1 University of Lausanne, Eye Hospital Jules Gonin and Institute for Research in
Ophthalmology, 15 avenue de France, 1004 Lausanne, Switzerland
2 University of Geneva, Sciences II, Biochemistry Department, 30 quai
Ernest-Ansermet, 1211 Geneva, Switzerland
3 Polish Academy of Sciences, Medical Research Center, Department of
Endocrinology, ul. Banacha 1a, 02-097 Warsaw, Poland
4 Medical Center of Postgraduate Education, Department of Clinical Biochemistry,
Marymoncka 99, 01-813 Warsaw, Poland
* Author for correspondence (e-mail: jean-marc.matter{at}biochem.unige.ch)
Accepted 28 June 2005
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SUMMARY |
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Key words: Retinogenesis, Retina patterning, Basic helix-loop-helix, Transcription, Chick embryo
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Introduction |
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The retina is one region of the central nervous system in which the
conversion of progenitor cells into particular classes of neural cells is
quite well understood (Cepko,
1999; Harris and Holt,
1990
; Reh and Levine,
1998
). Retina ontogenesis is geared to generate glia and six
classes of retinal neurons from an undifferentiated neuroepithelium, according
to a program that controls proliferation, specification, exit from the cell
cycle and differentiation. Cell differentiation initiates in the inner layer
of the central optic cup and progresses radially to the peripheral edge of the
retina. A characteristic feature of vertebrate retinogenesis is that the
different retinal cell types are generated in a fixed sequence. Retinal
ganglion cells (RGC) differentiate first, followed in overlapping phases by
amacrine cells, horizontal cells, cone photoreceptors, rod photoreceptors,
bipolar cells and, finally, Müller glial cells.
The generic programs of neuronal differentiation are regulated in
vertebrates as in Drosophila
(Anderson and Jan, 1997) by
members of the basic helix-loop-helix (bHLH) class of transcription factors.
The achaete-scute homologue ASH1 and the three neurogenins
(NGN1-NGN3) are among the earliest bHLH genes expressed in the developing
nervous system and they are thought to act as proneural genes. A spatial
complementarity between the expression of ASH1 and of the neurogenins appears
to be the rule in most proliferating neuroepithelia and these factors have a
role in the ontogeny of distinct classes of progenitors
(Bertrand et al., 2002
). In the
developing retina, most of the broadly expressed neurogenic bHLH proteins are
likewise implicated in the generation of distinct classes of neurons
(Inoue et al., 2002
;
Vetter and Brown, 2001
), but
it is unresolved whether these factors act individually or combine to promote
particular neuronal phenotypes. Likewise, the molecular mechanisms that
control the timing of their expression and/or function are poorly defined.
The atonal homologue ATH5 is almost exclusively expressed in the
developing retina. Initially cloned and analysed in Xenopus
(Kanekar et al., 1997), its
mouse (Brown et al., 1998
),
zebrafish (Masai et al.,
2000
), chicken (Liu et al.,
2001
; Matter-Sadzinski et al.,
2001
) and human (Brown et al.,
2002
) orthologues have also been identified. In the mouse,
inactivation of the Ath5 gene results in a retina lacking most RGCs
and, as a consequence, in optic nerve agenesis
(Brown et al., 2001
;
Wang et al., 2001
). Although
ATH5 is directly involved in its own regulation and is able to activate genes
that define neuronal identity traits (Liu
et al., 2001
; Matter-Sadzinski
et al., 2001
;
Skowronska-Krawczyk et al.,
2004
), the mechanism for integrating ATH5 expression into a
coherent program of RGC specification and differentiation has not been
elucidated.
Lineage tracing studies have led to the hypothesis that retinal progenitors
pass through a series of different competence states during which they
sequentially produce different types of neural cells
(Livesey and Cepko, 2001).
This model suggests that progenitors may be limited to producing certain types
of neurons at a given time in the course of retinogenesis. Here, we attempt to
define at the molecular level the time frame and cellular context in which
progenitors yield RGCs in the developing chick retina. Specifically, we
identify several of the stages along the pathway leading to the conversion of
progenitors into newborn RGCs. We show how the interplay between ATH5 and
other bHLH proteins controls the transitions between stages and coordinates
RGC specification with the patterning of progenitor cells. We highlight a
program that operates during the two main phases of ATH5 expression,
coordinating the selection of RGC precursors and the induction of RGC-specific
traits with cell cycle exit. The first phase involves crossregulatory
interactions between ATH5, NGN2, ASH1 and HES1 proteins that allow the
expansion of pools of progenitors, contribute to their progressive
intermingling and maintain ATH5 expression below the level required for
inducing RGC differentiation. The second phase initiates when RGC progenitors
are dispersed throughout the retina. The coordinated upregulation of NGN2 and
downregulation of HES1 contribute to the progression of progenitors through
the last cell cycle and create a suitable environment for efficient ATH5
autostimulation. The ATH5 protein upregulates its own expression and initiates
the transcription of RGC-specific traits. Cells committed to the RGC fate then
exit the cell cycle and express post-mitotic neuronal markers. In sum, we show
how a subset of progenitors is selected from the pool of ATH5-expressing cells
to enter the specification pathway at the proper time for RGC genesis.
