1 Division of Developmental Neurobiology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 Laboratory of Developmental Signalling and Patterning, Institute of Molecular
and Cell Biology, 1 Research Link, National University of Singapore, 117604,
Singapore
* Author for correspondence (e-mail: dwilkin{at}nimr.mrc.ac.uk)
Accepted 8 December 2004
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SUMMARY |
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Key words: Hindbrain, Boundary, wnt1, Neurogenesis, Lateral inhibition
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Introduction |
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In vertebrates, the segmentation of the hindbrain to form rhombomeres
provides an amenable model for studying regional specification and the
formation of precise patterns of cell differentiation. Each rhombomere has a
distinct anteroposterior identity regulated by Hox gene expression
that underlies the generation of segmentally organised neurons
(Lumsden and Krumlauf, 1996).
The sharp and straight interface between each rhombomere is stabilised by the
restriction of cell intermingling by Eph receptor-ephrin signalling
(Fraser et al., 1990
;
Mellitzer et al., 1999
;
Xu et al., 1999
). Interactions
between adjacent rhombomeres induce the formation of boundary cells at the
segment interfaces that have a distinct morphology from non-boundary cells and
express a number of specific molecular markers
(Guthrie et al., 1991
;
Guthrie and Lumsden, 1991
;
Heyman et al., 1995
;
Heyman et al., 1993
;
Lumsden and Keynes, 1989
;
Xu et al., 1995
). Because
boundary cells have reduced cell proliferation and interkinetic nuclear
migration compared with other neural epithelial cells
(Guthrie et al., 1991
), they
may act as a non-motile cell population that stabilises the interface of
segments. A further potential role of boundaries is suggested by studies of
cell organisation in the zebrafish hindbrain that reveal an anteroposterior
organisation of neuronal and glial cell types within each rhombomere
(Hanneman et al., 1988
;
Metcalfe et al., 1986
;
Trevarrow et al., 1990
). For
example, primary reticulospinal neurons are located at the centre of
rhombomeres, and a GFAP-expressing glial cell `curtain' forms adjacent to
segment boundaries (Trevarrow et al.,
1990
). The formation of this stereotypical pattern can most easily
be explained by a role of segment boundaries as signalling centres that
regulate the positioning of cell differentiation.
In recent work, we found that expression of radical fringe
(rfng), a modulator of Notch signalling, occurs in boundary cells in
the zebrafish hindbrain, and that Delta ligands are expressed in non-boundary
regions (Cheng et al., 2004;
Qiu et al., 2004
). Activation
of Notch receptor in boundary cells regulates their affinity such that they
remain segregated to segment interfaces, and mediates lateral inhibition that
prevents the premature neurogenesis of boundary cells
(Cheng et al., 2004
).
Rfng function is required to upregulate wnt1 expression in
hindbrain boundary cells but not in the roof plate
(Cheng et al., 2004
). To
identify molecular mechanisms that may mediate potential roles of hindbrain
boundaries in patterning, we set out to analyse functions of the Wnt1
signalling molecule. We show that Wnt1 regulates proneural and delta
gene expression in non-boundary regions in the zebrafish hindbrain, and that
this mediates lateral inhibition that prevents the spreading of hindbrain
boundaries. Wnt1 acts in a regulatory network that is strikingly similar to
that occurring at the dorsoventral compartment boundary in the
Drosophila wing imaginal disc.
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Materials and methods |
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Morpholino oligonucleotide and RNA injections
Morpholino oligonucleotides (MO) were purchased from Gene Tools (Oregon,
USA). One- to four-cell blastomeres were microinjected with 0.5-2.5 pmol of
MO. The following MO sequences were used:
Capped RNAs encoding full-length chick Wnt1 (cWnt1) and stabilised
ß-catenin (ß-cat), lacking 87 amino acids that include the
phosphorylation site for GSK3 (Domingos et
al., 2001), were synthesised as described previously
(Xu et al., 1995
). To achieve
mosaic expression, 0.2-1.0 ng of RNA was injected into one cell at the two- to
eight-cell stage. In the case of embryos injected with both wnt1 MO
and ß-catenin, the morpholino was injected at the one-cell stage, and
ß-catenin RNA was injected into one cell at the eight-cell stage,
together with membrane-targeted GFP.
