initial sequenc-ing and analysis of the human genome Nature 2001409860ndash921

50 Venter JC Adams MD Myers EW et al The sequence ofthe human genome Science 20012911304ndash51

51 Pennacchio LA Rubin EM Genomic strategies to identifymammalian regulatory sequences Nat Rev Genet 20012100ndash9

52 Plessy C Dickmeis T Chalmel F et al Enhancersequence conservation between vertebrates is favouredin developmental regulator genes Trends Genet 200521207ndash10

53 Sandelin A Bailey P Bruce S et al Arrays of ultra-conserved non-coding regions span the loci of key devel-opmental genes in vertebrate genomes BMC Genomics2004599

54 Woolfe A Goodson M Goode DK et al Highly conservednon-coding sequences are associated with vertebrate devel-opment PLoS Biol 20053e7

55 Bray N Dubchak I Pachter L AVID A global alignmentprogram GenomeRes 20031397ndash102

56 Brudno M Do CB Cooper GM et al LAGAN and Multi-LAGAN efficient tools for large-scale multiple alignment ofgenomic DNA Genome Res 200313721ndash31

57 Dubchak I Poliakov A Kislyuk A et al Multiple whole-genome alignments without a reference organism GenomeRes 200919682ndash9

58 Schwartz S Zhang Z Frazer KA et al PipMakerndasha webserver for aligning two genomic DNA sequences GenomeRes 200010577ndash86

59 Sanges R Kalmar E Claudiani P et al Shuffling of cis-regulatory elements is a pervasive feature of the vertebratelineage Genome Biol 20067R56

60 Kuntz SG Schwarz EM DeModena JA etal MultigenomeDNA sequence conservation identifies Hox cis-regulatoryelements GenomeRes 2008181955ndash68

61 Li Q Ritter D Yang N et al A systematic approach toidentify functional motifs within vertebrate developmentalenhancers Dev Biol 2010337484ndash95


62 Pennacchio LA Ahituv N Moses AM et al In vivo enhan-cer analysis of human conserved non-coding sequencesNature 2006444499ndash502

63 Sanges R Hadzhiev Y Gueroult-Bellone M et al Highlyconserved elements discovered in vertebrates are presentin non-syntenic loci of tunicates act as enhancers andcan be transcribed during development Nucleic Acids Res2013413600ndash18

64 Gross DS Garrard WT Nuclease hypersensitive sites inchromatin Annu Rev Biochem 198857159ndash97

65 Crawford GE Davis S Scacheri PC et al DNase-chip ahigh-resolution method to identify DNase I hypersensitivesites using tiled microarrays NatMethods 20063503ndash9

66 Song L Crawford GE DNase-seq a high-resolutiontechnique for mapping active gene regulatory elementsacross the genome from mammalian cells Cold Spring HarbProtoc 20102010pdb prot5384

67 Barski A Cuddapah S Cui K et al High-resolution profil-ing of histone methylations in the human genome Cell2007129823ndash37

68 Bernstein BE Kamal M Lindblad-Toh K et al Genomicmaps and comparative analysis of histone modifications inhuman and mouse Cell 2005120169ndash81

69 Heintzman ND Stuart RK Hon G et al Distinct andpredictive chromatin signatures of transcriptional promotersand enhancers in the human genome Nat Genet 200739311ndash8

70 Creyghton MP Cheng AW Welstead GG et al HistoneH3K27ac separates active from poised enhancers andpredicts developmental state Proc Natl Acad Sci USA 201010721931ndash6

71 Rada-Iglesias A Bajpai R Swigut T et al A uniquechromatin signature uncovers early developmental enhan-cers in humans Nature 2011470279ndash83

72 Zentner GE Tesar PJ Scacheri PC Epigenetic signaturesdistinguish multiple classes of enhancers with distinctcellular functions Genome Res 2011211273ndash83

73 Blow MJ McCulley DJ Li Z et al ChIP-Seq identificationof weakly conserved heart enhancers Nat Genet 201042806ndash10

74 Visel A Blow MJ Li Z et al ChIP-seq accurately predictstissue-specific activity of enhancers Nature 2009457854ndash8

75 Santos-Rosa H Schneider R Bannister AJ et al Activegenes are tri-methylated at K4 of histone H3 Nature2002419407ndash11

76 Wardle FC Odom DT Bell GW et al Zebrafish promotermicroarrays identify actively transcribed embryonic genesGenome Biol 20067R71

77 Bernstein BE Mikkelsen TS Xie X et al A bivalentchromatin structure marks key developmental genes inembryonic stem cells Cell 2006125315ndash26