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Materials and methods |
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Eukaryotic expression plasmids for ATH5, NGN2 and HES1
The pEMSV plasmid (Matter-Sadzinski et
al., 2001), which puts a cloned sequence under the transcriptional
control of the mouse sarcoma virus long terminal repeat, was used throughout
to express the ATH5, NGN2 and HES1 cDNAs in co-transfection and
electroporation experiments.
Northern blot
Ten electroporated central and peripheral retina explants were lysed in
guanidine thiocyanate. Total RNA was isolated, gel fractionated (2 µg/lane)
and hybridized as described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
2001).
In situ hybridization
35S-labelled antisense riboprobes were synthesized and in situ
hybridization on tissue sections were performed as described by
Matter-Sadzinski et al. (Matter-Sadzinski
et al., 2001). To correlate the expression level of a particular
gene with ATH5 or ß3 promoter activity, transfected retinal cells were
stained for ß-galactosidase and processed for in situ hybridization as
described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
2001
). For quantification (Fig.
1O), silver grains were counted in 50 radial sectors (
1300
µm2 each) corresponding to a visual angle of
3°.
[3H]-thymidine and BrdU labelling
To label the S phase, transfected cells were incubated in medium containing
5 µCi/ml [3H]-thymidine for 3 hours (stage 22-23) or 1 hour
(stage 29-30) at the end of the 24 hours expression period. They were stained
for ß-galactosidase and processed for autoradiography
(Matter-Sadzinski et al.,
2001). Neuroretinas were dissected and incubated for 45 minutes in
medium containing 100 µM BrdU and chased for 15 minutes. The explants were
fixed, embedded in paraffin wax, sectioned and processed for in situ
hybridization and for immunodetection of BrdU
(Roztocil et al., 1997
).
Cell cultures, transfection, CAT and ß-galactosidase assays
Chick embryos were staged according to Hamburger and Hamilton (Hamburger
and Hamilton, 1951). Neuroretina from stage 22-23 to stage 38 embryos were
dissected and dissociated into single cells that were transfected with CAT,
lacZ or GFP reporter plasmids. All transfections were carried out using the
lipofectin reagent (InVitrogen), as described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
1992). In all instances, the ratio of DNA to lipofectin was 1:4.
In transfection experiments using a single construct, we transfected 1 µg
of reporter plasmid per 106 cells. In co-transfection experiments
using two or three constructs, 1 µg of reporter plasmid was mixed,
respectively, with 0.5 µg or 2x0.5 µg expression vectors per
106 cells. Negative controls consisted of 1.0 µg reporter
plasmid and 1.0 µg empty expression vector per 106 cells.
Quantification of the chloramphenicol acetyl transferase (CAT) activity
obtained with pATH5-CAT and identification of ß-galactosidase-positive
cells (lacZ) were as described by Matter et al.
(Matter et al., 1995
) and
Matter-Sadzinski et al. (Matter-Sadzinski
et al., 2001
).
Electroporation of genetic material in the retina
Retinas were prepared from embryonic eyes collected at stages 22-23.
Electroporations were as described by Matter-Sadzinski et al.
(Matter-Sadzinski et al.,
2001). Briefly, whole retinas or dissected peripheral and central
sectors were immersed at room temperature in phosphate-buffered saline
containing a reporter plasmid and/or expression vectors (100 µg/ml of each
construct). Electroporation consisted of five 50 V/cm pulses of 50 mseconds
duration spaced 1 second apart. The electroporated tissues were cultured as
floating explants for 24 hours at 37°C. GFP- and
ß-galactosidase-positive cells were revealed or tissues were frozen in
liquid nitrogen prior to RNA extraction.
Single-cell collection and RT-PCR
Cells transfected with the ATH5-promoter/GFP reporter plasmid were plated
into poly-DL-ornithine-coated plastic petri dishes (30 mm in diameter).
