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First published online 11 June 2008
doi: 10.1242/dev.013482
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Centre de Biologie du Développement, UMR 5547 CNRS/UPS, Université Paul Sabatier Bât. 4R3, 118 route de Narbonne, 31062 Toulouse Cedex 9, France.
Author for correspondence (e-mail:
blader{at}cict.fr)
Accepted 15 May 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Notch, Neural specification, Zebrafish, Epiphysis, Photoreceptor, Projection neuron
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Basic helix-loop-helix transcription factors (bHLH) homologous to the
Drosophila proneural genes also play a role in defining neuronal
subtype identity (Brunet and Ghysen,
1999). For example, within the V2 domain (one of the five ventral
domains defined by the graded effect of Shh), Mash1 specifies the identity of
Chx10+ interneurons (Parras et
al., 2002). Finally, specific transcription factors are dedicated
to the specification of peculiar neurotransmitter phenotypes. For instance,
the bHLH transcription factor Ptf1a is necessary for the specification of
GABAergic neurons in the mouse cerebellum, spinal cord and retina, and is
sufficient to drive GABAergic traits when overexpressed in the dorsal
telencephalon (Fujitani et al.,
2006; Glasgow et al.,
2005; Hoshino,
2006). However, our understanding of the acquisition of the
various identities that make up the nervous system is far from complete.
The Notch signaling pathway plays a central role in the generation of
diversity within the Drosophila nervous system. Upon binding by
ligands from the DSL family (for Delta, Serrate, Lag2), Notch is proteolyzed
and its intracellular domain (Notch-intra) translocates to the nucleus, where,
together with co-factors, it activates transcription
(Bray, 2006). Notch is required
at several stages during neural development in the fly. First its activity is
crucial for the selection of a neural progenitor from a pool of competent
cells, a process referred to as lateral inhibition. In this context, Notch
signaling functions as a feedback loop in which the activation of target genes
by Notch-intra leads to a diminished expression of proneural genes. As
proneural genes control the expression of the Delta ligands, activation of
Notch within a cell leads to a reduced signaling to neighboring cells. The
Notch pathway thus provides a mechanism by which small differences in
proneural gene expression between neighboring cells can be readily amplified,
thus singling out one cell expressing a higher level of proneural genes from a
pool of equivalent cells (Simpson,
1997). Once this selection has occurred, the expression of the
proneural genes endows the cell with neural potential, a process referred to
as neural determination.
A second role of Notch in the fly nervous system concerns cell fate
diversification. For instance, in the eye, cell-cell communication via Notch
allows sorting of two distinct photoreceptor subtype identities called R3 and
R4. In this context, Notch signaling is initially biased by the activity of a
polarizing signal acting through the Frizzled receptor that leads to stronger
expression of Delta in the presumptive R3
(Fanto and Mlodzik, 1999).
Similarly, mechanosensory organ precursors generate cells that communicate
through Notch to specify the four distinct identities that compose the sensory
organ. These identities are generated through sequential binary decisions.
First, the sensory organ precursor divides to generate two intermediate
progenitors (pIIb and pIIa) that communicate via Notch to establish their
respective identities. These cells divide again to generate four cells, the
identities of which are established through communication between sister cells
via Notch. In this case, Notch signaling is biased by the asymmetric
segregation of Notch interactors during cell division
(Bardin et al., 2004). In
addition, the activity of Notch in such binary decisions involves targets
distinct from proneural genes (Guo et al.,
1995; Okabe et al.,
2001).
In vertebrates, the Notch pathway plays a clear role in the selection of a
neural progenitor from a pool of competent cells through the regulation of
proneural genes (Lewis, 1996).
By controlling this process, Notch signaling affects the timing of cell birth
and differentiation. A correlation has been observed between the timing of
cell birth and the identity of the neural cell produced in many neuronal
lineages (Temple, 2001).
