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First published online 23 April 2008
doi: 10.1242/dev.010751
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1 Department of Ophthalmology, University of Rochester, Rochester, NY 14642,
USA.
2 Center for Neural Development and Disease, University of Rochester, Rochester,
NY 14642, USA.
3 Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY
14642, USA.
* Author for correspondence (e-mail: lin_gan{at}urmc.rochester.edu)
Accepted 31 March 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: LIM-homeodomain, POU domain, MATH5, ATOH7, POU4F2, RGC, Retinal development, Transcription factor
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Young,
1985). During RGC development, the basic helix-loop-helix (bHLH)
transcription factor (TF), MATH5 (ATOH7 - Mouse Genome Informatics), is
expressed in post-mitotic retinal precursors and provides them with the
competency to become RGCs (Yang et al.,
2003). MATH5 regulates the expression of RGC-specific
differentiating TFs such as the POU-homeodomain (POU-HD) factor BRN3B (POU4F2
- Mouse Genome Informatics) and the LIM-homeodomain (LIM-HD) factor ISL1
(Wang et al., 2001;
Yang et al., 2003). In
Math5-deficient mice, fewer than 5% of RGCs are generated
(Brown et al., 2001;
Wang et al., 2001). In
contrast to the early function of MATH5 in RGC genesis, BRN3B expression
starts in nascent RGCs and is required for their terminal differentiation,
including axon outgrowth and pathfinding, and for cell survival
(Erkman et al., 2000;
Gan et al., 1999;
Pan et al., 2005;
Xiang, 1998). In
Brn3b-null mice, RGCs are initially generated but 80% of them undergo
apoptosis before birth (Erkman et al.,
1996; Gan et al.,
1996). Moreover, the compound mutation of Brn3b and
Brn3c (Pou4f3 - Mouse Genome Informatics) results in the
further reduction of RGCs in adult mice
(Wang et al., 2002a). Although
previous studies have shown clearly the importance of BRN3 factors in RGC
development, it remains poorly understood how RGC differentiation is regulated
and whether other TFs control this process in parallel to BRN3 factors.
Studies of CNS development in both vertebrates and invertebrates have shown
that TFs, often acting in distinct combinatorial manner, play important roles
during neurogenesis (Bang and Goulding,
1996; Castro et al.,
2006; Lee and Pfaff,
2001). The POU-HD and LIM-HD TFs appear to function primarily at
the later stages of neurogenesis. For example, in the spinal cord, the unique
combinatorial expression of LIM-HD TFs confers motoneuron subtypes with
specific axon targeting pathways (Appel et
al., 1995; Kania et al.,
2000; Sharma et al.,
2000; Sharma et al.,
1998; Tsuchida et al.,
1994). BRN3B is required in the development of RGCs (discussed
above). In Drosophila, Acj6 and Drifter (Vvl - FlyBase), both POU-HD
TFs, are required for the dendritic targeting in olfactory system
(Komiyama et al., 2003).
Moreover, LIM-HD and POU-HD TFs have been shown to cooperate in regulating
neuronal differentiation. One such example is the regulation of touch receptor
differentiation in C. elegans by POU-HD factor UNC-86 and LIM-HD
factor MEC-3. UNC-86 dimerizes with MEC-3 on the mec-3 promoter,
which is required for the maintenance of mec-3 expression and touch
cell differentiation (Lichtsteiner and
Tjian, 1995; Xue et al.,
1993). UNC-86 and MEC-3 also synergistically activate the touch
cell-specific genes mec-4 and mec-7
(Duggan et al., 1998).
ISL1, one of the founding members of LIM-HD TFs in the Islet subgroup, has
been intensively studied in the spinal cord. ISL1 is expressed in all
motoneurons immediately after they exit cell cycle and is essential for the
genesis of motoneurons (Pfaff et al.,
1996). By contrast, Drosophila Islet is not required for
the genesis of motoneurons, but for the axonal trajectory selection and
neurotransmitter expression (Thor and
Thomas, 1997). Here, we demonstrate the co-expression of BRN3B and
ISL1 in post-mitotic, differentiating RGCs. To investigate the role of ISL1 in
RGC development, we generate Isl1-lacZ knock-in and Isl1
conditional knockout mice. We demonstrate that in Isl1-null retinas,
RGCs appear to be generated normally but 67% RGCs subsequently undergo
apoptosis and RGC axon growth is defective, a phenotype strongly resembling
that of Brn3b mutants (Erkman et
al., 2000; Gan et al.,
1999). In Isl1 and Brn3b double null retinas,
greater than 95% nascent RGCs die of apoptosis, suggesting their cooperative
relationship in RGC development. Furthermore, chromatin immunoprecipitation
(ChIP) and in vitro transactivation assays demonstrate that both factors bind
to and regulate the expression of RGC-specific genes. Our data strongly argue
for the involvement of both parallel and cooperative functions of ISL1 and
BRN3B in RGC development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gan et al., 1999).