Twenty-four or 48 hours after transfection, individual GFP-positive cells were
collected by aspiration with a glass micropipette mounted on a
micromanipulator. Single-cell RT-PCRs were performed according to Brady and
Iscove (Brady and Iscove,
1993). Each cell was collected in 10 µl of a buffer containing
50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT
supplemented with 10 U of RNAsin (Promega), and immediately frozen in liquid
nitrogen. Single cells were thawed on ice, vortexed for 10 seconds, spun down,
heated to 65°C for 1 minute, vortexed again and then incubated on ice for
1 minute. For reverse transcription, dNTPs and Nonidet P-40 were added to
final concentrations of 0.5 mM each dNTP, 0.5% NP-40, supplemented with 50 ng
random DNA hexamers and 10 U RNAsin. 5 U of DNAse 1 (Gibco) was added and the
mix was incubated at 37°C for 30 minutes to destroy genomic DNA. DNAse 1
was inactivated by 10 minutes incubation at 65°C. The mix was aliquoted in
two fractions, both of which were again treated with 10 U of RNAsin and one of
which received 200 U of Superscript II reverse transcriptase (Gibco). Both
samples were incubated at 25°C for 10 minutes, at 37°C for 1 hour and
at 68°C for 10 minutes. The whole reaction mixes were then used as
templates in a first PCR performed with ExTaq polymerase (TaKaRa) and the
complete set of external primers (0.2 mM final concentration of each primer)
designed for amplifying the genes of interest (see Table S1 in the
supplementary material). Initial denaturation at 94°C for 3 minutes was
followed by 35 cycles consisting of denaturation at 94°C for 40 seconds,
annealing at 56°C for 40 seconds, elongation at 72°C for 1 minute, and
a final elongation at 72°C for 3 minutes. The second PCR was performed
separately for each gene of interest using the internal primers specific for
this gene (4 mM final concentration of each primer) and 0.1 volume of the
first PCR as template. PCR conditions were as described above. When using the
ATH5 internal primers (see Table S1 in the supplementary material), PCRs were
performed with templates originating from both the actual and the mock reverse
transcriptions. Only those cells that yielded no amplification of the negative
control sample were selected for expression of the other genes of
interest.
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Results |
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Dynamic changes in spatial patterning of progenitor cells precede RGC differentiation
The concentric expression pattern of the bHLH genes is maintained until
stage 26 (E5) (Fig. 2).
However, from stage 23 onwards, dynamic changes are taking place. Between
stages 18 and 26, the retina diameter increases about threefold and expansion
of the ATH5 and NGN2 expression domains parallels the growth of the whole
retina (Fig. 2A-E). HES1
expression, on the whole, remains complementary to that of ATH5, with
transcript levels maintained very high at the periphery and low in the centre
(Fig. 2C). At stage 23, the
central retina still contains isolated cells expressing HES1 at a high level
(Fig. 2L). Contrasting with the
mutually exclusive domains established at earlier stages, the anterior margin
of the ATH5 domain now overlaps the posterior HES1 region, where the levels of
HES1 transcripts are decreasing (Fig.
2H,I). Moreover, from stage 23 onwards, the NGN2 domain expands
more peripherally than that of ATH5, suggesting that NGN2 expression is less
sensitive than ATH5 to inhibition by HES1
(Fig. 2J,K), and thus precedes
the onset of ATH5 expression as both domains expand to the periphery.
Expansion of the NGN2 and ATH5 domains is paralleled by changes in the
expression pattern of ASH1. At stages 23-26, an annular ASH1 expression region
is still surrounding the ATH5 domain but ASH1- and ATH5/NGN2-expressing cells
are intermingled in the centre (Fig.
2E,F). At stages 28-30 (E6), distinct progenitor domains are no
longer detected and ATH5-expressing cells are distributed throughout the
retina, except at the ciliary margin (Fig.
2G). Expansion of the ATH5 domain to the periphery is paralleled
by a strong increase in accumulation of ATH5 mRNA
(Fig. 2G) and coincides with
the downregulation of HES1 in central and peripheral retina
(Fig. 2M). Thus, dynamic
changes in the expression profile of progenitor cells at domain boundaries and
in the central retina lead to a blending of different precursor sets, to a
progressive blurring of borders and to the merging of formerly discrete
domains.
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Single-cell transcription analysis reveals the stages of a progression along the RGC specification and differentiation pathway
The isolated ATH5 promoter region provides a unique means of identifying
ATH5-expressing progenitor cells, some of which will become committed to the
RGC lineage. Whereas in situ hybridization suffices to colocalize promoter
activity and expression of a single gene in individual cells
(Matter-Sadzinski et al.,
2001), the single-cell RT-PCR approach is necessary for detecting
the co-expression of multiple genes. To monitor the dynamics of bHLH
expression in individual cells during the period of RGC specification and
differentiation, acutely dissociated cells from stage 22-23 (E3.5) and 26 (E5)
retinas were transfected with the ATH5-promoter/GFP-reporter plasmid singly or
in combination with a NGN2 expression vector, plated into tissue culture
dishes and cultured for 24 or 48 hours. The time of cell collection thus
approximately corresponded to E4.5, E5.5 and E6 in vivo. One-hundred and sixty
GFP-positive cells were collected and single-cell RT-PCRs were performed to
produce collections of cDNA fragments representing the mRNA of single
ATH5-expressing cells. To examine the combinations in which the selected genes
were expressed, cells from the five groups generated by the experiment
(Fig. 4B) were independently
tested by second rounds of PCR, using appropriate (see Table S1 in the
supplementary material) sets of primers
(Fig. 4A,
Fig. 5A,B). All GFP-positive
cells expressed ATH5, as expected (Fig.