Therefore, it remains controversial whether the Notch pathway diversifies cell
fate through the regulation of the timing of neurogenesis or acts directly in
specifying neuronal subtype identity. For example, the Notch pathway plays a
role in specifying two distinct populations of GABAergic interneurons (called
KA' and KA'') at the expense of motoneuron fate in the ventral
spinal cord of the zebrafish. However, although in the case of the KA''
cells the effect of Notch is primarily to control the timing of neurogenesis
(Yeo and Chitnis, 2007), Notch
directly controls the KA' cell fate
(Shin et al., 2007). Recent
work performed in the mouse retina also suggests a direct role for Notch in
the specification of neuronal subtype identity. Conditional inactivation of
the Notch1 gene induces the production of an excess of photoreceptors
at the expense of other cell fates. Interestingly, this effect is independent
of the timing of Notch1 inactivation, which suggests a direct
activity for Notch in cell fate specification
(Jadhav et al., 2006;
Yaron et al., 2006). Finally,
Notch plays a role in specifying two distinct subtypes of interneurons in the
ventral spinal cord (Peng et al.,
2007). These two neuronal types are born from Lhx3+
progenitors and seem to be produced simultaneously, as judged by the
expression of molecular markers. However, in absence of birthdating studies,
the possibility remains that Notch indirectly influences cell fate by
controlling the timing of neuronal differentiation.
We have begun to analyze the mechanisms that govern cell fate
diversification in the zebrafish epiphysis, or pineal gland, a small
diencephalic structure involved in light detection and the regulation of
circadian rhythms (Foster and Roberts,
1982; Natesan et al.,
2002). This simple structure contains only two neuronal types:
photoreceptors and projection neurons
(Masai et al., 1997).
Precursors for epiphysial neurons arise from a subdomain of the dorsal
diencephalon that expresses the homeodomain transcription factor floating
head (flh). Flh is required for the expression of two proneural
genes, achaete/scute homolog 1a (ascl1a) and
neurogenin1 (ngn1), which are in turn redundantly required
for neuronal production within the epiphysis. However, genetic perturbation of
this Flh/proneural genes cascade affects both photoreceptors and projection
neurons, indicating that flh, ascl1a and ngn1 are not
involved in the specification of neuronal subtype identity
(Cau and Wilson, 2003;
Masai et al., 1997). Thus,
although we understand how neurons are produced in the epiphysis, the
mechanism by which these neurons acquire their identity remains to be
discovered.
In this paper, we examine the role of the Notch pathway in specifying the two neuronal subtypes of the epiphysis. We show that a reduction or a gain of Notch activity modifies the proportion of the two cell types compared with wild type. This effect is independent of cell birthdate as projection neurons and photoreceptors are born simultaneously. We propose that Notch controls the specification of neuronal subtype identity independently of its role on the timing of neurogenesis in this simple neuronal lineage.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos homozygous for mibta52b
(Itoh et al., 2003) or
dlAhi781 (Amsterdam et
al., 2004) and after-eight
(Holley et al., 2002)
mutations were obtained by intercrossing heterozygous carriers. Tg(HuC:GFP),
Tg(AANAT2:GFP), Tg(hs:Gal4) and Tg(UAS:Nintra) transgenic lines have
been described previously (Park et al.,
2000; Scheer et al.,
2002; Gothilf et al.,
2002) as has the sequence for dlD MO
(Holley et al., 2002).).
Sequence for dlDm MO and dlD 5'MO are
5'-aaaGagctatGattaCtcCtccGat-3' and
5'-agaggatctgaactgttgtgaaact-3', respectively. Although
dlD and dlDm MO were injected at 2.5 ng/µl, dlD
5'MO was injected at 3 ng/µl.
DAPT treatments
Embryos were raised in embryo medium containing DAPT (Calbiochem) at 100
µM and DMSO (1%), as previously described
(Geling et al., 2002). Control
embryos were incubated in an equivalent concentration of DMSO.