In situ hybridisation and immunohistochemistry
In situ hybridisation probes have previously been described as follows:
rfng (Cheng et al.,
2004; Qiu et al.,
2004
), foxb1.2 (Moens
et al., 1996
), wnt1
(Molven et al., 1991
),
asha and ashb (Allende
and Weinberg, 1994
), ngn1
(Blader et al., 1997
;
Kim et al., 1997
;
Korzh et al., 1998
),
p27xic1-a (Geling et
al., 2003
), cyclind1
(Yarden et al., 1995
),
deltaA and deltaD
(Haddon et al., 1998
),
dbx1a (Fjose et al.,
1994
), tbx20 (Ahn et
al., 2000
), and gfap
(Nielsen and Jorgensen, 2003
).
Digoxigenin-UTP labeled riboprobes were synthesized according to the
manufacturer's instructions (Roche) and in situ hybridisation was performed as
described previously (Xu et al.,
1994
). The colour reaction was carried out using NBT/BCIP
substrate (Roche). Embryos were then re-fixed in paraformaldehyde and mounted
for photography on a Zeiss Axiovision microscope, or processed for
immunohistochemistry.
Primary antibodies used are as follows: anti-EphA4 [1:500
(Irving et al., 1996)],
anti-HuC/HuD (1:200, Molecular Probes), anti-phosphohistone H3 (1:250,
Upstate) and anti-neurofilament (RMO-44, 1:200, Zymed). Antibodies were
diluted in PBS-Tween containing 2% goat serum, and embryos were blocked in 5%
goat serum. Detection of primary antibodies was carried out using Alexa
Fluor-488, -594 or -647 goat anti-mouse or anti-rabbit IgG (1:500, Molecular
Probes), or HRP-conjugated goat anti-rabbit IgG (1:250, Dako). Fluorescent
images were captured using a Leica TCS SP2 confocal microscope.
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Results |
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In recent work, we have shown that modulation of Notch activation by Rfng
is required for expression of wnt1 at hindbrain boundaries
(Cheng et al., 2004). As roof
plate expression of wnt1 is unaffected by rfng knockdown, this
allowed us to assess the role of the boundary expression of wnt1. We
find that in rfng MO embryos, the boundary expression domains of
rfng and of foxb1.2 expand
(Fig. 2I,J), as happens in
wnt1 MO embryos. Taken together with the wnt1 MO
experiments, these findings suggest that the boundary expression of
wnt1 is required to prevent boundary marker expression from
broadening. It is possible that rfng is also required for the
expression of other boundary signals that contribute to the restriction of
boundary spreading.
Previous studies have suggested that tcf3b, a mediator of
canonical Wnt signalling, is required for the formation of hindbrain
boundaries, as tcf3b MO-injected zebrafish embryos lacked
morphological hindbrain boundaries, and foxb1.2 expression at
boundaries appeared to be absent (Dorsky
et al., 2003). However, the uniform expression of foxb1.2
observed in this study could be due to ectopic hindbrain boundary expression,
rather than an absence of boundary expression and persistence of the
non-boundary expression of foxb1.2. We therefore examined the role of
tcf3b in more detail. Knockdown of tcf3b causes an expansion
of the expression domains of both rfng and foxb1.2
(Fig. 2K,L), as occurs in
wnt1 knockdowns. Our observation of the same phenotype following
knockdown of two distinct genes in the same pathway is strong evidence for the
specificity of this effect of the morpholino oligonucleotides, and suggests
that the action of Wnt1 in the hindbrain is mediated by Tcf3b.
There are several possible explanations for the expansion of boundary
marker expression following knockdown of wnt1 or tcf3b.