4A), but there was a high degree of heterogeneity and striking
temporal changes in the expression profiles of the unselected genes HES1,
Delta 1, Neuro M, ß3nAChR and BRN3C. At early stages (E4.5), we did not
find any cells co-expressing ASH1 and ATH5 (0/20), confirming that these two
proneural genes are initially expressed in separate sets of early progenitors.
At later stages (E5.5-E6), three out of 28 cells expressed both genes (e.g.
cell 149), two of them expressing Neuro M as well (data not shown). The large
majority (
80%) of cells transfected at stage 22-23 and collected 24 hours
later were expressing HES1, evidence that most ATH5-expressing cells are
proliferating progenitors. This proportion decreased to
5% when stage
22-23 cells were kept for 48 hours in culture or when cells were transfected
at stage 26 (Fig. 4C).
Conversely, 10% of cells transfected at stage 22-23 were found to express ATH5
and Neuro M after 24 hours in culture. This proportion increased to
60%
when stage 22-23 cells were cultured for 48 hours (e.g. cell 128), or when
stage 26 cells were cultured for 24 hours (e.g. cell 54). The expansion of the
population expressing Neuro M is paralleled by a proportional decrease in the
number of HES1-expressing cells as RGC precursors exit the mitotic cycle, a
process culminating at E6. Because no cells co-expressing HES1 and Neuro M
were found in the collection (0/60), whereas numerous single cells expressed
neither HES1 nor Neuro M (22/60), these two proteins must be expressed at
distinct stages separated by a lag. Interestingly, overexpression of NGN2 at
stage 22-23 leads, after 24 hours, to a drastic decrease in the proportion of
HES1-positive cells, but does not induce the precocious generation of Neuro
M-positive cells (Fig. 4C),
resulting instead in the accumulation of cells that express neither HES1 nor
Neuro M. The proportion of Neuro M-positive cells increased to
60% when
stage 22-23 cells overexpressing NGN2 were cultured for 48 hours. Some cells
co-expressed Delta 1 and HES1 (e.g. cell 114) and Delta 1 expression was seen
both in the presence of Neuro M (e.g. cell 126) and in cells that expressed
neither Neuro M nor HES1 (e.g. cell 116), indicating that the Notch ligand is
expressed soon after the downregulation of HES1 but prior to the onset of
Neuro M expression, a result that is consistent with previous in situ
hybridization studies (Henrique et al.,
1997
; Roztocil et al.,
1997
). The small proportion of Delta 1-positive cells (4/39;
Fig. 4A) suggests that this
gene is expressed very transiently in differentiating RGCs.
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A rapid decrease in the activity of the ATH5 promoter was detected between E6 and E9, marking the transition between the second and third phases in the regulation of ATH5 (Fig. 6A). At stages 34 and 37, the promoter is poorly active and no longer responds to overexpression of ATH5 or NGN2. Thus, it appears that the ability of the ATH5 protein efficiently to stimulate its own expression in a subset of competent progenitors is restricted to the narrow time window (E5-E7) when most RGC precursors are born.
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These results suggest that ATH5 and NGN2 may compete for the same
regulatory elements, and that ATH5 is the dominant activator in the absence of
HES1. At early stages, because of the presence of HES1, ATH5 cannot upregulate
its own expression but can efficiently compete with NGN2. Mutational analysis
showing that the two proteins are using the same E-box elements to mediate
their effects (Fig. 1S) and
chromatin immunoprecipitation (ChIP) experiments demonstrating that both the
ATH5 and NGN2 proteins bind the ATH5 promoter in vivo at stage 22-23 and stage
29-30 (Skowronska-Krawczyk et al.,
2004) are in favour of this hypothesis. In addition, the
co-transfection results suggest that autostimulation is in large part
responsible for the upregulation of ATH5 expression at stage 30, whereas at
earlier stages ATH5 may contribute to the downregulation of its own
expression.