Birthdating of neurons with 5-bromo-2-deoxyuridine
Embryos were incubated in embryo medium with 10 mM BrdU and 8% DMSO for 20
minutes on ice followed by 2 hours at 28.5°C. BrdU incorporation was
detected by immunohistochemistry using an anti-BrdU antibody (G3G4, 1/1000,
Developmental Studies Hybridoma Bank).
In situ hybridization
In situ hybridization was performed using an in situ hybridization robot
(Intavis AG, protocol available upon request). The following
digoxigenin-labeled antisense riboprobes were used: flh
(Talbot et al., 1995),
lhx3 (Glasgow et al.,
1997), ascl1a (Allende
and Weinberg, 1994), ngn1
(Blader et al., 1997),
islet1 (Appel et al.,
1995), dlA, dlD, dlB
(Haddon et al., 1998) and
exorhodopsin, red opsin and rhodopsin
(Forsell et al., 2001;
Mano et al., 1999).
Immunostaining
Antibody staining was performed as previously described
(Masai et al., 1997) using the
following primary antibodies: FRet43, 1/200
(Larison and Bremiller, 1990),
anti-Islet1 (39.4D5; 1:200, Developmental Studies Hybridoma Bank), anti-GFP
(1/1000, Torrey Pines Biolabs), anti HuC/D (1/400, Molecular Probes), Pax6
(1/1000) (Carriere et al.,
1993) and caspase 3 (1/200; BD Pharmigen); and the following
secondary antibodies (Molecular Probes): Alexa 488-conjugated goat anti-rabbit
IgG (1/1000), Alexa 546-conjugated goat anti-mouse IgG (1/1000), Alexa
647-conjugated goat anti-mouse IgG (1/1000), Alexa 546-conjugated goat
anti-mouse IgG1 (1/100) and Alexa 647-conjugated goat anti-mouse IgG2
(1/100).
Image acquisition and counts
Confocal acquisition was performed using a Leica (SP2) and ImageJ software
was used for cell counting. For each condition a minimum of three embryos was
analyzed.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Glasgow et al., 1997;
Masai et al., 1997). We
noticed that the total number of FRet43 and lhx3-expressing cells at
48 hours represents fewer than half of the total number of Islet1+ epiphysial
neurons, suggesting either that these markers are transiently expressed or
label distinct subtypes of projection neurons and photoreceptors
(Fig. 1B). Thus, to allow us to
quantify the total number of projection neurons and photoreceptors, we
searched for other markers of these two cell populations.
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). To
confirm this, we performed double-labeling experiments with photoreceptor
markers, such as FRet43 and a complex opsin probe (containing exorhodopsin,
rhodopsin and red opsin), or projection neuron markers, such as lhx3
and Pax6 (Masai et al., 1997).
As expected, although we observed co-labeling of FRet43 or opsin with GFP in
Tg(AANAT2:GFP) embryos, the expression of lhx3 and Pax6 was largely
excluded from cells expressing the AANAT2 transgene
(Fig. 1C-C'',E-E'';
see Fig. S1A,A',C,C' in the supplementary material).
Interestingly, however, we observed a few GFP+ cells that express
lhx3 or Pax6 in some Tg(AANAT2:GFP) embryos
(Fig. 7C; see Fig. S1C,C'
in the supplementary material). This either reflects the occasional activation
of lhx3 and Pax6 in photoreceptors or the presence of cells with a
transient mixed identity.
The RNA-binding protein HuC is a marker of newly born neurons and a
transgenic line containing regulatory elements upstream of the huC
gene driving GFP, Tg(HuC:GFP), has been reported to reproduce this pattern
(Park et al., 2000). To our
surprise, however, we observed that GFP from the Tg(HuC:GFP) transgenic line
only labels a subset of epiphysial neurons. The lateral position of these
GFP-expressing cells suggests that they are projection neurons
(Fig. 1D-D''). Consistent
with this, we observed that the vast majority of lhx3+ and Pax6+
cells co-express the HuC:GFP transgene
(Fig. 1F-F''; see Fig.
