1 Department of Human Anatomy and Cell Science, University of Manitoba,
Winnipeg, Manitoba, R3E 3J7, Canada
2 Department of Pediatrics and Child Health, University of Manitoba, Winnipeg,
Manitoba, R3A 1S1, Canada
3 Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba,
R3E 0V9, Canada
4 Department of Psychiatry, University of California, San Francisco, CA 94143,
USA
5 Department of Ophthalmology, University of Manitoba, Winnipeg, Manitoba, R3E
0V9, Canada
* Author for correspondence (e-mail: eisensta{at}cc.umanitoba.ca)
Accepted 3 November 2004
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SUMMARY |
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Key words: Apoptosis, Brn3b, BrdU birthdating, Chx10, Crx, Dlx1, Dlx2, Homeobox, Mouse, Ocular retardation, Retina
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Introduction |
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The transition from uncommitted multipotent to lineage-restricted
progenitors may be regulated by basic helix-loop-helix (bHLH) transcription
factors (Marquardt and Gruss,
2002). Expression of bHLH genes is controlled by lateral
inhibition through Delta/Notch signaling pathways, resulting in mosaic-like
expression patterns in the retina and other tissues
(Artavanis-Tsakonas et al.,
1999
; Kuroda et al.,
1999
; Fode et al.,
2000
; Marquardt et al.,
2001
). These repressive interactions may result in a heterogeneous
pool of progenitors with distinct retinogenic potentials
(Marquardt and Gruss, 2002
).
Terminal differentiation of these progenitors to particular retinal neurons is
accomplished, in part, through specific sets of transcription factors,
particularly homeobox genes. In the mouse, null mutation of genes encoding
homeodomain transcription factors such as Chx10 and Prox1
has resulted in abnormal retinal morphogenesis and the loss of specific cell
types (Burmeister et al.,
1996
; Dyer et al.,
2003
).
The vertebrate Distal-less (Dlx) homeobox gene family consists of
six known murine members (Panganiban and
Rubenstein, 2002) organized in three bigenic gene clusters
(McGuinness et al., 1996
;
Sumiyama et al., 2002
;
Ghanem et al., 2003
). Four Dlx
family members have been implicated in neurogenesis: Dlx1, Dlx2, Dlx5
and Dlx6 (Bulfone et al.,
1993
; Anderson et al.,
1997a
; Liu et al.,
1997
). Dlx1 and Dlx2 demonstrate similar
expression in the forebrain, and subtle defects in forebrain differentiation
of the Dlx2 single knockout suggest functional redundancy
(Qiu et al., 1995
;
Eisenstat et al., 1999
). Mice,
in which both Dlx1 and Dlx2 have been knocked out, die at
birth and display severe craniofacial (Qiu
et al., 1997
) and central nervous system defects
(Anderson et al., 1997a
;
Anderson et al., 1997b
;
Marin et al., 2000
;
Anderson et al., 2001
). Cells
born after embryonic day (E) 12.5 in the striatum do not fully differentiate
(Anderson et al., 1997a
),
resulting in a loss of migration of GABAergic interneurons to the neocortex
and olfactory bulb (Anderson et al.,
1997b
; Bulfone et al.,
1998
). Dlx1 and Dlx2 are both expressed in the
developing retinal neuroepithelium by E12.5
(Eisenstat et al., 1999
).
Expression of Dlx1 is largely restricted to the ganglion cell layer
(GCL); perinatally its expression is downregulated. Expression of
Dlx2 is maintained throughout the lifetime of the mouse with
expression restricted to RGC, amacrine and horizontal cells
(de Melo et al., 2003
).
In this study we assess the retinal phenotype of the Dlx1/Dlx2 null mouse. We demonstrate a loss of approximately one-third of RGCs in the mutant while other retinal neuronal classes appear unaffected. We further demonstrate that late-born RGCs are dependent on Dlx1 and Dlx2 function for their terminal differentiation, unlike the initial population of RGCs, which properly differentiate and migrate in the absence of Dlx1 and Dlx2. The observed decrease in RGCs in Dlx1/Dlx2 mutants is partly due to increased apoptosis among late-born RGCs.