78 Pan G Tian S Nie J et al Whole-genome analysis ofhistone H3 lysine 4 and lysine 27 methylation in humanembryonic stem cells Cell Stem Cell 20071299ndash312

79 Kane DA Kimmel CB The zebrafish midblastula transitionDevelopment 1993119447ndash56

80 Vastenhouw NL Zhang Y Woods IG et al Chromatinsignature of embryonic pluripotency is established duringgenome activation Nature 2010464922ndash6

81 Bogdanovic O Fernandez-Minan A Tena JJ et alDynamics of enhancer chromatin signatures mark the

transition from pluripotency to cell specification duringembryogenesis Genome Res 2012222043ndash53

82 Blader P Lam CS Rastegar S et al Conserved andacquired features of neurogenin1 regulation Development20041315627ndash37

83 Rastegar S Hess I Dickmeis T et al The words of theregulatory code are arranged in a variable manner in highlyconserved enhancers Dev Biol 2008318366ndash77

84 Kim TK Hemberg M Gray JM et al Widespreadtranscription at neuronal activity-regulated enhancersNature 2010465182ndash7

85 Li W Notani D Ma Q et al Functional roles of enhancerRNAs for oestrogen-dependent transcriptional activationNature 2013498516ndash20

86 Lai F Orom UA Cesaroni M et al Activating RNAsassociate with Mediator to enhance chromatin architectureand transcription Nature 2013494497ndash501

87 Wang KC Yang YW Liu B etal A long noncoding RNAmaintains active chromatin to coordinate homeotic geneexpression Nature 2011472120ndash4

88 Howe K Clark MD Torroja CF et al The zebrafishreference genome sequence and its relationship to thehuman genome Nature 2013496498ndash503

89 Hunter S Apweiler R Attwood TK et al InterPro theintegrative protein signature database Nucleic Acids Res200937D211ndash5

90 Domazet-Loso T Tautz D A phylogenetically basedtranscriptome age index mirrors ontogenetic divergencepatterns Nature 2010468815ndash8

91 Gordan R Murphy KF McCord RP et al Curatedcollection of yeast transcription factor DNA bindingspecificity data reveals novel structural and gene regulatoryinsights Genome Biol 201112R125

92 Taylor JS Braasch I Frickey T et al Genome duplicationa trait shared by 22000 species of ray-finned fish GenomeRes 200313382ndash90

93 Naef F Huelsken J Cell-type-specific transcriptomics inchimeric models using transcriptome-based masks NucleicAcids Res 200533e111

94 Ravasi T Suzuki H Cannistraci CV et al An atlas ofcombinatorial transcriptional regulation in mouse andman Cell 2010140744ndash52

95 Zhang W Morris QD Chang R et al The functionallandscape of mouse gene expression J Biol 2004321

96 Niehrs C Pollet N Synexpression groups in eukaryotesNature 1999402483ndash7

97 Karaulanov E Knochel W Niehrs C Transcriptionalregulation of BMP4 synexpression in transgenicXenopus EMBOJ 200423844ndash56

98 Albagli O Dhordain P Deweindt C et al The BTBPOZdomain a new protein-protein interaction motif commonto DNA- and actin-binding proteins Cell Growth Differ199561193ndash8

99 Kelly KF Daniel JM POZ for effectndashPOZ-ZF transcrip-tion factors in cancer and development Trends Cell Biol200616578ndash87

100 Stogios PJ Downs GS Jauhal JJ et al Sequence andstructural analysis of BTB domain proteins Genome Biol20056R82

101 Takeuchi JK Lou X Alexander JM et al Chromatinremodelling complex dosage modulates transcription


factor function in heart development NatCommun 20112187

102 Link BA Fadool JM Malicki J et al The zebrafish youngmutation acts non-cell-autonomously to uncouple differ-entiation from specification for all retinal cells Development20001272177ndash88

103 Gregg RG Willer GB Fadool JM et al Positional cloningof the young mutation identifies an essential role for theBrahma chromatin remodeling complex in mediatingretinal cell differentiation Proc Natl Acad Sci USA 20031006535ndash40

104 Le Dreau G Marti E Dorsal-ventral patterning of theneural tube a tale of three signals Dev Neurobiol 2012721471ndash81

105 Dessaud E McMahon AP Briscoe J Pattern formation inthe vertebrate neural tube a sonic hedgehog morphogen-regulated transcriptional network Development 20081352489ndash503

