The Enhancer of split transcription factor Her8a is a novel dimerisation partner for Her3 that controls anterior hindbrain neurogenesis in zebrafish.

Abstract

Neurogenesis control and the prevention of premature differentiation in the vertebrate embryo are crucial processes, allowing the formation of late-born cell types and ensuring the correct shape and cytoarchitecture of the brain. Members of the Hairy/Enhancer of Split (Hairy/E(spl)) family of bHLH-Orange transcription factors, such as zebrafish Her3, 5, 9 and 11, are implicated in the local inhibition of neurogenesis to maintain progenitor pools within the early neural plate. To better understand how these factors exert their inhibitory function, we aimed to isolate some of their functional interactors.
We used a yeast two-hybrid screen with Her5 as bait and recovered a novel zebrafish Hairy/E(spl) factor--Her8a. Using phylogenetic and synteny analyses, we demonstrate that her8a evolved from an ancient duplicate of Hes6 that was recently lost in the mammalian lineage. We show that her8a is expressed across the mid- and anterior hindbrain from the start of segmentation. Through knockdown and misexpression experiments, we demonstrate that Her8a is a negative regulator of neurogenesis and plays an essential role in generating progenitor pools within rhombomeres 2 and 4--a role resembling that of Her3. Her8a co-purifies with Her3, suggesting that Her8a-Her3 heterodimers may be relevant in this domain of the neural plate, where both proteins are co-expressed. Finally, we demonstrate that her8a expression is independent of Notch signaling at the early neural plate stage but that SoxB factors play a role in its expression, linking patterning information to neurogenesis control. Overall, the regulation and function of Her8a differ strikingly from those of its closest relative in other vertebrates--the Hes6-like proteins.
Our results characterize the phylogeny, expression and functional interactions involving a new Her factor, Her8a, and highlight the complex interplay of E(spl) proteins that generates the neurogenesis pattern of the zebrafish early neural plate.

Full text

Page 1

RESEARCH ARTICLEOpen Access
The Enhancer of split transcription factor Her8a is a
novel dimerisation partner for Her3 that controls
anterior hindbrain neurogenesis in zebrafish
Katharine J Webb1,5,6*†, Marion Coolen1,4,10†, Christian J Gloeckner2,3, Christian Stigloher1,7, Brigitte Bahn1,8,
Stefanie Topp1,9, Marius Ueffing2,3and Laure Bally-Cuif1,4,10*
Abstract
Background: Neurogenesis control and the prevention of premature differentiation in the vertebrate embryo are
crucial processes, allowing the formation of late-born cell types and ensuring the correct shape and
cytoarchitecture of the brain. Members of the Hairy/Enhancer of Split (Hairy/E(spl)) family of bHLH-Orange
transcription factors, such as zebrafish Her3, 5, 9 and 11, are implicated in the local inhibition of neurogenesis to
maintain progenitor pools within the early neural plate. To better understand how these factors exert their
inhibitory function, we aimed to isolate some of their functional interactors.
Results: We used a yeast two-hybrid screen with Her5 as bait and recovered a novel zebrafish Hairy/E(spl) factor -
Her8a. Using phylogenetic and synteny analyses, we demonstrate that her8a evolved from an ancient duplicate of
Hes6 that was recently lost in the mammalian lineage. We show that her8a is expressed across the mid- and
anterior hindbrain from the start of segmentation. Through knockdown and misexpression experiments, we
demonstrate that Her8a is a negative regulator of neurogenesis and plays an essential role in generating
progenitor pools within rhombomeres 2 and 4 - a role resembling that of Her3. Her8a co-purifies with Her3,
suggesting that Her8a-Her3 heterodimers may be relevant in this domain of the neural plate, where both proteins
are co-expressed. Finally, we demonstrate that her8a expression is independent of Notch signaling at the early
neural plate stage but that SoxB factors play a role in its expression, linking patterning information to neurogenesis
control. Overall, the regulation and function of Her8a differ strikingly from those of its closest relative in other
vertebrates - the Hes6-like proteins.
Conclusions: Our results characterize the phylogeny, expression and functional interactions involving a new Her
factor, Her8a, and highlight the complex interplay of E(spl) proteins that generates the neurogenesis pattern of the
zebrafish early neural plate.
Keywords: zebrafish, primary neurogenesis, midbrain-hindbrain, Hairy/E(spl), Her/Hes
Background
Neurogenesis in the early vertebrate neural plate
begins at stereotyped loci - termed proneural clusters
-, which prefigure the localization of the earliest neu-
ronal groups and the architecture of the primary
embryonic neuronal scaffold. These proneural clusters
consist of spatially defined progenitor groups engaged
in active neurogenesis, within which committed
precursors expressing higher levels of proneural genes
(such as neurogenin or achaete-scute-like genes,
respectively neurog1 and ascl1 in zebrafish) are singled
out to differentiate first. An identical scaffold is found
in all vertebrate embryos, highlighting the robustness
and functional relevance of this organization [1-4].
Dissecting the regulatory cascades involved in this pro-
cess is therefore of universal importance.
The control of neurogenesis progression within pro-
neural clusters relies on Hairy/Enhancer of split (E(spl))
factors (Hes in mouse; Her in zebrafish). These
* Correspondence: webb@helmholtz-muenchen.de; bally-cuif@inaf.cnrs-gif.fr
† Contributed equally
Full list of author information is available at the end of the article
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
© 2011 Webb et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