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Materials and methods |
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Retinal explant cultures
Eyes were dissected from embryos in sterile 1x PBS and transferred to
dishes containing DMEM/F12 media (Gibco/Invitrogen). Retinas were dissected
under sterile conditions from eyes with the lens and iris in situ and
transferred onto Millicell-CM 0.4 µm filters (Millipore) with the lens
facing away from the membrane. Filters were transferred to 6-well culture
plates containing media enriched with 1x N2 supplement, 1x MEM
sodium pyruvate, 2 mmol/l L-glutamine (all Gibco/Invitrogen), 5 µg/ml
insulin (Sigma), and 1 U/ml penicillin/1 mg/ml streptomycin (Sigma). Explants
were cultured at 37°C with 5% CO2 in a humidified incubator for
7 days.
Histological staining, immunofluorescence and combined immunohistochemistry/in-situ hybridization
Tissues stained with Cresyl Violet dye were immersed for 2 minutes and then
transferred through graded alcohol washes before mounting with Permount
(Fisher Chemicals) and coverslips. Immunofluorescence was performed on
cryosections as described (de Melo et al.,
2003). Primary antibodies used were: mouse anti-BrdU (1:200,
Chemicon), rabbit anti-BRN3a (1:100, courtesy of Dr E. Turner), rabbit
anti-BRN3b (1:200, Babco), goat anti-BRN3b (1:200, Santa Cruz), rabbit
anti-calretinin (1:3000, Chemicon), rabbit anti-caspase-3 (1:60, Cell
Signalling Technologies), mouse anti-Chat (1:100, Chemicon), rabbit anti-CHX10
(1:700, courtesy of Dr T. Jessell), rabbit anti-CRALBP (1:250, courtesy of Dr
J. C. Saari), mouse anti-cyclinD1 (1:100, Cell Signalling Technologies),
rabbit anti-DLX1 (1:700), rabbit anti-DLX2 (1:250), rabbit anti-GFAP (1:3000,
DAKO), mouse anti-islet-1 (ISL1) (1:600 Developmental Studies Hybridoma Bank,
University of Iowa), rabbit anti-L1 (1:5000, courtesy of Dr C. F. Lagenaur),
mouse anti-NF165 (1:50, Developmental Studies Hybridoma Bank, University of
Iowa), rabbit anti-phosphohistone H3 (1:1000, Upstate), rabbit anti-PROX1
(1:500, courtesy of Dr M. Nakafuku), rabbit anti-PAX6 (1:800, Sigma), mouse
anti-Rho4D2 (1:80, courtesy of Dr R. Molday), rabbit anti-SIX3 (1:500,
courtesy of Dr G. Oliver), mouse anti-syntaxin (1:6000, Sigma), rabbit
anti-VSX1 (1:10, courtesy of Drs R. L. Chow and R. R. McInnes). Peanut
agglutinin (1:2000, Vector Laboratories) was also used. Secondary antibodies
and fluorescent tertiary molecules used were FITC-conjugated goat anti-rabbit
(1:100, Sigma), Biotin-SP-conjugated goat anti-rabbit (1:200),
Biotin-SP-conjugated goat anti-mouse (1:200), Biotin-SP-conjugated rabbit
anti-goat (1:200, all Jackson ImmunoResearch), Streptavidin conjugated Oregon
Green-488 (1:200, Molecular Probes), and Streptavidin conjugated Texas Red
(1:200, Vector Laboratories). Negative controls omitted the primary antibody.
Non-radioactive in-situ hybridization was performed using digoxigenin-UTP
labeled Crx riboprobes combined with immunohistochemistry utilizing
CHX10 antibodies. The Crx cDNA was obtained from Dr C. Cepko. Single
and combined in-situ hybridization and immunohistochemistry was performed with
sense probe used as controls (Eisenstat et
al., 1999
). TUNEL staining was performed using the In Situ Cell
Death Detection Kit, TMR red (Roche Diagnostics) as per the manufacturer's
instructions.
BrdU labeling and birthdating
Timed pregnant animals were injected with BrdU (5 mg/µl). For pulse
labeling experiments, animals were sacrificed after 1 hour. For birthdating
experiments, animals were sacrificed at E18.5. Sections were treated with 50%
formamide/2x SSC for 2 hours at 65°C, 2x SSC for 5 minutes at
65°C, 2N HCl at 37°C for 30 minutes followed by 0.1 mol/l boric acid
pH 8.5 at RT for 10 minutes.