S1B,B' in the supplementary material), whereas no co-expression was
observed with opsin or FRet43 (Fig.
7C and data not shown). We thus consider Tg(HuC:GFP) to be a
marker of projection neurons.
Finally, to confirm that the GFP expression from the transgenes is stably detected in epiphysial neurons, we quantified Tg(HuC:GFP)+ and Tg(AANAT2:GFP)+ cells at 48 hours of development. We observed an average of 42.4±7 Tg(AANAT2:GFP)+ cells and an average 19.2±1.06 Tg(HuC:GFP)+ cells per embryo (Fig. 1B). At the same stage, the epiphysis contains 69.5±5.32 Islet1+ neurons. Thus, unlike FRet43 and lhx3, these transgenes stably label the entirety of the two neuronal subtypes in the epiphysis.
deltaB and deltaD are expressed specifically in projection neurons
Notch signaling has been implicated in cell fate choice in invertebrates,
but its role is more controversial in vertebrate neural lineage specification
(Bardin et al., 2004;
Fanto and Mlodzik, 1999;
Jadhav et al., 2006;
Shin et al., 2007;
Yaron et al., 2006;
Yeo and Chitnis, 2007). A role
for Notch in binary decisions is often associated with an asymmetric
expression of Notch ligands (Fanto and
Mlodzik,1999). Interestingly, two ligands deltaB and
deltaD show preferentially lateral expression in the zebrafish
epiphysis (Cau and Wilson,
2003) (Fig. 2B,C).
By contrast, deltaA shows widespread expression in the epiphysial
territory (Fig. 2A). We
hypothesized that the lateral expression of deltaB and
deltaD corresponds to projection neurons and performed
double-labeling experiments to confirm this idea. At 24 hours, cells
double-labeled with a deltaA probe and GFP were observed in both
Tg(HuC:GFP) and Tg(AANAT2:GFP) embryos
(Fig. 2D,D',G,G').
Thus, deltaA is expressed in both photoreceptors and projection
neurons. By contrast, deltaB and deltaD co-expressed GFP in
Tg(HuC:GFP) (Fig.
2E,E',F,F') but not in Tg(AANAT2:GFP)
(Fig. 2H,H',I,I').
These results show an enrichment of the expression of Delta genes in
projection neurons with deltaB and deltaD being expressed
selectively in projection neurons.
Reducing Notch signaling increases neurogenesis in the epiphysis
The asymmetry in the expression of deltaB and deltaD led
us to test a role for Notch signaling in the zebrafish epiphysis using embryos
mutant for mindbomb (mib); mib encodes a ring
ubiquitin ligase that modifies Delta, thereby potentiating its activity as a
ligand for Notch (Itoh et al.,
2003; Le Borgne and
Schweisguth, 2003). As other ring-ubiquitin ligases of the Mib or
Neuralized (Neur) families could partially compensate for mutations in
mib (Lai et al.,
2005; Le Borgne et al.,
2005; Zhang et al.,
2007a; Zhang et al.,
2007b), we also used the 
-secretase inhibitor DAPT, as a
more general way of inhibiting the Notch pathway
(Geling et al., 2002).
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Expression of the prepattern transcription factor floating head
(flh) is detected in the epiphysial anlage from 9 hours of
development and its activity is required for epiphysial neurogenesis as it has
been shown that few Islet1+ neurons are formed in flh mutant embryos
(Masai et al., 1997). To
determine whether the increased neuronal production observed in mib
mutant and DAPT-treated embryos resulted from an increase in the size of the
presumptive epiphysial territory, we assayed flh expression in
mib mutant and DAPT-treated embryos; flh expression was
analyzed at the stage where the first post-mitotic neurons can be detected
(Masai et al., 1997). No
change was detected in the size of the presumptive epiphysial territory in
embryos with reduced Notch signaling (Fig.