transcription factors belong to the basic-helix-loop-helix
(bHLH) family, characterized by a DNA-binding basic
domain and an HLH domain composed of two alpha
helices intervened by a loop of a few amino acids [5]. In
addition, Hairy/Enhancer of split (E(spl)) factors contain
an Orange domain, which is most probably involved in
protein-protein interactions, and a WRPW C-terminal
tetrapeptide, which mediates transcriptional repression
(reviewed in [6,7]). During the so-called process of lat-
eral inhibition, the expression of Notch ligands in com-
mitted precursors activates Notch signaling in
neighbouring progenitors, which in turn induces expres-
sion of Hes/Her factors. The latter down-regulate pro-
neural genes, hence maintaining Notch-receiving cells in
a progenitor state. Reflecting the intermingled distribu-
tion of committed and transiently inhibited progenitors,
the proneural and E(spl) genes are expressed in a salt-
and-pepper fashion within proneural clusters. E(Spl) fac-
tors expressed in proneural clusters in zebrafish include
her4.1 [8-10], hes5/her15, her2 and her12 [11]. In agree-
ment with the lateral inhibition model, her4.1 expression
is positively regulated by Notch, and inhibits expression
of neurog1 [10].
Recent work has demonstrated that proneural clusters
are delimited negatively, through a process of active
neurogenesis suppression taking place in surrounding
areas (reviewed in [12]). These “inhibited” areas, so-
called “progenitor pools”, are transiently maintained in
an refractory state to be recruited in later events of neu-
ronal production, and are organized as tight groups of
adjacent cells at stereotyped positions within the neural
plate. Major progenitor pools can be found at the pre-
sumptive midbrain-hindbrain boundary (MHB) [3,13]
and in longitudinal stripes separating the columns of
presumptive moto- and lateral neurons in the hindbrain,
or moto-, inter- and sensory neurons in the spinal cord
[11,14,15]. At the least, the MHB pool is maintained
until adulthood in zebrafish, where it participates in the
generation of adult-born neurons and oligodendrocytes
[16]. Embryonic progenitor pools are characterised by
the expression of a specific set of transcription factors,
including Zic, BF1/Anf and Rx family members [17] as
well as Hes/Her proteins. In zebrafish, the combinatorial
expression of a distinct set of her genes - which to date
includes her3, her5, her9 and her11 - characterises all
progenitor pools [12], while in mouse the genes Hes1,
Hes3 and Hes5 share sustained expression in adjacent
cells of the MHB pool, for example [18-20]. These her/
Hes genes exhibit functional similarities and have been
implicated in progenitor pool maintenance: their misex-
pression inhibits neurogenesis, whereas loss-of-function
causes premature expression of proneural genes in at
least part of their expression domains [11,13,15,19-22].
In addition, her3/5/9/11 as well as Hes1 at the mouse
MHB all demonstrate an irregular association with
Notch: while Hes/her genes in proneural clusters are
activated by Notch signaling, the expression of her3/5/9/
11 and Hes1 at the MHB is controlled in a Notch-inde-
pendent manner. The mechanisms accounting for these
specific features remain unknown.
The HLH region of Hes/Her factors functions as a
dimerisation domain, and the formation of hetero- and
homodimers as well as further possible interactions
through the Orange domain are key components of the
specificity of the actions of these proteins. Heterodimer-
isation can involve closely related members of the Her/
Hes family, or several different transcription factors or
transcriptional cofactors [7]. In order to better under-
stand the mechanism of action of Her factors expressed
in progenitor pools, and the pathways regulating their
activity, we performed a yeast two-hybrid screen using
the HLH and Orange domains of Her5 as bait. This led
to the recovery of Her8a, a novel Her factor of the Hes6
subfamily expressed in a broad manner at the presump-
tive midbrain-hindbrain domain of the early zebrafish
neural plate. Morpholino-mediated knockdown and mis-
expression studies establish Her8a as a negative-regula-
tor of neurogenesis playing an essential role in
maintaining progenitor pools of rhombomeres (r) 2 and
4. her8a knockdown produces a similar phenotype to
that of her3 knockdown, and co-purification demon-
strates that Her8a dimerises with Her3. At the MHB
however, we show that the predominant activity is
exerted by the combination of Her3, 5, 9 and 11.
Together, our results identify a new player in progenitor
pool formation and highlight the region-specific combi-
natorial activity of E(spl) factors in this process.
Results
Identification of Her8a as a potential binding partner for
Her proteins
To recover binding partners for Her proteins, we used a
yeast two-hybrid screen where a 181-amino acid frag-
ment of Her5 (excluding the basic domain and the
WRPW motif) was screened against an 18-20 hpf
embryo zebrafish library. This screen returned 280 posi-
tive clones, from a total of 76.1 million tested interac-
tions. These 280 positive clones represented 75 unique
protein-protein interactions. The quality of these inter-
actions was graded using a PBS scoring system - where
A is the highest score of confidence, B is very good, C is
good, D is low and N/A is used when no score could be
assigned (see Materials and Methods). Our screen
returned 6 As, 9 Bs, 2 Cs, 49 Ds and 9 N/As (see Addi-
tional file 1, Table S1 for a detailed description of all
recovered candidates). Gene ontology enrichment analy-
sis of the recovered binding partners revealed an enrich-
ment of proteins involved in protein transport and also
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 2 of 19

Page 3

heterodimerisation (see Additional file 2, Table S2).
Among these candidates, we note the presence of 7 dis-
tinct Her factors, in agreement with the postulated capa-
city for protein heterodimerisation within this family [7].
As further indication of the validity of the assay, Her5
was found to bind with Her11 with a score of B, an
interaction that had been shown previously in our
laboratory [22]. Her8a, scored A and corresponding to a
new E(spl) family member, proved very strongly
expressed in the midbrain-hindbrain (MH) area (see
below), and we consequently focused on this factor.
Her8a is a new Hes6-like protein, the ortholog of which
was lost in the mammalian lineage
The predicted Her8a protein displays a bHLH and an
orange domain, and harbors a WRPW motif at its C-
terminus - characteristics common to the Hairy/E(spl)
family. Sequence comparisons of the bHLH domain of
this family classify Her8a, together with zebrafish Her13
(previously Her13.1), Hes6 (previously Her13.2) and
Her8.2, within the subfamily showing highest homology
to mouse Hes6 [23]. Proteins of this subfamily exhibit a
shortened loop when compared to other Hairy-E(spl)
members [24], such as mouse Hes1 and Hes5 (Addi-
tional file 3, Figure S1). Hes6-like proteins also share
substantial similarity outside the bHLH domains (Addi-
tional file 3, Figure S1), allowing their phylogeny to be
studied using extended protein sequences. This con-
firmed the existence of two subfamilies of Hes6-like
proteins, Hes6.1 and Hes6.2, comprising respectively
Her13/Hes6 and Her8a/Her8.2 (Figure 1A) (and see
[23]), encoded by gene pairs (Figure 1B). Importantly, it
also resolved for the first time their relationship with
the single mammalian Hes6 protein, as sequence align-
ments directly assign mammalian Hes6 to the Hes6.1
subfamily (Figure 1A). hes6.2-like genes are found
neither in eutherian mammals nor in marsupials, but
exist in all other phyla, suggesting a late secondary loss
of this gene shortly after the divergence of eutherians
and marsupials from the monotreme lineage. Finally,
and as expected from the whole genome duplication
undergone in the teleost fish lineage subsequent to its
divergence from other vertebrates [25], followed by sec-
ondary gene loss, teleost species exhibit three or four
Hes6-like genes. Synteny analyses (Figure 1C) indeed
identify a conserved orthologous gene pair (locus 1) as
well as a conserved duplicate (locus 2). This duplicate
only contains the hes6.1-like member and, based on the
situation in other vertebrates, most likely lost the
hes6.2-like gene. Strikingly, in zebrafish, the latter gene
(her8a) was kept and transferred onto a different geno-
mic location (locus 3). Together, these results indicate
that her8a is the ortholog of her8.2 and that it is located
in a unique genomic setting compared to other related
Figure 1 her8a encodes a Hes6-like E(spl) protein closely related to, but not directly orthologous to, mammalian Hes6. A. Phylogenetic
tree depicting protein relationship within the Hes6 subfamily, based on the bHLH and Orange domain sequences. Note the absence of Hes6.2
proteins in marsupials and eutherian mammals. In zebrafish, the closest relative to Her8a is Her8.2, and the closest relative to Her13 is Hes6. B.
Genomic organization of Hes6-like genes through evolution, confirming that the generation of the two Hes6.1 and Hes6.2 genes is ancestral and
that Hes6.2 was secondarily lost within the mammalian taxa subsequent to the divergence of marsupials and eutherian mammals from
monotremes. “?” indicates cases where genomic linkage cannot be resolved at present, in the absence of a genome sequence for the
corresponding species. C. Genomic organization of the areas surrounding Hes6-like genes in teleost species compared to mouse. With the
exception of zebrafish, for all of the teleosts studied, synteny analyses suggest a secondary loss of the hes6.2 duplicate (missing from locus 2)
after the duplication of locus 1. In zebrafish, this gene (her8a) has been relocated to a third locus on a distinct chromosome. 1-3: genomic loci.
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 3 of 19