NGN2 upregulates promoter activity in proliferating cells and drives ATH5-expressing cells beyond the last S phase
The presence of ATH5 transcripts in 26% of stage 18 neuroepithelial
cells (Fig. 1)
(Skowronska-Krawczyk et al.,
2004
) and of HES1 transcripts in most of the ATH5-expressing cells
at stage 22-23 (Fig. 4), taken
together with previous findings (Liu et
al., 2001
; Matter-Sadzinski et
al., 2001
), provide ample evidence that the endogenous
ATH5 gene is transcribed in proliferating cells. The differential
response of the ATH5 promoter to the NGN2 and ATH5 proteins in the course of
retina development (Fig. 6) and
the different sensitivities of these proteins towards HES1
(Fig. 7) suggest their
implication at distinct moments in progenitor commitment to the RGC fate. To
analyse how stimulation of the ATH5 promoter correlates with the proliferative
status, NGN2 or ATH5 expression vectors were transfected together with an ATH5
promoter/lacZ-reporter plasmid at the stages when each factor exerts
its major effect and the transfected cells were pulsed with
[3H]-thymidine at the end of a 24-hour culture period
(Fig. 8A). Forced expression of
NGN2 at stage 22-23 led to a 3.5-fold increase in double-labelled cells and to
a sevenfold enlargement of the non-radioactive lac+ cell
population. On the whole, NGN2 overexpression diminished by half the
[3H]-thymidine-labelling index of cells that had an active ATH5
promoter when compared with controls (i.e. from
34% to
17%). This
result reveals the dual effect of overexpressed NGN2, which both stimulates
ATH5 expression in proliferating cells and significantly increases the pool of
non-dividing cells that have an upregulated ATH5 promoter
(Fig. 8A), indicating that NGN2
drives cells out of the S phase.
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Discussion |
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Spatiotemporal progenitor patterning and RGC genesis
A specific feature of retinogenesis is that it proceeds from the centre to
the periphery such that all seven retinal cell types are distributed at the
proper ratio throughout the retina. At early stages of development, the
retinal neuroepithelium is subdivided into two developmentally distinct
territories. Low levels of HES1 transcripts outline a broad region of the
posterior retina where ATH5, NGN2 and ASH1 are expressed, whereas a robust
accumulation of HES1 transcripts throughout the anterior retina prevents the
onset of proneural gene expression. HES1 functions similarly at the onset of
neurogenesis in the olfactory placode, where it circumscribes a domain of
Mash1 expression (Cau et al.,
2000). It thus appears that HES1 is acting, much like
hairy in Drosophila, as a prepattern gene
(Skeath and Carroll, 1991
).
Neurogenesis starts within a rather broad central region defined by expression
of ATH5, NGN2 and Neuro M. Cells expressing ATH5 at a high level and Neuro
M-positive cells are evenly distributed throughout the neurogenic domain,
indicating that the first newborn RGCs are produced with similar frequency
throughout the central retina. In the posterior retina, cells that initiated
expression of proneural genes are initially organized in two separate domains
corresponding to two retinal lineages: cells that express NGN2/ATH5 constitute
the progenitor pools from which early-born retinal neurons will emerge,
whereas ASH1-expressing cells form a pool for late-born neurons
(Brown et al., 1998
;
Jasoni et al., 1994
;
Matter-Sadzinski et al.,
2001
). The opposite effects of NGN2 on ATH5 and ASH1 expression
combined with the inhibitory activity of ASH1 on ATH5 transcription
(Akagi et al., 2004
;
Fode et al., 2000
;
Matter-Sadzinski et al., 2001
)
account for the distribution of ASH1 and ATH5/NGN2 cells in two distinct
progenitor domains, the more peripheral expression of ASH1 perhaps reflecting
its lower sensitivity towards HES1. The initial patterning of the posterior
retina resembles the neuroepithelial partitioning detected in other areas of
the developing CNS (Bertrand et al.,
2002
; Jessell,
2000
). However, whereas in other CNS regions the refining of
borders is essential for the precise spatial generation of different classes
of neurons along the dorsoventral axis, the blurring of borders and
intermingling of initially distinct progenitor pools are necessary for a
proper spatial distribution of neurons and glia throughout the retina.
Although ATH5/NGN2 and ASH1 expressions are mutually exclusive, a small
fraction of ATH5-expressing cells co-express ASH1
(Fig. 4)
(Matter-Sadzinski et al.,
2001
), indicating that they are in a transient state prior to
acquiring a definite progenitor status. Because the ATH5, NGN2 and ASH1 genes
crossregulate and display different sensitivities towards HES1, we suppose
that various balances between these four factors may mediate alternate fate
choices. Such dynamic regulatory interactions are, in part, responsible for
the progressive loss of patterning in the posterior retina. The ATH5/NGN2
domain remains restricted to the posterior retina until E4 and expands to keep
pace with growth of the whole retina at a rate similar to that reported for
the differentiation of RGCs (McCabe et
al., 1999
). Despite significant changes in the expression pattern
of ATH5, similar proportions of retinal cells express this gene at stages 18
and 29-30, suggesting that ATH5-expressing cells propagate at a rate
comparable with that of the other progenitors during the period of
patterning.