3B,C; data not shown). The increase in the number of Islet1+ cells
detected in embryos with compromised Notch signaling might, alternatively,
result from increased neurogenesis within the epiphysial anlage. To explore
this possibility, we assayed the expression of the proneural genes
ascl1a and ngn1, which are redundantly required for the
production of epiphysial neurons downstream of Flh
(Cau and Wilson, 2003). In
contrast to flh expression, both mutation of mib and DAPT
treatment affect the expression of ascl1a and ngn1
(Fig. 3D-G; data not shown).
Indeed, in wild-type and mock-treated embryos, we observed 10-15
ascl1a+ cells compared with 20-25 cells in embryos with reduced Notch
signaling at 16 hours of development (Fig.
3D,E; data not shown). Similarly, we observed more ngn1+
cells in embryos with reduced Notch signaling than in wild-type embryos at 16
hours; 5-10 cells in Notch compromised versus fewer than five cells in
wild-type embryos (Fig. 3F,G;
data not shown). Thus, the Notch pathway is required to inhibit neuronal
production in the epiphysial anlage. This effect most probably reflects a role
for Notch in maintaining epiphysial cells in a progenitor state through the
downregulation of the expression of ascl1a and ngn1, in a
manner similar to that described in other areas of the nervous system.
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Reducing deltaA and deltaD function specifically affect neuronal identity
Next, we tested the functions of the Delta genes in the epiphysis. For
this, we used a retroviral insertion mutant in the deltaA gene
(Amsterdam et al., 2004). For
deltaD, we used a previously reported morpholino (dlD MO)
(Holley et al., 2002), as well
as a second morpholino directed further upstream in the 5' UTR of the
gene (dlD 5'MO). Embryos with reduced deltaA and/or
deltaD activity exhibit an increase in the number of Tg(HuC:GFP)+
cells (Fig.
5A,A',B,B',E). In parallel, we observed a decrease in
the number of Tg(AANAT2:GFP)+ cells upon reduction of deltaD or
deltaA and deltaD functions
(Fig. 5G). Thus, as for
mib mutants or early DAPT-treated embryos, the relative number of
photoreceptors falls from 72.4% to 62.9% and 63.5% in the absence of DeltaA
and DeltaD function, respectively. Interestingly, however, reducing
deltaA and/or deltaD activity had no effect on the total
number of Islet1+ neurons compared with wild type
(Fig. 5C). Although we observed
similar effects in both conditions of deltaD knock down, no phenotype
was obtained upon injection of a control morpholino in which the sequence of
the dlD MO harbors five mismatches (dlDm MO;
Fig. 5C-G). We also confirmed
the specificity of the deltaD morpholinos used in this study by
analyzing the number of Islet1+, HuC/D+ and FRet43+ cells in the
deltaD mutant after eight (aei). In all three
cases, results with aei mutant embryos were comparable with those
generated by morpholino injection (Fig.
5C,D,F). Thus, reducing deltaA and deltaD
function affects neuronal subtype identity. Furthermore, it does so without
modifying total neuronal numbers. These results suggest that the Notch effects
on neuronal number and on neuronal identity reflect two distinct
activities.
|
). To understand
whether the effect of reducing Notch signaling on the specification of
projection neurons and photoreceptors reflects a role in the time of birth of
these two populations, we performed birthdating experiments using
bromodeoxyuridine (BrdU) pulses in Tg(AANAT2:GFP) or Tg(HuC:GFP) transgenic
embryos. When BrdU was applied before or at 18 hours, most
transgene-expressing cells were also BrdU+, indicating that the majority of
epiphysial progenitors are still dividing at these stages
(Fig.
6A,A',B,B',E). By contrast, after a pulse at 20.5
hours, most GFP+ cells were BrdU negative
(Fig.