Cell counting and statistical analysis
For cryosections, pooled counts from a series of matched sections of paired
Dlx1/Dlx2 mutant and wild-type retinas were taken at regularly spaced
intervals to completely survey each retina. Six sets of eyes consisting of one
Dlx1/Dlx2 mutant and one wild-type eye from littermate pairs were
used for quantification at E18.5 (five sets for ISL1, BrdU and phosphohistone
H3 counts). Eyes were sectioned at 12 µm. Sections through the widest
region of the optic nerve head were used as a centered start point and were
matched histologically. The start section and sections 120 and 240 µm above
and below were used for immunohistochemistry. Results from five sections were
pooled to provide a count for each eye. BRN3b+ (Pou4f2 - Mouse Genome
Informatics) cells located in the GCL were counted as RGCs; PAX6+ cells in the
inner neuroblastic layer (NBL) but not the GCL or outer NBL, were counted as
amacrine cells; and NF165+ cells located in the outer NBL were counted as
horizontal cells. Comparisons between sets of count data were made using the
paired t-test to determine statistical significance. For cell death and cell
proliferation counts, sections were immunostained with antibodies to activated
caspase-3. Sections from E13.5 and 16.5 embryos 60 and 120 µm above and
below the start section were used due to smaller eye size. For BrdU
birthdating studies, the proportion of BRN3b-expressing RGCs labeled with BrdU
represents the number of RGCs born at the time of BrdU pulsing.
For retinal explants, sections from each mutant explant were histologically matched with those from a wild-type littermate and then immunoassayed with cell-type specific markers and with DAPI stain (Vector). Total cell numbers/section were determined by counting DAPI+ cells, then immunoreactive cells were counted and proportions were determined.
Morphometry
Six paired sets of littermate Dlx1/Dlx2 mutant and wild-type eyes
were processed. Sections were centered on the thickest region of the optic
nerve head, which was taken as the midpoint. Sections 12, 24 and 36 µm
above and below, including the middle section were immunostained with L1 N-CAM
antibody to visualize the optic nerve. Thickness of the optic nerve head was
measured in three regions (Fig.
3A) using Image-Pro Plus 4.5 software (Media Cybernetics), a mean
thickness was determined and comparisons were made using the paired
t-test.
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Results |
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As Dlx1/Dlx2 mutants die at P0, to determine whether late-born
cell classes were affected, retinal explant cultures were collected at E18.5
and cultured for 7 days (Fig.
2B; see Fig. S1A-H in the supplementary material). Expression of
peanut agglutinin, a marker for cone photoreceptors
(Chen et al., 1994) (see Fig.
S1A,B in the supplementary material), and Rho4D2, a marker for rod
photoreceptors (Davidson et al.,
1994
) (see Fig. S1C,D in the supplementary material), could not
discern any differences between mutant and wild-type tissues. Antibodies to
the transcription factors CHX10 and VSX1 were used to identify rod and cone
bipolar cells, respectively (Chow et al.,
2001
; Hatakeyama et al.,
2001
). No abnormalities in the number or histological placement of
these cells could be detected in the Dlx1/Dlx2 mutants (see Fig.
S1E-H in the supplementary material). No amacrine or horizontal cell
differences could be identified among mutant and wild-type explants consistent
with our findings in the intact E18.5 retina (see Fig. S1I-P in the
supplementary material). Hence, amacrine and horizontal interneurons, which
normally express Dlx1 and/or Dlx2 in the developing retina,
are unaffected by their absence. Finally, we used CRALBP and GFAP expression
as markers for Müller glia
(Bunt-Milam and Saari, 1983
;
Kuhrt et al., 2004
). No
difference was observed between wild-type and mutant retinas (data not shown).
Moreover, there were no significant differences between mutant and wild-type
explants in the proportion of late developing retinal cell classes: rods,
cones, bipolar interneurons or Müller glia
(Fig. 2B). These results
support a specific loss of RGC in the Dlx1/Dlx2 double mutant.
The optic nerve is reduced in Dlx1/Dlx2 mutant mice corresponding to RGC loss
The optic nerve of Dlx1/Dlx2 mutants displayed no gross anatomical
abnormalities. To determine whether the Dlx1/Dlx1 mutants displayed
aberrant optic nerve morphology, we performed morphometric measurements.