Page 4

genes. It also assigns her8a to a subfamily of Hes6-like
genes that is closely related to but not directly ortholo-
gous to mammalian Hes6.
her8a is expressed across progenitor pools and proneural
clusters in the anterior neural plate
Among hes6-like genes, we found that her8a was the
only one with expression in the anterior neural plate at
the 3-somite stage. At this stage, her13 was restricted to
a portion of the presumptive lateral proneural clusters
in the anterior spinal cord (Additional file 4, Figure S2).
As shown previously, hes6 was found to be expressed in
the tail bud and posterior paraxial mesoderm (Thisse
and Thisse, 2004), and her8.2 was very weakly or not
expressed (not shown). In contrast, following weak
expression during gastrulation [26], her8a displayed
strong expression in the neural plate from the tailbud
stage onwards. At tailbud, her8a is expressed through-
out the neural plate, with the exception of the eye field
and the midline, with strongest intensity at presumptive
midbrain and hindbrain levels (Figure 2A, bracket). At
early segmentation stages, the domain of strong her8a
expression overlaps with that of genes expressed in pro-
genitor pools within the presumptive mid- and hind-
brain, such as her3 (Figure 2B) and her5 (Figure 2C)
(purple arrows). It also encompasses the proneural clus-
ters located in the mid- and anterior hindbrain area (e.g.
compare with neurog1 expression, Figure 3A). At 10
somites her8a is expressed in stripes in the hindbrain,
with denser expression in rhombomeres (r) 1 and 3-5
(Figure 2D,E). Expression in the midbrain persists. From
24hpf onwards, her8a acquires a distinct expression pro-
file, highlighting neurogenic domains throughout the
central nervous system, with weaker staining at the mid-
brain-hindbrain boundary (mhb) (arrow) and at the
zona limitans intrathalamica (Figure 2F) (asterisk). Cross
sections of the brain at this stage reveal that her8a
expression is confined to the progenitor, ventricular
domain, largely complementary to the expression of the
post-mitotic neuronal marker HuC/D (Figure 2G). From
48hpf through to adult, her8a remains expressed in pro-
liferation/ventricular zones throughout the brain (Figure
2H,I, and data not shown).
Gain of Her8a function inhibits neurogenesis
Mouse and Xenopus Hes6 proteins are known as posi-
tive regulators of differentiation [24,27]. In this context,
the expression of her8a across both pro-neural and non-
neurogenic domains was puzzling and prompted us to
explore Her8a function.
In a first gain-of-function approach, embryos were
injected with her8a capped mRNA encoding the full-
length protein at the one cell stage. They were subse-
quently fixed and analyzed at 3 somites. We observed
that her8a misexpression caused a complete loss of neu-
rog1 expression throughout the embryo (Figure 3A,B)
(77% of cases, n = 22). Co-labeling with tp63 (previously
ΔNp63), which highlights the border of the epidermal
ectoderm juxtaposed to the neural plate [28] (Figure 3A,
white arrowheads), showed that the size or morphology
of the neural plate were not affected (100% of cases, n =
22), suggesting that overexpressing Her8a specifically
blocks neurogenesis without an effect on neural plate
formation. This was confirmed by expression analyses
for patterning markers such as barhl2, her5 and her9,
which highlight distinct domains along the entire
antero-posterior axis of the neural plate [11,29,30]
(Additional file 5, Figure S3). Together, these results
indicate that her8a is capable of inhibiting neurogenesis,
at least at non-physiological concentrations, and this
even across domains normally co-expressing neurog1
and her8a such as the proneural clusters of the mid-
and anterior hindbrain (vcc, r2MN, r2l, R4MN, r4l).
Her8a is required to maintain the proper neurogenesis
pattern in rhombomeres 2 and 4 and acts as binding
partner for Her3
To better appreciate the endogenous requirements for
Her8a, we next turned to a loss-of-function approach.
Embryos at the one-cell stage were injected with mor-
pholinos (MO) directed against the donor splice site of
her8a exon 1 (MO1), the acceptor splice site of her8a
exon 2 (MO2), or the her8a ATG (MO3), and were ana-
lyzed at 3 somites. Reverse transcription PCR was used
to reveal strong down-regulation of expression and
abnormal splicing of her8a transcripts with both MO1
and MO2, whereas other genes, such as bactin2,
remained unaffected (Figure 3G). These observations
substantiate that her8aMOs lead to knock-down of
her8a expression.
Blocking Her8a resulted in an ectopic expression of
neurog1 within the normally non-neurogenic area separ-
ating motor- and lateral proneural clusters of r2 and 4
(Figure 3C, arrowheads, Figure 3D, arrows) (80% of
cases, n = 60). Although the location of neurog1-positive
cells can be slightly variable from embryo to embryo,
this phenotype was robust and never observed in wild-
type animals. The results obtained with the three MOs
were strictly identical (Additional file 6, Figure S4 for a
comparison of MO1 and MO2, and data not shown),
confirming their specificity. We will compile these data
below. This knockdown phenotype strikingly resembles
the published effect of her3 knockdown [15], which we
further confirmed (Figure 3E) (56% of cases, n = 50).
Given that the r2/r4 area of ectopic neurogenesis is
where the intense expression of her8a overlaps with that
of her3 (Figure 1B, Figure 4B), these identical pheno-
types suggest a direct or indirect functional interaction
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 4 of 19