Because hindbrain boundary markers are upregulated by interactions between
cells with distinct segmental identity at segment interfaces
(Guthrie and Lumsden, 1991),
the broadening of boundary marker expression could be due to a mixing or
interleaving of interfaces. However, we detect no difference from uninjected
embryos in the formation of sharp segmental expression domains of
hoxb1a and krox20 in wnt1 MO embryos in which
ectopic boundary marker expression has occurred (data not shown). An
alternative possibility is that decreased wnt1 function leads to a
major increase in boundary cell proliferation and loss of non-boundary cells
during the period of boundary marker expression expansion. To examine this, we
detected phospho-histone H3, a marker of mitosis, and found that during
boundary marker expansion at 19 hours there is a decrease in mitotic index
(Fig. 2M,N,Q), both of boundary
cells (42% decrease) and of non-boundary cells (30% decrease). Furthermore, we
find that cyclinD1, a known downstream transcriptional target of Wnt1
signalling associated with cell proliferation in the spinal cord
(Megason and McMahon, 2002
),
is expressed at elevated levels in hindbrain boundaries
(Fig. 2O). In wnt1 MO
embryos, cyclinD1 expression at boundaries is decreased
(Fig. 2P), suggestive of a role
of Wnt1 in enabling rather than constraining boundary cell proliferation. We
therefore favour a third explanation for the broader expression domains of
hindbrain boundary markers: that Wnt1 signalling via Tcf3b is required to
prevent non-boundary cells from upregulating boundary markers.
Expression of proneural and delta genes in the hindbrain
A potential mechanism of Wnt1 action was suggested by the expression
patterns of achaete-scute homologue (ash) and neurogenin
(ngn) proneural genes, and of Notch ligands in the hindbrain
(Cheng et al., 2004). At 18
hours, the proneural genes asha and ngn1 are expressed in
cells in the dorsal and ventral half of each rhombomere, respectively, but are
excluded from boundaries. At the same stage, ashb is expressed at
high levels in cells in r4 (Fig.
3A-C). Subsequently, proneural gene expression resolves into
stripes that become established by 24 hours and persist until at least 48
hours of development. For example, at 26 hours ashb and ngn1
are expressed in presumptive neuroblasts adjacent to rhombomere boundaries,
with weaker expression throughout the ventricular zone except at boundaries
(Fig. 3G,H). At this stage,
asha expression occurs in scattered cells but is excluded from
boundaries (Fig. 3F), and from
30 hours is found in stripes adjacent to boundaries (data not shown).
Expression of these genes at 24-48 hours is patterned along the dorsoventral
axis, with ngn1 being most ventral, ashb medial, and
asha most dorsal. We found a similar progression in the expression
patterns of delta genes, which are downstream targets of proneural
genes, and of p27Xic1-a, which mediates cdk inhibition and
cell cycle exit in neuroblasts (Carruthers
et al., 2003
; Geling et al.,
2003
; Ohnuma et al.,
1999
). At 18 hours, expression of deltaA, deltaD and
p27Xic1-a occurs throughout the rhombomeres, except in boundary
cells, with stronger expression seen at lateral locations
(Fig. 3D,E;
Fig. 4A). By 26 hours, stripes
of high level deltaA, deltaD and p27Xic1-a expression have
formed adjacent to the boundaries, with weaker expression in the ventricular
zone in rhombomere centres (Fig.
3I,J and data not shown).
|
|
Effect of wnt1 knockdowns on neuronal differentiation
Analysis of deltaD gene expression in wnt1 MO embryos
revealed that at 18 hours neurogenesis appears unchanged from uninjected
embryos, but by 19.5 hours there is a major reduction in the number of
expressing cells (Fig. 4A-D).
Similarly, there is a major decrease in the number of cells expressing
p27Xic1-a in wnt1 MO embryos
(Fig. 4E,F). These results
suggest that Wnt1 is not required for early neurogenesis, but is required for
neurogenesis from 19.5 hours onwards. In support of this, we find that there
are fewer differentiated neurons (marked by expression of Hu;
Fig. 4Q,R) at 25 hours in
wnt1 MO embryos compared with in uninjected embryos. Counting of the
number of neural epithelial cells in hindbrain segments at 19 hours reveals
that wnt1 MO embryos have approximately 85% of the cell number of
uninjected embryos. Because many precursor cells remain, this decrease does
not account for the major deficiency in neurogenesis. The width and morphology
of the hindbrain was severely affected in wnt1 knockdowns at later
stages due to the major depletion of the mantle layer of post-mitotic
neurons.