Measurements of the thickness of the optic nerve were made at the region where
the nerve exited the retina (optic nerve head) in order to standardize the
region of measurement (Fig. 3A,
arrows). Antibodies to L1 were used to stain unmyelinated axons of the optic
nerve (Bartsch et al., 1989)
(Fig. 3A,B). Measurements
revealed a mean optic nerve thickness of 318.10±59.76 µm in
wild-type animals, while paired mutants had a mean thickness of
244.17±62.20 µm (Fig.
3C), a significant 23% decrease (t=3.99, P<0.005,
n=10).
There is increased apoptosis in developing RGCs in Dlx1/Dlx2 mutants
In order to explain the loss of RGCs, we assessed cellular proliferation
and apoptosis in the retina. BrdU pulse labeling experiments demonstrated no
significant differences between the populations of S-phase cells in mutants
compared with wild-type retinas (see Fig. S3B in the supplementary material).
In addition, studies using an antibody to cyclin D1 (data not shown), a
general proliferation marker (Tong and
Pollard, 2001) and an antibody to phosphohistone H3, an M-phase
marker (Ajiro et al., 1996
),
were unable to identify any differences between mutant and wild-type retinal
proliferation dynamics (see Fig. S3A in the supplementary material).
Antibodies specific to activated caspase-3, an effector caspase, were then
utilized to quantify apoptosis. Mutant retinas displayed significantly
increased numbers of activated caspase-3 positive cells beginning at E13.5, 1
day after DLX1 and DLX2 expression is normally established in the retina
(Eisenstat et al., 1999
).
Mutants at E13.5 displayed a significant 3-fold increase (t=5.96,
P<0.005, n=6) in apoptotic cells
(Fig. 4A, asterisk). Mutant
retinas at E16.5 had a significant 66% increase (t=6.04, P<0.005,
n=6) in activated caspase-3+ cells
(Fig. 4A, cross). However, by
E18.5 the number of apoptotic cells in Dlx1/2 mutants returned to
levels similar to those of wild-type littermates (t=1.81, P>0.05,
n=6) (Fig. 4A).
Virtually identical patterns of apoptosis were yielded by TUNEL assays,
confirming results generated using activated caspase-3 expression (see Fig.
S4A-I in the supplementary material).
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There is a complete loss of late-born RGCs in the Dlx1/Dlx2 double mutant
As the earliest BRN3b-expressing RGCs are established before the onset of
Dlx1 and Dlx2 expression in the developing retina, we
hypothesized that Dlx1/Dlx2 function is required for the
terminal differentiation of a subclass of late-born RGCs. In retinal explants,
there is loss of all pre-existing RGCs within 3 days of culture, due to RGC
axon severance resulting from optic nerve transection during tissue
preparation (Caffé et al.,
1989; Tomita et al.,
1996
). However, the GCL remains with its displaced amacrine cells.
Thus, any detected RGCs in explants cultured beyond 3 days are likely to have
differentiated ex vivo. Retinas were collected at E18.5, as any subsequent
terminally differentiated RGCs could be considered late-born relative to the
total population. Explants were cultured for 7 days to ensure that all
pre-existing RGCs were cleared. In wild-type explants, rare BRN3b expressing
cells could be identified after 7 days of culture
(Fig. 5A, box). These may
represent newly specified RGCs. However, in Dlx1/Dlx2 mutant
explants, BRN3b-expressing cells could not be detected
(Fig. 5B), suggesting that
late-born RGCs are present only in wild-type explants. Subsequently, BrdU
birthdating experiments were performed
(Fig. 5C-T). A single BrdU
pulse was delivered to timed-pregnant animals at E12.5, 13.5, 16.5 and 18.0
and embryos were collected at E18.5. BrdU expression marked cells born on the
date of the BrdU pulse. Co-labeling with BRN3b and BrdU identified RGCs in
mutant and wild-type retinas pulsed with BrdU at E12.5
(Fig. 5C-D,L-N), E13.5
(Fig. 5F-H,O-Q), and E16.5
(Fig. 5R-T,U). However, no
co-labeling was evident in either mutant or wild-type retinas that were BrdU
pulsed at E18.0. Proportions of RGCs born at the time of BrdU pulsing were
determined. RGCs generated at E12.5 formed a significantly larger proportion
of the population in mutant retinas (Fig.