Even though the population of ATH5-expressing cells is established at E2.5,
only a small fraction of these will differentiate into RGCs until E4
(Prada et al., 1991;
Rager, 1980
;
Waid and McLoon, 1995
).
Retinogenesis is controlled by components of the Notch pathway
(Perron and Harris, 2000
;
Vetter and Brown, 2001
), which
may employ two strategies to keep the majority of cells in the central retina
from differentiating during the patterning period. Cells that express
proneural genes may promote the upregulation of HES1 in neighbouring cells,
thereby preventing them from expressing proneural genes. The proximity in
central retina of individual cells that highly express HES1 or ATH5 is indeed
indicative of ongoing lateral inhibition. However, cells strongly expressing
Notch effectors are rare in the posterior retina
(Fig. 1I,
Fig. 2L), whereas a high
proportion of ATH5-expressing progenitors co-express HES1
(Fig. 4). Thus, it appears that
the low level of HES1 in cells that have already initiated NGN2 and ATH5
expression suffices to prevent the upregulation of these genes. The
proliferative state is thereby maintained in most ATH5-expressing cells, as
required to ensure the proper ratio of RGC progenitors in the posterior retina
and as expected of HES genes, which function to keep neuroepithelial cells
undifferentiated, thereby regulating the size and cell architecture of brain
structures and retina (Hatakeyama et al.,
2004
; Ishibashi et al.,
1995
; Takatsuka et al.,
2004
; Tomita et al.,
1996
). In anterior retina, progenitor cell patterning becomes
evident by E4 and the expansion of proneural gene expression proceeds, much as
in zebrafish (Masai et al.,
2000
), in a wave-like fashion as HES1 expression recedes to the
retinal margin. The ASH1 and NGN2 expression domains expand to the periphery
at similar rates, whereas the progression of the ATH5 domain is slightly
delayed (Fig. 2). The full
patterning of the retina accomplished around E6 coincides with the
upregulation of proneural gene expression throughout the retina and with the
peak of RGC production.
To analyse how ATH5 is regulated along the course of RGC specification, we
used a promoter region extending 775 bp upstream of the initiation codon. The
cloned sequence accurately reproduces the activity and the mode of regulation
of the endogenous promoter. It contains essential regulatory elements that are
well conserved across distant vertebrate species
(Brown et al., 2002;
Hutcheson et al., 2005
;
Skowronska-Krawczyk et al.,
2004
), but it is unclear whether the different species use similar
strategies to regulate ATH5 expression. Whereas a proximal cis-regulatory
region of the Xenopus Xath5 gene suffices, much as in the chick
retina, to drive retina specific reporter gene expression in a bHLH-dependent
manner, the mouse ATH5 promoter appears to be regulated differently
(Hutcheson et al., 2005
). It
is tempting to speculate that the different modes regulating ATH5 across
species may account for differences in the spatiotemporal progenitor
patterning of the retinal neuroepithelium. Differences in the developments of
the anterior and posterior retinas may have permitted the evolution of a
specialized structure such as the macula.
Multiple functions of NGN2 in the specification of RGCs
Our study reveals that NGN2 acts at different regulatory levels during RGC
specification. In early retina, NGN2 is a principal regulator of ATH5
expression and exerts this function through direct activation of ATH5
transcription and through crossregulatory interactions with HES1. In addition,
NGN2 drives ATH5-expressing cells out of S phase. Whereas the capacity of NGN2
to promote cell cycle arrest is part of its panneuronal activities and is in
evidence in other compartments of the developing CNS
(Farah et al., 2000;
Ma et al., 1996
;
Novitch et al., 2001
), its
capacity to activate ATH5 expression is largely retina specific. The
quasi-simultaneous onset of NGN2 and ATH5 expression in the central retina
shortly after formation of the eye cup
(Fig. 1), the capacity of NGN2
to activate ATH5 transcription (Figs
6,
7)
(Matter-Sadzinski et al.,
2001
) and to bind the ATH5 promoter
(Skowronska-Krawczyk et al.,
2004
) at the early stages of development suggest that NGN2 may be
directly involved in the activation of ATH5 expression. Our finding that the
expansion of the NGN2 domain towards the anterior edge of the retina precedes
that of ATH5 argues in favour of this interpretation. In the retina of the
Ngn2/ mouse, the much increased expression of ASH1
(Akagi et al., 2004
) and the
downregulation of ATH5 (D. Skowronska-Krawczyk and J.M.M., unpublished) when
compared with the wild type, may result from an increase in the population of
ASH1-expressing cells at the expense of the ATH5/NGN2 progenitors, thus
underlining the importance of NGN2 in establishing and maintaining a pool of
ATH5-expressing cells. Both the NGN2 and ATH5 genes fail to be activated in
the retinal precursors of the Pax6/ mouse and Pax6
has been proposed to regulate NGN2 directly in the mouse retina
(Brown et al., 1998
;
Marquardt et al., 2001
). There
are multiple E-boxes but no consensus Pax6 binding site in the chicken ATH5
promoter, and therefore we favour the idea that Pax6 regulates ATH5 via NGN2.