Page 5

Figure 2 Embryonic expression of her8a. Expression is revealed by whole-mount in situ hybridization at the stages indicated (som: somites,
hpf: hours post-fertilization) together with other positional marker genes (B,C,E, color-coded) or proteins (G, immunocytochemistry for the
neuronal marker HuC/D in green). A-E and H are dorsal views of flat-mounted embryos, F,I are lateral views, all embryos are viewed anterior left.
G is a cross section of a 24hpf embryo at midbrain levels (as indicated in F). At early neural plate stages, the domain of strongest her8a
expression covers the mid- and anterior hindbrain (bracket in A-C). It overlaps the presumptive midbrain-hindbrain boundary (her5-positive,
purple arrow in C) and the progenitor pools separating medial and lateral hindbrain neurons (her3-positive, purple arrow in B). It extends into
rhombomere 2 (white arrowhead in C), more posterior rhombomeres being more weakly labeled. At 10 somites, expression in the midbrain is
maintained. It resolves in stripes in the rhombencephalon (D,E). It avoids the midbrain-hindbrain boundary and zona limitans intrathalamica
(asterisk). From 24 hpf onwards, her8a characterizes the ventricular zone and progenitor domains of the neural tube (G-I, arrows in I point to the
ventricular zone and the arrowhead points to the progenitor domain of the optic tectum). Abbreviations: e: eye field, mid: presumptive
midbrain, mhb: midbrain-hindbrain boundary, r: rhombomere. Scale bars: 100 μm.
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 5 of 19

Page 6

Figure 3 Like Her3, Her8a activity maintains the non-neurogenic areas of rhombomeres 2 and 4. A,B. neurog1 expression highlights the
proneural clusters at the 3-somite stage (black arrows in A) and is eliminated upon her8a overexpression (B, embryo injected with her8a capped
mRNA). Expression of tp63, which highlights the neural plate border (white arrowheads in A), is unchanged. C-F. Compared expression of neurog1
in control embryos (C) and embryos injected with her8aMO (D), her3MO (E) or both MOs (F) shows ectopic neurogenesis between the medial and
lateral proneural clusters of r2 and r4 (blue arrows in D-F, compare with white arrowheads in C) when Her8a and/or Her3 activities are blocked. Few
“ectopic” neurog1-positive can sometimes be found between the vcc and mnr2; this is however highly variable between individuals and observed
in both control and morphant embryos. A-F are dorsal views of flat-mounted embryos, anterior left. Abbreviations: black arrows indicate proneural
clusters: IN: presumptive interneurons, MN: presumptive motoneurons, r2: rhombomere 2, r4: rhombomere 4, r2l: lateral neurons of rhombomere 2,
r4l: lateral neurons of rhombomere 4, SN: presumptive sensory neurons, vcc: ventro-caudal cluster. G. RT-PCR analysis of her8a expression (left and
middle panels) in embryos injected with her8aMO1 ("mo1”) and her8aMO2 ("mo2”) versus control embryos ("ctr”). Low levels of full length, normally
spliced her8a transcripts are detectable in morphants (left and middle panels, arrows) while abnormally spliced transcripts including all or part of
intron 1 become produced (stars). Expression of bactin2, used as RT-PCR control, is indentical in all samples (right panel). The scheme at the top
indicates the genomic structure of her8a, the position of exons (E, purple) and introns (black bars), the binding sites of her8aMO1 and MO2 (red),
the position of RT-PCR primers (blue arrows) and the length of amplified wild-type products (excluding introns) (blue bars).
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 6 of 19

Page 7

of Her3 and Her8a in controlling common progenitors.
As a first obvious possibility, we tested whether her3
and her8a influence each other’s expression. Ruling out
this scenario, her8aMO-injected embryos displayed a
normal expression of her3, and her3MO embryos a nor-
mal expression of her8a (not shown) (100% of cases, n
= 37 and 24, respectively). A following hypothesis is that
her3 and her8a act in a dose-dependent manner to com-
pensate for each other outside r2 and r4, with r2 and r4
showing highest sensitivity to the amount of “Her3
+Her8a” proteins. A similar situation was previously
demonstrated for Her5+Her11 at the MHB [22]. In this
case, we expect that the knockdown of both genes
would produce a phenotype of a greater magnitude than
Figure 4 The heterodimerisation potential of full-length Her8a and Her3 proteins may account for their identical loss-of-function
phenotypes. A. Co-affinity purification of full-length Her3 and Her8a proteins in HEK293T cells. When lysates from cells expressing both N-Strep/
Flag-Her3 (N-SF-Her3) and Myc-tagged Her8a (myc-Her8a) proteins are eluted from a STREP-Tactin resin, myc-Her8a is observed to co-purify with
N-SF-Her3 (lane 4). B-E. Schematized summary of the expression patterns (B) and loss-of-function phenotypes (C-E) of her8a and her3. Genes
expression are color-coded and her5/her11 expression is indicated as a landmark (see also Figure 5H). Abbreviations: as in Figure 3; tg: trigeminal
ganglion.
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 7 of 19