To determine whether Wnt1 signalling via Tcf3b is required for generation of all neurons or of specific subsets of neurons after 19.5 hours, we analysed expression of proneural and delta genes that mark distinct populations of differentiating cells. We found that by 24 hours, wnt1 or tcf3b knockdown led to a major decrease in the number of cells expressing deltaA (Fig. 3I, Fig. 4G,H), ashb (Fig. 4I-K), ngn1 (Fig. 4M-O), asha and deltaD (not shown) throughout r2, r3, r5 and r6. The greater decrease in neurogenesis following tcf3b knockdown compared with wnt1 knockdown (Fig. 4G,H) may be due to a contribution of other Wnts acting via Tcf3b. It is notable that knockdown of wnt1 or tcf3b consistently had a weaker effect on neurogenesis in the spinal cord, in the hindbrain posterior to r6, and in r4 (Fig. 4F,J-L,N,O). Importantly, we find that decreased proneural and delta gene expression occurs following knockdown of rfng that leads to loss of wnt1 expression at hindbrain boundaries (Fig. 4L and data not shown).
To analyse in more detail whether Wnt1 is selectively required for
generation of specific neural cell types, we analysed the organisation of the
hindbrain at 48 hours, when there is a well-characterised pattern of neuronal
and glial cell types (Trevarrow et al.,
1990). We found that following wnt1 knockdown, generation
of primary reticulospinal neurons located at rhombomere centres is unaffected
(Fig. 5A,B), motor neurons form
but in lower numbers (Fig.
5C,D), and there is a major decrease in the number of
dbx1a-expressing neurons adjacent to segment boundaries, whereas
dbx1a expression still occurs in progenitor cells
(Fig. 5E,F,I-L). Furthermore,
wnt1 knockdown leads to ectopic formation of GFAP-expressing glial
cells that in uninjected embryos are located adjacent to hindbrain boundaries
(Fig. 5G,H). This pattern is
consistent with the requirement for Wnt1 only for later neurogenesis
(subsequent to 18 hours of development) revealed by analysis of delta
gene expression (Fig. 4A-D):
most reticulospinal neurons are born prior to 15 hours, motor neurons
differentiate between 16-20 hours, and neurogenesis adjacent to hindbrain
boundaries occurs between 22-48 hours
(Chandrasekhar et al., 1997
;
Mendelson, 1986
;
Trevarrow et al., 1990
).
|
Effect of Wnt pathway activation on neurogenesis
Gain-of-function experiments suggest that in the mouse spinal cord
activation of the Wnt/ß-catenin pathway promotes proliferation and
inhibits differentiation of neuronal progenitors
(Megason and McMahon, 2002;
Zechner et al., 2003
). We
therefore further tested whether Wnt1 promotes neurogenesis in the hindbrain
by analysis of the effects of ectopic expression of Wnt1 or stabilised
ß-catenin (lacking sequences required for GSK3-induced degradation). We
found that overexpression of Wnt1 (Fig.
4S,T) or of stabilised ß-catenin
(Fig. 4U,V) led to an increase
in the number of differentiated neurons in comparison to uninjected control
embryos (Fig. 4Q). Furthermore,
expression of stabilised ß-catenin in wnt1 MO embryos
(Fig. 4W,X) rescues the
decrease in neurogenesis in embryos injected with wnt1 MO
(Fig. 4R). These findings
suggest that Wnt1 promotes rather than inhibits neuronal differentiation in
the hindbrain.
Role of proneural and delta genes in repressing boundary markers
We next analysed whether, analogous to the situation in the
Drosophila wing disc, proneural and delta gene function is
required to repress ectopic boundary marker expression. We find that expansion
of rfng and foxb1.2 expression occurs in asha MO
embryos, to a lesser extent in ashb MO injected embryos, and with a
stronger phenotype in asha/ashb double knockdowns
(Fig. 6A-D,I-K). Knockdown of
ngn1 does not lead to hindbrain boundary spreading, but synergises in
a double knockdown with ashb (Fig.
6E,F). These synergistic effects suggest that proneural genes
overlap in restricting boundary spreading. As found in wnt1 and
tcf3b knockdowns, boundary spreading does not occur in r4 in multiple
knockdowns of proneural genes. Remarkably this was the case even following
triple knockdown of asha, ashb and ngn1, which almost
completely blocks neurogenesis (Fig.