5U, asterisk). However, for RGCs generated at E13.5 and 16.5,
wild-type retinas displayed significantly larger proportions
(Fig. 5U, cross, #). The
difference was more pronounced at E16.5 than E13 (nearly 3-fold versus 60%).
These results support a loss of late-born RGCs in the
Dlx1/Dlx2 double mutant, with early-born RGCs constituting a
larger proportion of the total mutant RGC population compared with
controls.
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Discussion |
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No other retinal cell classes appeared to be affected by the loss of
Dlx1 and Dlx2. Amacrine cells and horizontal cells both
express Dlx2 during development and maintain expression of
Dlx2 in the mature adult retina
(de Melo et al., 2003). Most
amacrine cell subclasses, including those identified by tyrosine hydroxylase,
ChAT, GABA, PROX1 and calretinin immunoreactivity, and virtually all
horizontal cells also express Dlx2
(de Melo et al., 2003
) (J.d.M.
and D.D.E., unpublished). Therefore, we hypothesized that loss of
Dlx1/Dlx2 would play a significant role in the development and
maintenance of these retinal cell types. The extensive role of both
Dlx1 and Dlx2 in interneuron differentiation in the
developing forebrain (Anderson et al.,
1997a
; Anderson et al.,
1997b
) further substantiated this hypothesis. However, the present
study suggests that Dlx1 and/or Dlx2 are not required for
either the generation or differentiation of amacrine or horizontal cells. This
may be due, in part, to redundancy of function with other Dlx genes expressed
in the developing retina, such as Dlx5
(Zhou et al., 2004
). The onset
of Dlx5 expression at approximately E16.5 is several days after the
onset of expression of Dlx1 and Dlx2 (G.D. and D.D.E.,
unpublished). Dlx5 RNA expression is unaffected in the
Dlx1/Dlx2 mutant retina (data not shown). Similar genetic redundancy
is seen among members of the Brn POU domain homeobox gene family.
Brn3a, Brn3b and Brn3c are expressed in RGCs. Brn3b
knockouts display severe RGC loss (Gan et
al., 1996
; Erkman et al.,
1996
), while Brn3c knockout mice do not
(Xiang et al., 1997
).
Brn3b/Brn3c double knockout mice display a more severe RGC phenotype,
suggesting that Brn3c and Brn3b are partially redundant.
Brn3c is sufficient to initiate RGC development even though it is not
required for proper RGC genesis (Wang et
al., 2002
). Analysis of the retinal phenotype of Dlx5
knockout (Levi et al., 2003
;
Long et al., 2003
) and
Dlx5/Dlx6 double knockout mice
(Merlo et al., 2002
;
Robledo et al., 2002
) may
further illuminate the role of Dlx homeobox genes in amacrine and horizontal
cell development.
Dlx1 and Dlx2 function is necessary for the differentiation of late-born RGCs
As Dlx1/Dlx2 mutants die at birth, we established explant cultures
to study postnatal retinal differentiation. All RGCs generated prior to
establishment of the cultures were lost due to transection of the optic nerve.
Newly specified RGCs, while not abundant, were detected in explants from
wild-type retinas but not in mutant explants. We suspected that the inability
to detect RGCs in the Dlx1/Dlx2 mutants was due to a failure of RGC
terminal differentiation and/or survival during tissue culture, suggesting
that late-born RGCs require Dlx1 and Dlx2. Since cultures
were established from E18.5 retinas, all RGCs generated could be considered
late-born relative to the total birthdate distribution of RGCs. BrdU
birthdating experiments at select stages of embryogenesis labeled neurons
undergoing their final S-phase and exit from the cell cycle. Co-labeling with
BRN3b, a specific marker for RGCs, allowed us to identify RGCs born on the day
of the BrdU pulse. Dlx1/Dlx2 mutants contained a greater proportion
of RGCs born before E13.5, whereas late-born RGCs were more prevalent in
wild-type retinas. Hence, late-born RGCs may fail to terminally differentiate
due to a requirement for Dlx1 and/or Dlx2. These cells are
lost by apoptosis, as we have shown using activated caspase-3 and TUNEL
assays. This loss of late-born RGCs results in early-born RGCs comprising a
greater proportion of the RGC population in Dlx1/Dlx2 mutants.