Page 8

the ectopic expression of proneural markers in r2 and
r4. To test this possibility, we simultaneously blocked
Her3 and Her8a by the co-injection of her3MO and
her8aMO (at the same concentrations shown to produce
the individual phenotypes). This produced no additional
effect on neurog1 expression (Figure 3F) (80% of cases,
n = 20). Likewise, the co-injection of her3MO and her8-
aMO in amounts just below their effective doses (0.75
mM for her8a and 0.375 mM for her3) induced no phe-
notype in r2 and r4 (not shown). Together, these obser-
vations indicate that these two factors alone are not
compensating for each other to repress neurog1 in other
areas of the embryo.
As a final alternative hypothesis, and based on the
recovery of Her8a as binding to a Her bHLH domain in
yeast cells, we tested whether Her8a and Her3 could act
as necessary heterodimerisation partners. As no com-
mercial antibodies are available for Her3 and Her8a, and
attempts by our laboratory to have them manufactured
failed, we chose a co-purification approach using tagged
versions of the full-length zebrafish proteins recombi-
nantly expressed in HEK293T cells. By purifying Strep/
Flag-tagged Her3 via its Strep-tag II moiety, Myc-tagged
Her8a was successfully co-purified (Figure 4A), demon-
strating that both proteins interact with each other. This
interaction may be relevant to the maintenance of the
progenitor pools within r2 and r4, where both her3 and
her8a are strongly expressed, and hereby account for
the identical phenotypes of Her3 and Her8a loss of
function (summarized in Figure 4B-E).
In the absence of Her3, 5, 9 and 11 activity, endogenous
Her8a alone is insufficient to preserve neurog1-free
progenitor pools in the midbrain-hindbrain domain
The results above indicate that, although her8a is
expressed across the entire MH domain, it is only
strictly required to block neurogenesis in r2 and r4.
This raises the question of which her genes combination
encodes the endogenous pattern of neurogenesis inhibi-
tion in the MH domain, and whether this combination
involves her8a expression. To address this issue, we
used double in situ hybridization to re-analyze expres-
sion of the progenitor pools genes, comparing her3, 5, 9
and 11. Our data confirmed the full overlap of her5 and
her11 (Figure 5A,D) as well as the extension of her3
longitudinal stripes into the presumptive MHB domain
[15] (Figure 5B, arrows), and revealed a previously unre-
ported expression of her9 coinciding with the antero-lat-
eral aspects of the her5/11 territory (Figure 5C, arrows)
(summarized in Figure 5K,L). Previous loss-of-function
experiments of combinations of these genes never
achieved a full neurogenic phenotype: concomitantly
blocking her5 and her11 induced neurog1 medially but
only to a lesser extent in mediolateral and lateral MHB
domains [22] (Figure 5M,N), and the co-inhibition of
her3 and her9 largely recapitulated her3 loss-of-function
in the MH area, with a restricted induction of neurog1
within r2 and r4 [11] (Figure 5O,P). We found that the
down-regulation of all four factors together, through the
coinjection of the relevant gripNA antisense oligonu-
cleotides, was required to generate a large neurog1-posi-
tive domain across the presumptive MHB and r2
(Figure 5E,F) (74% of cases, n = 19) - although the most
lateral aspects of the neural plate remained neurog1-free
-. In these conditions however, her8a expression
remained unperturbed (Figure 5G,H) (100% of cases, n
= 39) (schematized in Figure 5Q). Together, these
results demonstrate that the endogenous activity of
Her8a, in the absence of other progenitor pools Her fac-
tors, is insufficient to inhibit neurog1 expression in the
MH area. her8a expression also appears insensitive to
the combined expression levels of Her3/5/9/11 and to
the neurogenic status of this neural plate domain. When
following the fate of ectopic neurog1-expressing progeni-
tors in the absence of Her3/5/9 and 11 activities, we
found however that only a subset were maintained until
24hpf. These were located ventrally across the midbrain-
hindbrain boundary, immediately posterior to vcc-
derived neurons (Figure 5I,J).
Endogenous her8a expression in the early neural plate is
independent of Notch signaling but requires the
expression of SoxB factors
Although many E(spl) transcription factors are down-
stream effectors of Notch signalling, previous work has
shown that zebrafish her genes expressed in progenitor
pools, such as her3, 5, 9 and 11 [11,13,31] exhibit a
non-canonical regulation by Notch: they do not require
Notch for their expression, and are insensitive to or
transcriptionally inhibited upon ectopic Notch activa-
tion. This is in contrast to other family members such
as her4.1 that are expressed in neurogenic zones and are
activated by Notch signaling [32].
Unusually, her8a is expressed across both progenitor
pools and proneural clusters in the early neural plate.
To analyze the effect of ectopic Notch activation, we
overexpressed the intracellular domain of zebrafish
Notch1a (NICD) [10] through capped mRNA injection
at the one-cell stage. We could replicate previously
published results [10] showing that NICD misexpres-
sion completely downregulates neurogenesis through-
out the early neural plate (Figure 6A,B) (88% of cases,
n = 16). We found that overexpression of NICD causes
ectopic or enhanced expression of her8a throughout
the embryo (Figure 6C,D) (100% of cases, n = 17),
although this induction was weaker at the caudal end
of the neural plate (Figure 6D, asterisk). The latter
observation was repeated when studying the her4.1
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 8 of 19

Page 9

Figure 5 The activities of Her3, Her5, Her9 and Her11 together account for the progenitor pool pattern of the midbrain-hindbrain
area and do not influence her8a expression. A-D. A comparison of the expression patterns of her3, her5, her9, her11 and neurog1 at 3
somites in the midbrain-hindbrain (MH) area using double in situ hybridization (color-coded), on dorsal views of flat-mounted embryos. Arrows
point to the her5/her11 domains co-expressing her3 or her9. E-J. Compared neurogenesis in control embryos (E,G,I) and embryos co-injected
with the four gripNA antisense oligonucleotides directed against her3, 5, 9 and 11 transcripts (see Methods) (F,H,J). E,F. Expression of neurog1
(revealed by in situ hybridization against gfp in -8.4neurog1:gfp transgenic embryos) [62]: the majority of the MHB/r2 area is induced to express
neurog1. G,H. The expression her8a is unaltered upon injection of the four gripNAs. I,J. Detection of GFP in -8.4neurog1:gfp embryos at 24 hpf
(sagittal view, confocal projection of a 20 μm section of the neural tube). Ectopic neurons are formed ventrally across the midbrain-hindbrain
boundary (position of the boundary indicated by the white bar), in a location normally devoid of GFP-positive cells (arrowheads). K,L.
Summarized compared expression of her3, 5, 9 and 11 (K), also together with her8a (L). M-Q. Summary of the combined loss-of-function results
for MH-expressed her genes, from Geling et al. [13] (M), Ninkovic et al. [22] (N), Bae et al. [11] (O,P) and the present paper (Q). Abbreviations: mid:
midbrain, MN: presumptive motoneurons, r: rhombomere, r2l: lateral neurons of rhombomere 2, vcc: ventro-caudal cluster.
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 9 of 19

Page 10

Figure 6 her8a expression is independent of endogenous Notch signaling in the early neural plate. Dorsal views of flat-mounted embryos
analyzed at the 3-somite stage for the expression of the genes indicated. A-F. Ectopic Notch activation (injection of capped NICD mRNA) abolished
neurog1 expression (B) and activates her8a (D) and her4.1 (F) compared to non-injected embryos (ctrl). G-J’. Notch blockade (incubation in the
gamma-secretase inhibitor DAPT) increases neurog1 expression within proneural clusters (H) but leaves her8a expression intact (J) at early neural
plate stages compared to embryos treated with a vehicle only (ctrl). G’-J’ are high magnification views of the areas boxed in G-J.
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 10 of 19