106 Briscoe J Ericson J Specification of neuronal fates in theventral neural tube Curr Opin Neurobiol 20011143ndash9

107 Jacob J Briscoe J Gli proteins and the control of spinal-cord patterning EMBORep 20034761ndash5

108 Hui CC Angers S Gli proteins in development anddisease Annu RevCell Dev Biol 201127513ndash37

109 Lewis KE How do genes regulate simple behavioursUnderstanding how different neurons in the vertebratespinal cord are genetically specified PhilosTrans RSoc LondB Biol Sci 200636145ndash66

110 Koudijs MJ den Broeder MJ Groot E et al Geneticanalysis of the two zebrafish patched homologues identifiesnovel roles for the hedgehog signaling pathway BMCDevBiol 2008815

111 England S Batista MF Mich JK et al Roles of Hedgehogpathway components and retinoic acid signalling in spe-cifying zebrafish ventral spinal cord neurons Development20111385121ndash34

112 Barth KA Wilson SW Expression of zebrafish nk22 isinfluenced by sonic hedgehogvertebrate hedgehog-1and demarcates a zone of neuronal differentiation in theembryonic forebrain Development 19951211755ndash68

113 Guner B Karlstrom RO Cloning of zebrafish nkx62 anda comprehensive analysis of the conserved transcriptionalresponse to HedgehogGli signaling in the zebrafish neuraltube Gene Expr Patterns 20077596ndash605

114 Schafer M Kinzel D Neuner C et al Hedgehog andretinoid signalling confines nkx22b expression to thelateral floor plate of the zebrafish trunk Mech Dev 200512243ndash56

115 Xu J Srinivas BP Tay SY et al Genomewide expressionprofiling in the zebrafish embryo identifies target genes

regulated by Hedgehog signaling during vertebratedevelopment Genetics 2006174735ndash52

116 Oosterveen T Kurdija S Alekseenko Z et al Mechanisticdifferences in the transcriptional interpretation of localand long-range Shh morphogen signaling Dev Cell 2012231006ndash19

117 Peterson KA Nishi Y Ma W et al Neural-specific Sox2input and differential Gli-binding affinity provide contextand positional information in Shh-directed neural pattern-ing Genes Dev 2012262802ndash16

118 Vokes SA Ji H McCuine S et al Genomic characteriza-tion of Gli-activator targets in sonic hedgehog-mediatedneural patterning Development 20071341977ndash89

119 Batista MF Jacobstein J Lewis KE Zebrafish V2 cellsdevelop into excitatory CiD and Notch signallingdependent inhibitory VeLD interneurons Dev Biol 2008322263ndash75

120 Kimura Y Satou C Higashijima S V2a and V2b neuronsare generated by the final divisions of pair-producingprogenitors in the zebrafish spinal cord Development20081353001ndash5

121 Park HC Shin J Appel B Spatial and temporal regulationof ventral spinal cord precursor specification by Hedgehogsignaling Development 20041315959ndash69

122 Wyart C Del Bene F Warp E etal Optogenetic dissectionof a behavioural module in the vertebrate spinal cordNature 2009461407ndash10

123 Schafer M Kinzel D Winkler C Discontinuous organiza-tion and specification of the lateral floor plate in zebrafishDev Biol 2007301117ndash29

124 Shin J Park HC Topczewska JM et al Neural cell fateanalysis in zebrafish using olig2 BAC transgenics MethodsCell Sci 2003257ndash14

125 Huang P Xiong F Megason SG et al Attenuationof Notch and Hedgehog signaling is required for fate spe-cification in the spinal cord PLoSGenet 20128e1002762

126 Li H de Faria JP Andrew P et al Phosphorylationregulates OLIG2 cofactor choice and the motor neuron-oligodendrocyte fate switch Neuron 201169918ndash29

127 Sun Y Meijer DH Alberta JA et al Phosphorylation stateof Olig2 regulates proliferation of neural progenitorsNeuron 201169906ndash17

128 Christian M Cermak T Doyle EL et al Targeting DNAdouble-strand breaks with TAL effector nucleases Genetics2010186757ndash61

129 Jinek M Chylinski K Fonfara I et al A programmabledual-RNA-guided DNA endonuclease in adaptive bacter-ial immunity Science 2012337816ndash21

130 Crocker J Stern DL TALE-mediated modulation of tran-scriptional enhancers in vivo NatMethods 201310762ndash7


silencer to its cognate gene promoter and interaction

of CCCTC-binding factor (CTCF) with insulators

has been demonstrated to be crucial to shield distal

enhancerssilencers [9] This classical view of CTCF

being a major marker for insulator elements has been

questioned by 4C experiments showing that many

of the CTCF-mediated long range interacting sites

correlate with enhancerndashpromoter interactions

Further experiments showed that CTCF facilitates

enhancerndashpromoter interactions directly through

cohesin-mediated DNA looping [10ndash12]