6C,C',D,D',E). The similarity between the
incorporation curves obtained for projections neurons and photoreceptors
indicate that the birthdate is the same for the two cell types. These results
rule out sequential production of neuronal types as a possible mechanism by
which Notch regulates neuronal specification in this system.
Notch controls neuronal numbers and specification in dividing progenitors
Reduction of Notch activity alters both the total number of epiphysial
neurons, as well as the identity of these neurons. We next searched for the
stages at which Notch activity was required for these two activities by
treating embryos with DAPT from various stages of development. Although an
increase in the total number of Islet1+ neurons was observed when DAPT was
administered from stages up to 14 hours, treatment starting at or after 16
hours did not affect neuronal number (Fig.
6F). Similarly, we observed a decrease in the number of
Tg(AANAT2:GFP)+ cells when DAPT was administered from up to but not after 14
hours (Fig. 6G). Surprisingly,
however, we observed a statistically significant increase in the number of
Tg(HuC:GFP)+ cells when DAPT treatment was started at stages up to 16 hours
(Fig. 6H). These results
suggest that cell fate can still be modified at a stage when inhibition of
Notch activity no longer affects the total number of neurons. We conclude that
the effect of Notch on neuronal numbers and on neuronal identity reflects
distinct sequential activities. Furthermore, as 98.03±3.4% of the
future Tg(HuC:GFP)+ and 83.4±14.4% of the future Tg(AANAT2:GFP)+ cells
have not exited their last S phase at 18 hours
(Fig. 6E), our results indicate
that Notch activity is required in cycling progenitors for both the control of
neuronal number and the specification of neuronal identity.
Notch signaling is required to resolve mixed identity
We noted that treatment with DAPT at 16 hours increases the number of
Tg(HuC:GFP)+ cells without a concomitant diminution of the number of
Tg(AANAT2:GFP)+ cells or an increase in the total number of neurons. One
possible explanation is that in the absence of a functional Notch pathway,
some cells are unable to choose between a photoreceptor or a projection neuron
identity and therefore retain markers for both identities. We performed double
staining with an antibody against HuC/D in a Tg(AANAT2:GFP) background to
assess this possibility; the HuC/D antibody recapitulates the expression of
Tg(HuC:GFP) except for a few ventrally located cells that are HuC/D+ but
Tg(HuC:GFP)- and that we interpret to be newly born projection neurons (data
not shown). In wild-type or mock-treated embryos, we observed a low occurrence
of HuC/D+/Tg(AANAT2:GFP)+ cells (4.1% of specified epiphysial
neurons). A similar frequency of huC+/Tg(AANAT2:GFP)+ cells was
observed in embryos double labeled for huC transcripts (data not
shown). Interestingly, double-labeled cells were more numerous in mib
mutants and embryos treated with DAPT at 16 hours (12.6 and 14.1%,
respectively; Fig. 7A-C). We
also observed an increase in the number of cells double-labeled for
Tg(AANAT2:GFP) and the projection neurons markers lhx3 or Pax6 upon
reduction of Notch signaling (Fig.
7C). Finally, we used a complex opsin probe to assess co-labeling
with the Tg(HuC:GFP) transgene. Although in a wild-type context these two
markers are exclusive, we observed rare co-labeled cells in mib
mutants (Fig. 7C, 50% of
mib mutant embryos show one double-labeled cell). These results
suggest that epiphysial neurons pass through a state where they express
markers for both subtype identities and that Notch is required for the
resolution of such mixed identity.
|
).
As shown in other studies, a graded response to Notch signaling is achieved
depending on the temperature of heat-shock activation
(Shin et al., 2007). Indeed,
whereas embryos subjected to a strong heat shock (0.5 hours at 40°C) at 9
hours show very few epiphysial neurons, upon milder activation (1 hour at
38°C) a wild-type number of Islet1+ neurons in the epiphysis was observed
(Fig. 8C and data not shown).