Page 11

target gene (Figure 6E,F) (100% of cases, n = 13), sug-
gesting a generally lower ability of this neural plate
area to respond to NICD overexpression, rather than a
her8a-specific feature in Notch response. Next, to
determine whether her8a expression depends on endo-
genous Notch signaling, we incubated embryos
between the 50% epiboly and 3-somite stages into the
gamma-secretase inhibitor DAPT, which blocks Notch
by preventing the cleavage of NICD and has a strong
neurogenic effect [33,34]. As anticipated from previous
studies [33], DAPT treatment increased the amount of
neurog1-positive cells within each proneural cluster
(Figure 6G-H>’) (79% of cases, n = 19). However, it
failed to reproducibly affect the expression of her8a
(Figure 6C,I-J’) (100% of cases, n = 15). Together,
these results indicate that endogenous Notch signaling
is not required for her8a expression in the early neural
plate.
In order to gain further insight into the endogenous
mechanisms controlling her8a regulation at these stages,
we scanned the her8a promoter (100 bp downstream
and 1000 bp upstream of the ATG start site) with the
ModelInspector program (Genomatix) [35]. This
revealed a potential Sox (Sry-related HMG box) binding
site (SORY_OCT1_01) at position 873-897(+). ModelIn-
spector uses Genomatix’s in-house Promoter Module
Library, which includes experimentally verified models
for functional promoter subunits. In this case the pro-
moter sequence was derived from a publication describ-
ing the activation of the mouse Fgf4 enhancer by Sox2
and Oct-3 [36]. This led us to investigate the possibility
that a member of the sox gene family is controlling
her8a expression. Mouse Sox2 is a member of Group
B1, a subdivision of Sox genes involved in neural devel-
opment [37]. Within this subgroup, we focused on the
zebrafish genes sox2 [37], sox3 [37], sox19a and sox19b
[38] (see [37] for phylogenetic description), excluding
sox1a and sox1b, which are not expressed at the MHB
at early embryonic stages [26,37]. In addition, we also
investigated sox21a (previously sox21 or sox30), a mem-
ber of the related subgroup B2 with specific MHB
expression at early embryonic stages [39]. We found
that expression of these different sox genes overlapped
all or part of the her8a-positive domain at the 5-somite
stage: sox2 and sox3 displayed strongest overlap with
her8a expression in the anterior hindbrain (mostly r3)
(Figure 7A,B), sox21a at the MHB (Figure 7E), while
sox19a and b were intensely expressed throughout the
MH (Figure 7C,D). In addition, sox2, 3, 19a and 19b all
displayed an expression identical to her8a in the pre-
sumptive telencephalon and ventral diencephalon,
excluding the eye field (Figure 7A-D, compare with F).
To analyze the role of these genes in controlling her8a
expression, we used MOs targeting their ATG start site
[40-43]. A single MO was used to inhibit sox2 and 3,
which share the sequence surrounding their ATG.
When injected individually at the one-cell stage, none of
these MOs produced a phenotype on her8a expression
at the 3-somite stage (not shown) (n = 25). However,
the combined knock-down of all five sox genes at once
caused reduced her8a staining in the MH area (Figure
7G-I) (100% of cases, n = 18), indicating that these fac-
tors cooperate, possibly in a dose-dependent manner, to
enhance her8a expression within the early neural plate.
In a reverse step, we analyzed whether these genes were
linked by a positive regulatory loop. We found however
that blocking Her8a function upon her8aMO injection
had no effect on sox genes expression at the 3-somite
stage (not shown) (100% of cases, n = 10 for each sox
gene tested).
Discussion
Her8a is a neurogenesis repressor in the early zebrafish
neural plate
Two lines of evidence demonstrate that Her8a can act
as a repressor of neurogenesis: firstly, the overexpres-
sion of full-length her8a causes a complete loss of neu-
rog1 expression in the early embryo; secondly, we show
that morpholino-mediated knockdown of her8a causes
ectopic neurog1 expression in rhombomeres 2 and 4.
These results are surprising, since the Hes6-like factors
studied to date tend to exhibit neurogenesis-promoting
activity. When ectopically expressed, Hes6 promotes
neurogenesis in the Xenopus embryo [27], the differen-
tiation of cortical neurons at the expense of astrocytes
in the mouse [44,45], and the differentiation of retinal
precursor cells into photoreceptors in mouse retinal
explants [24]. These activities at least in part involve
functionally antagonizing Hes1, since it was shown that
Hes6 alone cannot bind the canonical E(spl) binding
site (N box) [24,45]. Rather, Hes6 dimerises with Hes1
and modifies its DNA binding properties [24], its capa-
city for recruiting the co-repressor Groucho or its stabi-
lity [44]. Interestingly at least some of these properties
appear controlled by the loop domain of Hes6, which is
five amino acids shorter than that of other E(spl) pro-
teins (see Additional file 3, Figure S1). Indeed, the addi-
tion of five amino acid residues into the loop of Hes6
confers Hes1-like repressor activity on the N box, while
conversely, the removal of five amino acid residues
from the loop of Hes1 completely ceases repression
activity and confers Hes6-like activity [24]. We observed
that Her8a has an intermediate loop-length compared
to Hes6 and Hes1 (Additional file 3, Figure S1).
Although the functional significance of this feature
remains a matter for investigation, it is possible that it
confers specific mechanistic properties to Her8a that
distinguish it from Hes6 and bring it closer to the
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 11 of 19

Page 12

mode of action of Hes1-like proteins. In addition, our
phylogenetic and synteny analyses revealed the complex
evolution of Hes6-like genes and, contrary to previous
belief, that Her8a is not a direct ortholog of Hes6.
Rather, Hes6 orthologs comprise zebrafish Her13 and
Hes6, both of which have the same loop length as
mouse Hes6. We show here that zebrafish her13 is spe-
cifically expressed in a pattern coincident with neuro-
genesis (Additional file 4, Figure S2) reminiscent of
Hes6 expression in the developing nervous system of
both mouse and Xenopus, highlighting committed pro-
genitors or early neurons [24,27]. Thus we would pre-
dict that zebrafish Her13, rather than Her8a, shares
functional properties with mammalian Hes6. The func-
tion of Hes6.2 subfamily proteins, to which Her8a
belongs, has not been thoroughly tested, largely due to
their absence in mammals. In the chicken neural tube
however, Hes6.2 exerts a neurogenesis promoting activ-
ity [46], suggesting that Hes6.2 proteins may differ in
their activities. We further propose that the splitting of
Figure 7 her8a expression in the early neural plate is partially dependent on the expression of SoxB factors. Dorsal views of flat-
mounted embryos analyzed at the 5-somite stage (A-F) and 3-somite stage (G-H) for the expression of the genes indicated (color-coded). A-F.
her8a expression overlaps with that of the Sox family members sox2, sox3, sox19a, sox19b and sox21a, including the mid- and anterior hindbrain
(red brackets). G-I. the simultaneous knockdown of sox2/3/19a/19b and 21a causes a reduction of her8a expression in the MH area of the early
neural plate (white arrows).
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 12 of 19