6G). Finally, we analysed the effect of decreased delta
gene function. Hindbrain boundary expansion is not detected in homozygous
after eight mutant embryos that have a null mutation in the deltaD
gene (data not shown). However, knockdown of deltaA leads to a major
expansion of rfng and foxb1.2 boundary expression
(Fig. 6H,L).
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Discussion |
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Roles of wnt1 in proliferation and neurogenesis
A key question is the nature of the relationship between Wnt1 and the
control of neurogenesis in the zebrafish hindbrain. A number of studies have
found that activation of the Wnt/ß-catenin pathway promotes neural
precursor proliferation and inhibits neuronal differentiation
(Chenn and Walsh, 2002;
Megason and McMahon, 2002
;
Zechner et al., 2003
). For
example, Wnt1 expression in the roof plate of the mouse spinal cord acts via
upregulation of cyclinD1 to drive cell proliferation, which expands the
neuronal precursor population and inhibits neuronal differentiation in dorsal
regions (Megason and McMahon,
2002
). Indeed, the observation that neurogenesis occurs first in
the centre of zebrafish hindbrain segments
(Trevarrow et al., 1990
) is
suggestive of the situation in the spinal cord in which neurogenesis is
initiated distal from the source of wnt1 expression. However, we find
that rather than suppressing neurogenesis, after 18 hours of development
wnt1 is required for neurogenesis in the zebrafish hindbrain.
One possible explanation for our findings is that, as in the spinal cord,
wnt1 promotes cell proliferation, and that wnt1 knockdown
leads to a subsequent lack of neurogenesis in the hindbrain because of a
depletion of precursor cells available to differentiate into neurons at 19
hours and later stages. Indeed, wnt1 knockdown leads to decreased
cyclinD1 expression and cell proliferation in the hindbrain. However,
there is only an approximate 15% decrease in the number of neural epithelial
cells at 19 hours in wnt1 knockdowns, with many neural precursors
remaining that in r2, r3, r5 and r6 downregulate proneural gene expression and
upregulate boundary cell markers. As boundary cells are themselves neural
progenitors (with delayed differentiation), the decrease in neurogenesis
following wnt1 knockout is in large part due to a switch of
progenitors from non-boundary to boundary identity. In support of an
inhibition of neuronal differentiation rather than loss of neural progenitors,
we observe that wnt1 knockdown leads to inhibition of the
differentiation of dbx1a-expressing neuronal precursors. Furthermore,
the ectopic differentiation of GFAP-expressing glial cells in rhombomere
centres following wnt1 knockdown could be due to a block in neuronal
differentiation of precursors (Nieto et
al., 2001), and/or to the ectopic formation of hindbrain boundary
cells. By contrast, proneural gene expression is maintained in the spinal
cord, with a sharp anterior boundary at the r6/r7 interface in wnt1
knockdowns, suggestive of a distinct role of wnt1 in neurogenesis in
the spinal cord compared with in the hindbrain. Intriguingly, the spatial
relationship between wnt1 and cyclinD1 expression is
different in the spinal cord and hindbrain. In the spinal cord,
cyclinD1 is expressed in the dorsal part of neural tube adjacent to
the wnt1-expressing roof plate, whereas in the hindbrain
cyclinD1 expression occurs at high levels in boundary cells,
suggesting that Wnt1 has an autocrine as well as a paracrine role.
We therefore favour a model in which, in addition to a role in regulating cell proliferation, Wnt1 promotes neurogenesis in the hindbrain. Because in wnt1 or tcf3b knockdowns, proneural gene expression is decreased and boundary markers upregulated throughout r2, r3, r5 and r6, Wnt1 seems to act throughout these segments to enable neurogenesis. The expression of wnt1 in both the roof plate and hindbrain boundaries raises the question of the relative contribution of these signalling sources. We find that loss of the boundary expression but not roof plate expression of wnt1 in rfng knockdowns leads to boundary spreading and decreased neurogenesis. As it is likely that the roof plate expression of Wnt1 also contributes to the promotion of neurogenesis and inhibition of hindbrain boundary spreading, expression by hindbrain boundaries may achieve a threshold level of signal required to drive sufficient proneural gene expression to block boundary spreading.