The expression of NGN2 in many regions of the nervous system anlage where ATH5
is not detected and the demonstration that recruitment of NGN2 on the ATH5
promoter is retina specific
(Skowronska-Krawczyk et al.,
2004
) provide evidence that a retina-specific context accounts for
the capacity of NGN2 to activate ATH5 expression. The ability of bHLH factors
to regulate the development of distinct neurons has been proposed to depend
upon the cellular contexts in which they function
(Perron et al., 1999
). In
retina, this context may be determined, among other possibilities, by the
balance between NGN2 and HES1, as we show that HES1 inhibits the NGN2-mediated
activation of ATH5 in a dose-dependent manner
(Fig. 7). Likewise, the
upregulation of NGN2 correlates with the dowregulation of HES1
(Fig. 2C)
(Matter-Sadzinski et al.,
2001
). Moreover, single cell transcriptional analysis reveals that
overexpressing NGN2 diminishes the pool of cells that co-express ATH5 and HES1
(Fig. 4C), an indication that
NGN2 may contribute to the downregulation of HES1 in early neural progenitors,
thereby providing a cellular environment permissive for ATH5
autostimulation.
The upregulation of both NGN2 and ATH5 occurs later in development, around
E6, but by then ATH5 has become the main regulator of its own transcription
(Fig. 6)
(Matter-Sadzinski et al.,
2001). NGN2 occupies the ATH5 promoter similarly at E3 and at E6
(Skowronska-Krawczyk et al.,
2004
), suggesting that it still directly participates in the
control of ATH5 transcription. However, its main contribution to ATH5
expression may occur through other, indirect regulatory pathways. As
ATH5-expressing progenitors exit the cell cycle, NGN2 promotes the expression
first of Neuro M and then of Neuro D
(Novitch et al., 2001
;
Perron et al., 1999
;
Roztocil et al., 1997
) both
stimulators of ATH5 promoter activity
(Matter-Sadzinski et al.,
2001
). These distinct functions of NGN2 in the ontogenesis of RGCs
illustrate how, depending on specific combinations of transcription factors
and of other cellular components, neurogenic proteins may contribute to
neuronal identity.
Molecular interactions between bHLH transcription factors control ATH5 expression during progenitor patterning
How does the retina prevent the differentiation of cells that have
initiated ATH5 expression and preserve an expanding pool of progenitors during
a period of highly dynamic patterning and considerable tissue growth? The
interplay between the molecular mechanisms underlying patterning and those
controlling the rate of RGC differentiation appears to be an integral part of
retina development. Among the different regulatory pathways that are involved,
the transcriptional network regulating ATH5 has a pivotal role. In early
retina, the ATH5 gene is transcribed at a low rate in ATH5-expressing
progenitors and its forced expression initiates the precocious transcription
of ß3 in these cells
(Matter-Sadzinski et al.,
2001), indicating that they are competent for the expression of
this early RGC marker. The appropriate ATH5 dose is controlled through a
complex interplay between positive (NGN2) and negative (HES1) regulators. We
show that individual progenitors co-express HES1 and ATH5, and that HES1
represses the ATH5 promoter, thereby demonstrating that an effector of Notch
helps maintain a low rate of ATH5 transcription during progenitor patterning,
in agreement with the role of Xnotch pathway components in the regulation of
Xath5 function (Schneider et al.,
2001
). The precise dose of HES1 is crucial during this period;
when the level of HES1 is too high expression of ATH5 is suppressed, whereas
the lack of HES1 leads to the precocious differentiation of retinal cells. In
the posterior retina, HES1 is expressed at levels that are high enough to
prevent ATH5 autostimulation, but low enough to allow NGN2-mediated expression
of ATH5 (Fig. 9). As revealed
by overexpression experiments, NGN2 is a potent activator of ATH5 in retinal
progenitors, but its low expression level normally leads to low ATH5 levels.