Page 13

her8a from locus 2 to a distinct genomic location (Fig-
ure 1C) permitted the acquisition of a unique expres-
sion profile for this gene in zebrafish.
Combined Her activities generate the midbrain-hindbrain
progenitor pool pattern through different modalities
Our loss-of-function studies demonstrate that Her8a
activity is necessary to prevent neurogenesis within the
mediolateral territory of r2 and 4, hence keeping the
proneural clusters for moto- and lateral neurons spa-
tially separated within the anterior hindbrain. Combined
with the fact that endogenous her8a expression does
not depend on Notch at this developmental stage (Fig-
ure 6), this function is typical of a “pre-patterning”
activity, comparable to that exhibited by the other E(spl)
factors Her3, 5, 9 and 11 that delimit the territories
competent for neurog1 expression within the neural
plate [11,13,15]. Despite this functional relevance, the
phenotype of her8a morphants appears very restricted
compared to the broad expression of her8a, which
encompasses the entire mid- and anterior hindbrain
(Figure 4B). Functional redundancy and dosage effects
have been described for other members of the E(spl)
family in the mouse neural tube [20] and the zebrafish
early neural plate [11,22]. For example, Her5 and Her11
act in an equivalent and dose-dependent manner to
block neurog1 expression in the medial and lateral
aspects of the presumptive MHB [22], and Her3 and
Her9 also cooperate to inhibit neurogenesis within the
longitudinal stripe separating the presumptive moto-
and interneuron clusters of the spinal cord [11]. We
found that all her genes analyzed here (her3, 5, 9, 11
and 8a) were at least partially co-expressed within the
presumptive MH (Figure 5L), strongly suggesting that
redundancy may account for the normal development of
this area in her8a morphants. The situation in the hind-
brain, however, appears different. The only two her
genes highlighting progenitor pools in r2-4 are her3 and
her8a. While morphant embryos for each of these genes
have an identical phenotype, our results argue against a
dose-dependent mechanism involving Her3 and Her8a.
Indeed, we found that the co-injection of her8aMO and
her3MO at active doses did not produce an additional
phenotype (Figure 3F) and that, if both morpholinos
were injected together in amounts just below their effec-
tive concentration, ectopic neurog1 expression was not
observed. Given that the two factors do not regulate
each other’s expression, these experiments suggest that
the presence of each factor individually, rather than
their overall dose, is relevant to maintain neurogenesis
inhibition within r2 and r4. Although Her8a was isolated
as a binding partner for Her5 in yeast cells, co-purifica-
tion shows that the full length Her8a and Her3 proteins
heterodimerise (Figure 4A), and the overlapping
expression of her8a and her3 makes it possible that this
interaction occurs in vivo. Her proteins can dimerise
with a variety of partners, as also supported by our yeast
two-hybrid results (Additional file 1, Table S1), and
Her-Her heterodimers display enhanced stability over
homodimers [7,22]. A parsimonious interpretation of
our results is therefore that the heterodimerisation of
Her8a and Her3 is required for sufficient activity of
these factors in r2 and r4. Alternatively, the individual
activities of Her3 and Her8a may control complemen-
tary properties necessary to maintain the progenitor
pool cell state.
In spite of the high level of her8a expression across
the MHB progenitor pool, the results of the present
paper also identify that the decisive inhibition of neu-
rog1 expression in this location is played by other fac-
tors, namely Her3, 5, 9 and 11. Her5 and 11 were
known for their dose-dependent redundant functions,
accounting for neurogenesis inhibition across part of
this domain [22]. Through knocking-down all four
genes, we could achieve for the first time the transfor-
mation of most of the MHB into a neurogenic domain
(Figure 5), while leaving her8a expression intact. Collec-
tively, our findings show that the progenitor pool pat-
tern of the midbrain and anterior hindbrain is
established by the joined activities of five prepatterning
E(spl) factors which act in different combination in the
MHB and rhombomere domains. They also suggest that
distinct mechanisms of action of these factors may be
involved in these two domains.
Importantly however, we observed that the massive
neurogenic phenotype induced upon blocking Her3/5/9/
11 E(spl) activities is only partially followed by neuronal
differentiation (Figure 5). In fact, ectopic neurons are
restricted to the ventrolateral aspects of the midbrain-
hindbrain boundary, like upon blocking Her5 function
alone [21]. The corresponding progenitor population
may be particularly prone to neuronal differentiation.
For all other progenitors, our observations suggest the
need for a further commitment event, independent of
Her3/5/9/11 activities, to achieve neuronal differentia-
tion following neurog1 induction. Blocking Notch signal-
ing concomitantly to Her3/5/9/11 did not allow further
neurogenesis progression (C. Stigloher, unpub.). Persis-
tent her8a expression in this context may contribute to
neurogenesis reversion, although it was not possible to
evaluate this possibility as embryos blocked for the
activities of all five E(spl) factors developed abnormally.
her8a expression in the early neural plate is controlled by
Sox transcription factors but not Notch signaling
Two types of her genes have been recently distinguished
based on their Notch response profile: those acting as
Notch mediators, depending on Notch signaling for
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 13 of 19

Page 14

their expression and overexpressed upon Notch activa-
tion, and “non-canonical” her genes endogenously inde-
pendent of Notch and repressed when Notch is
experimentally activated (reviewed in [12]). The former
class comprises zebrafish her4.1 and her15, expressed in
active neurogenic domains such as proneural clusters of
the early neural plate [9-11]; the latter class is composed
of her3, 5, 9 and 11, expressed in progenitor pools
[11,13,15,21,22]. Our results illustrate that her8a is unu-
sual in its expression pattern, which overlaps both pro-
neural clusters and progenitor pools. This property is
shared with its ortholog Hes6.2 in chicken [46]. her8a
also shows a distinctive response to Notch signaling
among hairy/E(spl) genes within the early neural plate,
since it endogenously does not depend on Notch signal-
ing but responds positively to the experimental activa-
tion of Notch (Figure 6). In agreement with the latter
observation, we could identify Su(H) binding sites in the
upstream regulatory sequence of her8a. However, this
potential appears not to be used within proneural clus-
ters of the early neural plate, demonstrating that Her8a
is not a mediator of lateral inhibition. This is also in
agreement with its uniform rather than salt-and-pepper
expression profile. We found nevertheless that overex-
pressing her8a abolishes neurog1 expression even in
proneural clusters where the two genes are normally co-
expressed. Although we cannot ascertain that high over-
expression levels mimic endogenous Her8a activity, one
hypothesis reconciling this different information is that
Her8a function within proneural clusters may generally
dampen neurog1 expression, contributing to the func-
tion of other Her factors in Notch-inhibited precursors,
and ensuring a proper differentiation schedule in com-
mitted progenitors. Although not further analyzed in
this paper, we noted also that her8a expression becomes
dependent on Notch signaling at later developmental
stages (K. Webb, unpublished).
Our analyses of her8a expression in morphant con-
texts for other Her factors did not highlight cross-regu-
lations, although we found several consensus N and E
boxes within the 600 bp upstream of the her8a start
site. Previous work demonstrated the positive regulation
of Xenopus Hes6 by proneural bHLH proteins, in parti-
cular Neurogenin [27]. Given the presence of E boxes
on the her8a promoter, and the co-expression of her8a
and neurog1 in proneural clusters, it will be interesting
to test whether her8a expression is also positively con-
trolled by proneural factors in these locations. The
Ngn1/Hes6 cascade is positively reinforcing proneural
activity in Xenopus [27], but our functional data would
predict an opposite outcome for a Neurog1/Her8a regu-
lation in zebrafish.
Finally, our results show that the levels of her8a
expression are under control of a combination of SoxB1
and B2 factors (Sox2/3/19a/19b and Sox21a, respec-
tively) that display intense and partially overlapping
expression within the anterior neural plate (Figure 7). In
a recent study, Okuda et al. [41] demonstrated that
SoxB1 factors function redundantly to control several
successive aspects of zebrafish nervous system develop-
ment, including neural plate patterning and primary
neurogenesis. Although single morphants do not harbor
a visible phenotype, her3 expression fails to be induced
in quadruple SoxB1 morphants, strongly suggesting that
the four factors act redundantly to activate her3 tran-
scription [41]. In a comparable manner, we found that
individual SoxB1/B2 morphants display no phenotype,
while her8a expression is reduced in the MH domain
when all five SoxB1/B2 proteins are abolished. Although
we have not tested all possible knock-down combina-
tions, and in particular did not assess the individual
relevance of Sox21a in the context of the quadruple
knock-out for SoxB1 proteins, these results demonstrate
that her8a expression levels are under control of the
activity of at least partially redundant SoxB proteins.
Expression of these factors is an integral part of the
mechanisms patterning the early embryo [40,41], linking
her8a expression with neural plate regionalization. The
identification of a Sox2 binding site within the her8a
enhancer, and the fact that all SoxB proteins recognize a
similar binding motif in vitro, further suggests that part
of this control may be direct. In support of this hypoth-
esis, direct binding of SoxB1 factors onto the her3
enhancer has been demonstrated [41]. Like several other
SoxB2 proteins, Sox21b was shown to act as a transcrip-
tional inhibitor during dorsoventral patterning of the
zebrafish gastrula [40] and generally promotes neuro-
genesis [47]. It can however act as an activator in other
contexts [48], and its specific effect on MH neurogenesis
and her genes needs to be directly evaluated. It was
recently proposed that SoxB1 transcription factors and
Notch cooperate through distinct mechanisms in their
control of neurogenesis inhibition, including the inhibi-
tion of proneural protein activity and the transcriptional
upregulation of Hes/her genes, respectively [49]. Our
results and those of Okuda et al. [41] suggest yet
another level of regulation, where SoxB proteins directly
control the level of expression of some her genes.
Whether this is limited to Notch-independent contexts,
such as with the regulation of her3 and her8a, remains
to be addressed.
Conclusions
In this work, we identify the Hairy/E(spl) transcription
factor Her8a as a local inhibitor of neurogenesis in the
developing hindbrain. Specifically, we show that Her8a
function, like Her3 [11], is required to generate the
non-neurogenic progenitor pools normally separating
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 14 of 19