Brn3b expression is established well before the onset of
Dlx1 and Dlx2 in the retinal neuroepithelium. However, at
birth, all BRN3b-expressing cells co-express DLX2
(de Melo et al., 2003). We
suggest that the lost late-born RGCs in the Dlx1/Dlx2 mutants are
Dlx-dependent and may not require Brn3b, although expression of BRN3b
protein is established later as these neurons develop
(de Melo et al., 2003
). Of
significance, Brn3b knockout mice lose approximately 70-80% of RGCs
(Erkman et al., 1996
;
Gan et al., 1996
;
Lin et al., 2004
), leaving
20-30% of RGCs specified through alternative mechanisms. Of interest, there is
increased Dlx1 and Dlx2 expression in Brn3b null
mice (Mu et al., 2004
). This
compensatory increase in Dlx gene expression may be required for
differentiation of the remaining RGC pool in the BRN3b mutant. In the
Dlx1/Dlx2 mutants, one-third of RGCs are lost, indicating that nearly
70% of RGCs develop unhindered by the loss of Dlx1/Dlx2. We suggest
that the surviving RGC population in Brn3b knockout mice may comprise
late-born RGCs with terminal differentiation that requires Dlx gene
expression, while surviving RGCs in the Dlx1/Dlx2 knockouts are
Dlx-independent (refer to model, Fig.
9). Dlx-dependent RGCs may derive from distinct retinal progenitor
pools as specified by bHLH genes. The bHLH transcription factor Math5
(Atoh7 - Mouse Genome Informatics) identifies a subpopulation of
retinal progenitors, in which Brn3b expression commits cells to an
RGC fate (Liu et al., 2001
;
Wang et al., 2001
;
Yang et al., 2003
). By
contrast, Dlx1/Dlx2-expressing lineages originate from progenitor
pools defined by expression of the bHLH gene Mash1 in the developing
central nervous system (Cassarosa et al., 1999;
Andrews et al., 2002
;
Letinic et al., 2002
;
Yun et al., 2002
). However,
the genetic interaction between Dlx genes and Mash1 remains to be
defined in the developing retina.
|
RGCs undergo increased apoptosis in the Dlx1/Dlx2 mutant retina
Increased and ectopic Crx expression in the Dlx1/Dlx2
mutant suggests that upon loss of Dlx1 and/or Dlx2, some
retinal progenitors may commit to photoreceptor differentiation pathways as an
alternative to cell death. Math5 mutant mice feature an absence of
RGCs and an increase in the number of cone photoreceptors, possibly due to a
binary fate switch (Brown et al.,
2001). A similar cell fate switch may partly explain the decrease
in RGCs in the Dlx1/Dlx2 mutant retina. Unlike Math5
mutants, the Dlx1/Dlx2 mutant is not viable beyond birth. As a
consequence, we cannot characterize photoreceptors in a mature retina.
However, in explant cultures no significant differences were determined when
quantifying rods and cones. Hence, aberrant Crx expression in the
Dlx1/Dlx2 mutant may be transient or may result indirectly from a
loss of Dlx1 and/or Dlx2 function.
In the Dlx1/Dlx2 mutants there were increased apoptotic cells. We
attribute this increase in cell death to a loss of late-born RGCs that require
Dlx1/Dlx2 expression for their terminal differentiation. The
utilization of caspase-mediated apoptotic pathways in the modulation of RGC
number has been demonstrated in chick
(Mayordomo et al., 2003). Our
results suggest that similar mechanisms are involved in the clearance of RGCs
with incomplete differentiation. Failure of RGC development in the
Dlx1/Dlx2 mutants may be due to several mechanisms. Interestingly,
these cells express BRN3b protein at the time of death, suggesting that
lethality results from a failure of later developmental processes. The direct
transcriptional downstream targets of Dlx1 and Dlx2 remain
largely undefined except for the Dlx5/Dlx6 intergenic enhancer
(Zerucha et al., 2000
;
Zhou et al., 2004
). Regulation
of survival factors and/or apoptosis may be mechanisms by which specific
retinal neuronal classes are maintained. Characterization of the genetic
networks regulated by Dlx1 and Dlx2 presents a challenging
direction to further define the role of Dlx genes in the developing
retina.
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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/132/2/311/DC1
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