Our findings could be explained by two alternative roles of Wnt1
signalling. First, that Wnt1 induces a proneural state of non-boundary cells
and is a permissive factor for neurogenesis, whereas other factors control the
spatial patterns of differentiation. In support of this are the observations
that neurogenesis occurs adjacent to the full dorsoventral extent of hindbrain
boundaries, whereas wnt1 expression is restricted to the dorsal part
of hindbrain boundaries, and that neurogenesis does not occur in a stripe
adjacent to wnt1 expression in the roof plate of the hindbrain. A
second possibility is that in addition to Wnt1 being required for a proneural
cell state through the segment, high levels of Wnt1 induce the differentiation
of neurons adjacent to boundaries. This situation is similar to the
Drosophila wing disc, in which cells adjacent to the source of Wg
express proneural genes and, as a consequence, undergo mitotic arrest and
differentiate (Couso et al.,
1994; Johnston and Edgar,
1998
; Phillips and Whittle,
1993
). Consistent with this, recent studies in the mouse neocortex
and in in vitro cell culture have found that Wnt signalling promotes
neurogenesis and directly regulates neurogenin 1 gene expression
(Hirabayashi et al., 2004
;
Israsena et al., 2004
;
Otero et al., 2004
).
Furthermore, Wnt signalling may have stage-specific effects, in which it
initially promotes proliferation and later induces neuronal differentiation
(Hirabayashi et al., 2004
).
However, the spatial pattern of neurogenesis in the hindbrain cannot be
explained based solely on Wnt1 signalling; for example, since neurogenesis
adjacent to boundaries persists as wnt1 expression becomes
increasingly dorsally restricted in boundaries. Therefore, other factors are
required to cooperate with or overlap functionally with any instructive role
of Wnt1 in spatial patterning of neuronal differentiation.
During completion of this work, another study has analysed roles of Wnt
signals in the hindbrain (Riley et al.,
2004). There are significant differences in the conclusions of
these authors compared with the current work. First, it is proposed that Wnt
signals from hindbrain boundaries have a purely organising role in the
anteroposterior pattern of neurogenesis, whereas we demonstrate a temporal
requirement for Wnt signals in neurogenesis that underlies a selective loss of
later neurons that differentiate adjacent to boundaries. The data of these
authors are consistent with a loss rather than a spatial disorganisation of
neurogenesis following knockdown of wnt genes or of tcf3b.
Second, Riley et al. (Riley et al.,
2004
) propose that Wnt signals are required to maintain hindbrain
boundaries, whereas we show that they are required to prevent spreading of
boundaries. This reflects that these authors did not have appropriate markers
available to identify ectopic boundary cells, although, in agreement with our
findings, GFAP-expressing glial cells that normally flank boundaries were
observed in ectopic locations following decreased Wnt activity. A third
difference is that Riley et al. (Riley et
al., 2004
) observe only subtle phenotypic changes in a deletion
mutant that removes wnt1 and wnt10b, but knockdown of
further wnt genes (or of tcf3b) leads to a similar effect on
neurogenesis as we observe following knockdown of wnt1. The reason
for the quantitative difference is unclear, but might be due to the large
deletion that removes wnt1 and wnt10b also removing a
gene(s) that has a compensatory effect on neurogenesis.
A conserved pathway of neural differentiation and lateral inhibition
A potential pathway by which Wnt1 might inhibit ectopic expression of
boundary cell markers was suggested by the similarity of gene expression
patterns in the zebrafish hindbrain to those adjacent to the dorsoventral
boundary of the Drosophila wing imaginal disc. In the wing imaginal
disc, expression of Fringe and Serrate in the dorsal
compartment, and of Delta in the ventral compartment, leads to a
stripe of Notch activation at the dorsoventral boundary
(Panin et al., 1997), and
Notch activation upregulates wg expression
(Diaz-Benjumea and Cohen,
1995
; Kim et al.,
1995
; Rulifson and Blair,
1995
). Wg protein acts on adjacent cells in the anterior
compartment to upregulate expression of as-c proneural genes, which
specify a post-mitotic sensory hair cell fate, and upregulate Delta gene
expression (Couso et al., 1994
;
Johnston and Edgar, 1998
;
Phillips and Whittle, 1993
).