Page 15

the presumptive moto- and lateral neurons of r2 and 4.
We demonstrate that Her8a is a binding partner for
Her3 and we propose that this interaction may be func-
tionally relevant in r2 and r4. We further show that
Her8a alone is not sufficient to inhibit neurog1 expres-
sion in the presumptive MHB area; this event depends,
in contrast, on the combined activities of four other E
(spl) factors, Her3, 5, 9 and 11. Unlike canonical E(spl)
genes, we demonstrate that her8a does not depend on
Notch signaling for its expression at early neural plate
stages, and we identify a combination of SoxB factors
that together enhance her8a expression. Finally, using
phylogenetic analyses, we show that Her8a belongs to a
Hes6-like subfamily that was recently lost in the mam-
malian lineage. This observation provides a context for
the strikingly divergent functions of Her8a from Hes6;
Hes6, which was previously believed to be the mamma-
lian ortholog of Her8a, displays proneural activity.
Together, our results characterize the phylogeny, expres-
sion and functional cascades involving a new Her factor,
and highlight the complex interplay of E(spl) proteins
that generates the neurogenesis pattern of the zebrafish
midbrain-hindbrain area.
Methods
Yeast Two-Hybrid Analysis
Yeast two-hybrid screening was performed by Hybri-
genics, S.A., Paris, France (http://www.hybrigenics.com).
The coding sequence for amino acids 20 to 201 of the
Danio rerio Her5 protein (GenBank proteic accession
number gi: 18858797)
(amino acid sequence DRINQSLETLRMLLLENTN
NEKLKNPKVEKAEILESVVHFLRAEQASETDPFQITR
VKRARTEESDEDVESPCKRQSYHDGMRTCLLRVSN-
FITGKSHEFGQELEKACENIHK SHSRQVQLLSTPSLI
EPQVHLYEDPSQQHLAHVQL SNSCTPSGCSKLAQRT
VPAMTSSPKQPVMLCDPV)
was PCR-amplified and cloned into pB29 as an N-
terminal fusion to LexA (N-Her5-LexA-C). The con-
struct was checked by sequencing the entire insert and
used as a bait to screen a random-primed Danio rerio
embryo (stages 18-20 hpf) cDNA library constructed
into pP6. pB29 and pP6 derive from the original
pBTM116 [50] and pGADGH [51] plasmids, respec-
tively. 76 million clones (7.6 -fold the complexity of the
library) were screened using a mating approach with
Y187 (mata) and L40DGal4 (mata) yeast strains as pre-
viously described [52]. 280 His+ colonies were selected
on a medium lacking tryptophan, leucine and histidine,
and supplemented with 2 mM 3-aminotriazole to handle
bait autoactivation. The prey fragments of the positive
clones were amplified by PCR and sequenced at their 5’
and 3’ junctions. The resulting sequences were used to
identify the corresponding interacting proteins in the
GenBank database (NCBI) using a fully automated pro-
cedure. For each interaction, a Predicted Biological
Score (PBS) was computed to assess interaction reliabil-
ity. This score represents the probability of an interac-
tion being nonspecific. PBS relies on two different levels
of analysis; the algorithm and methods used in the cal-
culation are described in detail in Formstecher et al.
[53]. Briefly, at first a local score takes into account the
redundancy and independency of prey fragments (i.e.
the times the interaction was detected with different
independent clones and whether it was detected with
different or the same fragments), as well as the distribu-
tion of reading frames and stop codons in overlapping
fragments. Thus, interactions detected with several and
different fragments are ranked with a very high confi-
dence score and interactions detected with a single inde-
pendent fragment are ranked with a moderate
confidence score. Secondly, a global score takes into
account the interactions found in all the screens per-
formed at Hybrigenics using the same library. This glo-
bal score represents the probability of an interaction
being nonspecific. For practical use, the scores were
divided into four categories, from A (highest confidence)
to D (lowest confidence). A fifth category (E) specifically
flags interactions involving highly connected prey
domains previously found several times in screens per-
formed on libraries derived from the same organism.
Finally, several of these highly connected domains have
been confirmed as false-positives of the technique and
are now tagged as F. The PBS scores have been shown
to positively correlate with the biological significance of
interactions [54,55].
Gene ontology analysis
Gene ontology enrichment analysis was performed on
the recovered yeast-2-hybrid candidates from the cate-
gories A, B and C using the AmiGO “Term Enrichment
tool” [56] (available at http://amigo.geneontology.org/
cgi-bin/amigo/term_enrichment), using the following
settings: ZFIN database as a background set, the maxi-
mum p-value set at 0.05 and a minimum number of
gene products of two.
Sequence alignment, protein domain identification,
phylogenetic and synteny analyses
Protein sequences were retrieved by using a tblastn
search [57] against the non redundant database on
NCBI or on Ensembl genomic data (current release of
genomes, July 2010). For non-annotated sequences and
to further support the expression of the predicted gene,
a search for expressed sequence tags was also performed
by tblastn on the EST database of the NCBI server. A
list of all sequences used for the molecular phylogeny
and their genomic locations is provided in Additional
Webb et al. BMC Developmental Biology 2011, 11:27
http://www.biomedcentral.com/1471-213X/11/27
Page 15 of 19