Enhancers have been traditionally defined as a

class of cis-regulatory elements that are able to acti-

vate and maintain gene expression in a location- and

orientation-independent manner [13 14] They are

found in the up- and downstream regions as well as

in introns of the regulated gene However enhancers

can also overlap with coding exons [15] or the

50-untranslated region (UTR) [16] They may be

located as far as 1 megabase from the target gene

as has been shown for example for a limb-specific

enhancer of the sonic hedgehog gene [17]

Enhancers are thought to represent islands of open

chromatin that act as docking stations for TFs [18]

By a combination of distinct TFs that bind to the

same enhancer various signalling inputs can be inte-

grated and interpreted thereby leading to specific

gene expression outputs In the prevailing model

the physical interaction of enhancers with their

target promoters is facilitated by a decondensation

of the chromatin structure enabling the DNA to

loop out from the nuclear periphery to the centre

to form enhancerpromoter interactions [19ndash21]

A recent publication suggests CTCFcohesin-

mediated DNA looping is a determinant for pro-

moter choice [22] However not all loci require

looping-out for transcriptional activity [23]

Promoters can be functionally subdivided into the

proximal promoter and the core promoter Proximal

promoters are located in the immediate vicinity of

the transcription start site (TSS) and contain like

enhancers or silencers recognition sites for DNA-

binding factors that are involved in transcriptional

regulation The core promoter is usually defined as

a 100ndash150 bp stretch of DNA encompassing the TSS

that is sufficient to direct initiation of transcription by

the RNA polymerase II machinery [24] Core pro-

moters can differ considerably in motif composition

(reviewed in [25ndash27]) and can be bound by a multi-

tude of promoter recognition complexes [28 29]

Not all enhancers are able to interact with every

type of core promoter [18 30 31] providing

another level of regulatory control

LCRs are like enhancers activating CRMs They

possess chromatin domain-opening activity and are

able to confer lsquocopy number-dependentrsquo expression

on a linked gene ie the number of integrated trans-

genes determines the expression level and not the

influence of the chromatin surrounding the trans-

genes (reviewed eg in [32 33])

Interestingly enhancers and silencers share im-

portant properties such as DNase I hypersensitivity

active chromatin marks and interaction with tran-

scription factors and RNA polymerase II It has

therefore been proposed that it depends on the

gene target and the developmental stage whether a

given CRM activates or represses gene transcription

[34]

CRMs contain the information required for

precise transcriptional regulation The implementa-

tion of this information is carried out by TRs

through sequence-specific interactions with CRMs

of their target genes As described above TFs activate

or repress gene transcription by binding to enhancers

or silencers respectively In addition general TFs

are involved in the basic processes of transcription

such as initiation elongation and termination of

transcription However they may be less lsquogeneralrsquo

than previously thought as recent evidence suggests

gene-specific employment of these factors [35 36]

Finally chromatin remodelling proteins alter the

state of the chromatin and thereby affect transcrip-

tion by regulating access to the CRMs

In summary GRNs can be defined as the imple-

mentation of the regulatory information encoded

in the DNA of CRMs via their interactions with

TRs Modelling a GRN related to any development

process requires knowledge of which TFs and signal-

ling molecules are involved when and where the

genes are expressed and how these factors interact

with each other and with CRMs [8] The zebrafish

is an ideal model to study the components and com-

position of GRNs Its fast and external development

and the high number of transparent embryos obtain-

able have proven very useful to address questions in

transcriptional regulation Zebrafish and other fish

species have been utilized to analyse developmental

GRNs [37 38] and promoterenhancer interactions

[31] to validate and analyse enhancer function on

a large scale [39ndash44] to address the regulatory

potential of short sequences [45] and to identify

regulatory regions driving tissue-specific expression














































































































level [81]



















































the CRMs
































































































































tional regulation

























ations of TFs






































evolution [109]




















































[105]


































































































































studies of TRs

Key Points





































































































































































































































































level [81]



















































the CRMs
































































































































tional regulation

























ations of TFs






































evolution [109]




















































[105]


































































































































studies of TRs

Key Points





























































































































































































































































tional regulation

























ations of TFs






































evolution [109]




















































[105]


































































































































studies of TRs

Key Points






































































































































































ations of TFs






































evolution [109]




















































[105]


































































































































studies of TRs

Key Points































































































































































[105]


































































































































studies of TRs

Key Points

















































































































































































































































studies of TRs

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Gene transcription in the zebrafish embryo: regulators and networks

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Transcript of Gene transcription in the zebrafish embryo: regulators and networks