Moreover, NGN2 is counteracted by ATH5 itself, which acts as a
dominant-negative inhibitor of NGN2 in cells expressing HES1, thereby directly
contributing to the negative control of its own expression. This competition
between NGN2 and ATH5 may occur at the promoter level as both factors bind the
ATH5 promoter in stage 22-23 retinas
(Skowronska-Krawczyk et al.,
2004
) and require the E-boxes E2 and E4 (see Fig. S1 in the
supplementary material). Although HES1 represses the ATH5 promoter and
prevents efficient autostimulation in retinal progenitors
(Fig. 7), it does not prevent
the binding of either NGN2 or ATH5 to the ATH5 promoter. We surmise that ATH5
activity at early stages may be repressed by heterodimerization with HES1, as
reported for other bHLH proteins (Alifragis
et al., 1997
). This mechanism of inhibition is consistent with the
fact that overexpression of ATH5 in retinal progenitors does not overcome
HES1-mediated inhibition of the ATH5 promoter, whereas NGN2 does so in a
dose-dependent manner (Fig. 7).
Thus it appears that a subtle balance and interplay between NGN2, ATH5 and
HES1 is responsible for maintaining ATH5 expression below the level needed to
trigger cell commitment and direct expression of RGC markers.
|
Our results position HES1 as an important prompt at distinct stages in the sequence of events leading to the specification of RGCs. By interacting with the pathways that regulate ATH5 transcription, it prevents high-level ATH5 expression at the time of progenitor patterning, a function essential for establishing and maintaining the pool of ATH5-expressing cells. Its timely downregulation during the last cell cycle releases the autostimulatory activity of the ATH5 protein in a subset of progenitors and thus controls the timing of their commitment and perhaps the size of their pool.
Cells that upregulate ATH5 exit the cell cycle
(Fig. 8) and start expressing
the post-mitotic factor Neuro M. There is a time lag between the
downregulation of HES1 and the expression of Neuro M
(Fig. 4). Overexpression of
NGN2 in early retina leads to the precocious accumulation of ATH5-positive
cells expressing neither HES1 nor Neuro M, and drives these progenitors out of
S phase, presumably in G2 or in G1, until they withdraw from the cell cycle
and start expressing Neuro M. Thus, although the onset of Neuro M expression
coincides with the transient upregulation of NGN2 and ATH5 in the developing
retina (Roztocil et al., 1997;
Matter-Sadzinski et al.,
2001
), it appears not to be regulated directly by these proteins,
a notion supported by the absence of ATH5 and NGN2 binding on the Neuro M
promoter in developing retina
(Skowronska-Krawczyk et al.,
2004
) (D. Skowronska-Krawczyk and J.M.M., unpublished) and by the
finding that induction of Xath3 transcription by Xngn1 requires de novo
protein synthesis (Perron et al.,
1999
). This also suggests that additional factors are required for
Neuro M expression, which may not yet be present in early ATH5-expressing
cells. Overexpression of NGN2 prevents early ATH5 progenitors from re-entering
the S phase but is not sufficient to promote their precocious cell cycle exit.
Likewise, overexpression of Xath5 at early stages of Xenopus
retinogenesis produces extra RGCs that are all born at the appropriate time
(Ohnuma et al., 2002
). Thus,
it is only when the coordinated upregulation of NGN2 and ATH5 coincide with
the build up of cdk inhibitors (Dyer and
Cepko, 2001
; Ohnuma and
Harris, 2003
) that progenitors may leave the cell cycle in G1,
enter G0 and begin expressing Neuro M. Expression of ATH5 remains strong in
newborn RGCs and at this stage Neuro M contributes to its regulation
(Fig. 9).
About two-thirds of ATH5-expressing cells fail to upregulate ATH5
expression and to acquire RGC traits. We reason that although these cells
suppressed HES1, they only accessed their last S phase on E6, too late for
properly upregulating ATH5 and thus missing the time-window when most RGCs are
produced. Cell-fate tracing experiments suggest that they may become other
retinal cell types (Yang et al.,
2003) and the finding that a fraction of progenitors co-express
ATH5 and ASH1 on E6 indicates ongoing alternate fate opportunities
(Fig. 4)
(Matter-Sadzinski et al.,
2001
). Even though all uncommitted ATH5-expressing progenitors are
competent to upregulate ATH5 (Fig.
6C), not all of them can be made to adopt the RGC fate by forced
expression of ATH5 (Liu et al.,
2001
). Overexpression of Xath5 at later stages of Xenopus
retinogenesis does not change the proportion of RGCs and increases the number
of photoreceptor and bipolar cells (Moore
et al., 2002
). It has been proposed that the ability of bHLH
factors to promote the development of distinct retinal neurons depends upon
the timing of their expression and/or function
(Moore et al., 2002
;
Morrow et al., 1999
). If this
was the case, the set of genes regulated by ATH5 would change over time to
comprise genes specific for later-born neurons. Establishing the compendium of
ATH5 transcriptional targets should help answer the question of whether ATH5
is dedicated solely to the production of RGCs or whether it also promotes the
development of other retinal subtypes.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/17/3907/DC1
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