Nonetheless, mild activation of Notch signaling produced a strong decrease in
the number of cells labeled with projection neurons markers
(Fig. 8A,B,D). Consistent with
results from late treatment with DAPT, the reduction of the numbers of
Tg(HuC:GFP)+ cells was not observed when heat shock was induced at 24 hours, a
stage where the majority of epiphysial progenitors have passed their last S
phase (Fig. 8D). Unexpectedly,
the number of photoreceptors remains unchanged regardless of the stage at
which Notch signaling is activated (Fig.
8A,B,E; data not shown). Thus, although neurons are produced
normally under mild Notch activation, projection neurons fail to be specified.
Furthermore, preventing the specification of projection neurons is not
sufficient to induce the transformation of unspecified cells into
photoreceptors. We conclude that Notch signaling represses the projection
neuron fate but is not instructive for photoreceptor identity, which
presumably requires other inducing signals.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Notch regulates cell number and identity in a short time window in cycling epiphysial progenitors
Our results indicate that Notch signaling plays two distinct roles in the
epiphysis: it regulates the number of neurons produced and the balance between
projection neuron and photoreceptor identity. Such a dual role for Notch
signaling has been already described in other areas of the vertebrate nervous
system (see Shin et al.,
2007). Interestingly, our DAPT time course and birthdating studies
suggests that these two decisions occur in dividing epiphysial progenitors.
Furthermore, the very short delay observed between the two Notch-driven
decisions raises the issue of the how epiphysial progenitors adapt to such
rapid changes in the level of Notch activation. One attractive hypothesis is
that determination and specification of neuronal subtype identity employ
different Notch signaling components. Indeed, we have shown that reduction of
deltaA and deltaD functions alters the balance between the
projection neuron and photoreceptor fates without affecting the total number
of neurons. Alternatively, the control of neuronal numbers and identity could
involve the same Notch ligands with the effects on neuronal number and
identity reflecting differences in the sensitivity of the two processes to the
absolute levels of ligand present. In this case, determination and
specification of neuronal subtype identity might employ different
intracellular components downstream of Notch.
|
; Peng et al.,
2007). Here, we present evidence that cells of a mixed
photoreceptor/projection neuron identity can be observed in the wild-type
epiphysis, albeit with a low frequency. Furthermore, cells expressing markers
of both neural subtypes are more numerous in the epiphysis of embryos with
reduced Notch activity. These results suggest that epiphysial progenitors pass
through a transient phase of double identity and that Notch is required to
resolve this. As we observe an increase in the number of cells expressing
markers of projection neurons in Notch compromised embryos, it appears that
the increase in the number of cells with mixed identity reflects that Notch is
required to repress projection neuron identity in these cells. However,
although reduction of Notch activity promotes the formation of projection
neurons, the constitutive activation of the pathway inhibits the projection
neuron fate but does not induce the transformation of these neurons into
photoreceptors. Although we cannot rule out that activation of the
photoreceptor fate requires a different threshold or mode of Notch activity
than the Tg(hs:Gal4); Tg(UAS:Nintra) system provides, our data suggest that
the Notch pathway does not play an instructive role in specifying the
photoreceptor fate. In this regard, the situation found in the epiphysis is
strikingly different from the previously described cases of Notch triggering a
`binary fate decision' both in Drosophila and vertebrates
(Fanto and Mlodzik, 1999;
Guo et al., 1995;
Shin et al., 2007). For
example, in the ventral spinal cord, loss of Notch activity induces the
production of an excess of motoneurons at the expense of KA'
interneurons and a reciprocal excess of KA' interneurons at the expense
of motoneurons is induced upon constitutive activation of the pathway
(Shin et al., 2007). As activating Notch is not sufficient to activate the photoreceptor fate in the epiphysis, we postulate the existence of a photoreceptor inducing signal. We propose that mixed identity cells have received the postulated photoreceptor-inducing signal but have not yet downregulated the projection neuron program via Notch signaling. Three possibilities can be envisaged for what happens to cells with a mixed identity when Notch activity is impaired: they die, they retain markers of both identities or they finally adopt one of the two fates in a stochastic manner. We have shown that reduction of Notch activity induces cell death in both Tg(HuC:GFP) and Tg(AANAT2:GFP)+ cells. Interestingly, a negative correlation is observed between the presence of dying cells and the presence of cells with a mixed identity upon DAPT treatment. Indeed, we observe a relatively high frequency of mixed identity cells and no significant increase in apoptosis upon late DAPT treatment, while the opposite is observed upon early DAPT treatment. However, it is not possible to ascertain whether dying cells correspond to mixed identity cells and thus to establish a causal link between the failure to resolve such identity and apoptosis. By contrast, as we observe an excess of projection neurons at the expense of photoreceptors in embryos expressing reduced levels of Delta ligands, we would predict that at least some cells with mixed identity downregulate the photoreceptor program and adopt a projection neuron fate.