Delta acts cell autonomously to inhibit Notch activation, and because Notch
activation is required to activate wg expression, this mediates a
lateral inhibition that prevents spreading of the wg expression
domain (de Celis and Bray,
2000
; Rulifson et al.,
1996
) (Fig.
7A).
|
We have thus uncovered a striking parallel with the mechanisms that in the
Drosophila wing disc control formation of sensory hair cells and
prevent ectopic boundary cell formation. As the zebrafish hindbrain and
Drosophila wing are not homologous structures, we propose that this
reflects an independent recruitment during evolution of a regulatory network
of genes that underlies an activity of boundaries both in patterning adjacent
tissue and in preventing the spread of the signalling source. There are
several differences in the Notch-Wnt-proneural network between the hindbrain
and wing imaginal disc; for example, in the expression patterns of
fng and Notch ligands that establish a stripe of Notch activation at
boundaries. This may be due to the larger number of fng genes in
vertebrates that have distinct expression domains in hindbrain segments and
boundaries (Cheng et al., 2004;
Prince et al., 2001
), and may
have diverged in their function (Dale et
al., 2003
). Another difference is that in the hindbrain, Wnt1 has
a long-range role in promotion of neurogenesis, whereas in the wing imaginal
disc, neural cells only form immediately adjacent to the Wg signalling
source.
A model of boundary and proneural patterning in the hindbrain
Taken together with previous work, our findings reveal a regulatory loop
between boundary cells and non-boundary regions that stabilises the pattern of
each cell population via bidirectional lateral inhibition
(Fig. 7B). Rfng-mediated
modulation of Notch activation upregulates wnt1 expression in
boundary cells (Cheng et al.,
2004). Notch activation also regulates the affinity properties of
boundary cells (Cheng et al.,
2004
), thus maintaining their segregation to the interfaces of
segments. Wnt1 expressed by boundary cells promotes proneural and
delta gene expression in non-boundary regions, which enables neuronal
differentiation and laterally inhibits the spread of boundary marker
expression. In addition, roof-plate expression of Wnt1, which is independent
of Rfng function, contributes to the promotion of neurogenesis, but is not
sufficient to prevent hindbrain boundary spreading. Delta expression by
non-boundary cells activates Notch in boundary cells, and thus laterally
inhibits boundary cells from expressing proneural genes and undergoing
neuronal differentiation (Cheng et al.,
2004
).
As wnt1 knockdown affects neurogenesis only after 18 hours of
development, this model predicts that other signals from boundaries regulate
neurogenesis and restrict boundary spreading at earlier stages of
differentiation in the hindbrain. In addition, it is striking that
specifically in r4 there is less downregulation of proneural gene expression
following wnt1 or tcf3b knockdown, suggesting that other
factors regulate neurogenesis in r4. This continued proneural gene expression
in r4 could provide an explanation for why there is no boundary spreading into
r4 following wnt1 or tcf3b knockdown. However, we find that
even following an almost complete block of neurogenesis in a triple knockdown
of proneural genes, hindbrain boundary spreading did not occur into r4. The
mechanism underlying the restriction of boundary spreading in r4 is not known,
but a potential functional significance is suggested by the findings that this
rhombomere is the first to form and differentiate, and acts as an early
signalling centre to pattern adjacent segments
(Maves et al., 2002). This
early role of r4 may require that other signals regulate the restriction of
boundary spreading and neurogenesis independently of the later upregulation of
wnt1 gene expression in boundaries.
Finally, our findings have implications for mechanisms by which boundary cells form at the interface of adjacent hindbrain segments. An attractively simple model is that boundary cells are specified by interactions that only occur at segment interfaces; for example, by complementary segmental expression of a cell surface receptor/ligand system. However, our finding that after blocking the Wnt1/Tcf3 pathway most or all cells (except in r4) can express boundary markers shows that boundary cell formation does not need to be located adjacent to the segment interface. We therefore propose that interactions at segment interfaces instead provide a bias that ensures that boundary cell specification is initiated at that location, and that lateral inhibition then prevents further cells from becoming boundary cells.
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ACKNOWLEDGMENTS |
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