Towards a model of epiphysial cell type specification
Our results show that Notch controls both neuronal numbers and neuronal
subtype identity in the zebrafish epiphysis and a model summarizing how this
might be achieved is presented in Fig.
9. First, Notch effects on neuronal number and fate appear to
occur in dividing precursors. However, impairing Notch activity at 16 hours
modifies cell fate without modifying neuronal number. Thus, the choice between
neuronal subtype identities is made slightly later than the decision to
differentiate. Therefore, the first role of Notch results in the selection of
one neural progenitor from a pool of equipotent cells. As the choice of a
subtype identity is slightly delayed, we suggest that cells having chosen to
differentiate change neighbors between the two Notch-dependent decisions. We
speculate that such a change occurs as a result of interkinetic nuclear
movements that neural progenitors undergo within the neuroepithelium
(Frade, 2002;
Sauer, 1935). Cells first
decide whether they will stop dividing after they have completed their last
cycle (Fig. 9A1). Then, they
migrate basally where they encounter other neural progenitors which have
already been selected to differentiate
(Fig. 9A2). Communication
between these cells would allow them to choose a fate before the completion of
their last S-phase with Notch signaling inhibiting the projection neuron fate
in cells having received the photoreceptor inducing signal
(Fig. 9A3).
Our model implies a role for the Notch pathway in establishing cell fate
through communication between cells expressing high levels of Delta (the
progenitors for projection neurons) and cells expressing lower levels of Delta
(the progenitors for photoreceptors). Indeed, two Notch ligands,
deltaB and deltaD, are specifically expressed in projection
neurons. Interestingly, the restriction of deltaB expression to
projection neurons requires a functional Notch pathway as in mib
mutants, we observed the expression of deltaB in photoreceptors
(E.C., A.Q. and P.B., unpublished). This suggests that cell-cell communication
via Notch is required to restrict the expression of certain Notch ligands to
projection neurons (see Fig.
9B), in a manner similar to that described in the fly proneural
clusters (Simpson, 1997).
Conclusion
Although the effect of Notch signaling on the spatio-temporal control of
neurogenesis has been extensively studied, comparatively little is known about
the role of Notch on the specification of neuronal subtype identity in
vertebrates. Our results highlight a novel role for Notch. Indeed, acquisition
of the photoreceptor fate in the epiphysis involves two distinct events: the
induction of a photoreceptor program and the inhibition of projection neurons
traits. However, although Notch is required to resolve fate choice by
inhibiting the undesired genetic program, in contrast to other models in which
Notch has been studied, it is not sufficient for the induction of the
appropriate program. Further studies will show whether induction of other
neuronal subtype identities similarly involves two distinct signals one for
the induction of the appropriate fate and the other for the inhibition of
inappropriate traits.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/14/2391/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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