Isl2lacZ knock-in mice (L. Gan, unpublished) were
generated by replacing all the Isl2-coding sequences with a
lacZ-SV40 polyA-Neo cassette. The Isl1lacZ
targeting construct was generated by inserting the Isl1 2.6 kb
5'- and 4.2 kb 3'-flanking sequences into the 5' and
3' multiple cloning sites of PKII-lacZ vector (L. Gan, unpublished),
where a cassette with lacZ-SV40 polyA-Neo replaced Isl1 exon
1-2 and adjacent intron sequences. To make the Isl1CKO
targeting construct, we inserted a neomycin resistance gene along with a loxP
site at the 5' of exon 2, and another loxP site at the 3' of exon
2. The targeting vectors were electroporated into W4 mouse embryonic stem
cells (Auerbach et al., 2000)
to generate Isl1lacZ/+ and Isl1CKO/+
mice. Isl1loxP/+ mice were obtained by crossing
Isl1CKO/+ mice with the ROSA26-FLPe mice (The Jackson
Laboratory, Stock Number: 003946) to remove FRT-flanked neomycin resistance
gene. The PCR genotyping of these animals was performed as follows: 5'-AGGGCCGCAAGAAAACTATCC and 5'-ACTTCGGCACCTTACGC TTCTTCT to detect a 404 bp product of Isl1lacZ allele; 5'-GGTGCTTAGCGGTGATTTCCTC and 5'-GCACTTTGGGATGGTAATTGGAG to detect a 452 bp product of WT Isl1 allele and a 512 bp product of Isl1loxP allele; and 5'-GTGGAATCGCTGAATCTTGAC and 5'-GCCCAAATGTTGCTGGATAGT to detect Six3-cre allele. The mouse strains were maintained in the C57BL/6J and 129S6 mixed background. Embryonic day 0.5 (E0.5) was defined as the day when the vaginal plug appeared. University Committee of Animal Resources at the University of Rochester approved all animal procedures.
Hematoxylin and Eosin (H&E) staining, immunohistochemistry, X-Gal staining and in situ hybridization
Embryos were harvested from E11.5 to E18.5, decapitated and fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS) for several hours and were
processed for paraffin sections or cryosections. Horizontal retina sections
across optic disc collected from controls and mutant littermates were mounted
side by side for comparisons. BrdU labeling, H&E and X-Gal staining were
carried out as previously reported (Pan et
al., 2005). The primary antibodies used in immunohistochemistry
were: mouse anti-BRN3A (POU4F1 - Mouse Genome Informatics) (Santa Cruz,
1:200), goat anti-BRN3B (Santa Cruz, 1:200), mouse anti-ISL1 (Developmental
Studies Hybridoma Bank, 1:200), mouse anti-bromodeoxyuridine (BrdU) (Becton
Dickinson, 1:400), rabbit anti-phosphorylated histone 3 (Santa Cruz, 1:400),
mouse anti-SMI32 (Sternberger Monoclonals, 1:1000) and rabbit anti-activated
caspase 3 (R&D Systems, 1:100). The Alexa-conjugated secondary antibodies
(Molecular Probes) were used at 1:1000 dilution. Non-radioactive in situ
hybridization was performed using digoxigenin-UTP labeled riboprobes
(Radde-Gallwitz et al., 2004).
The specific cDNA sequences used to generate riboprobes were: Brn3a
(3'UTR); Olf1 (L12147, nucleotides 865-1180); Irx4
(NM_018885, nucleotides 1556-2265); Ablim1 (AF316037, nucleotides
12-31); L1cam (NM_008478, nucleotides 3083-3748). Isl1 and
Isl2 probes were described previously
(Yang et al., 2003). The
Shh probe was a generous gift from Dr Valerie A. Wallace
(Jensen and Wallace, 1997).
Confocal images were acquired on an Olympus microscope (BX50WI) with Fluoview
4.3 laser scanning. Other pictures were taken with a Nikon Eclipse TE2000-U
inverted microscope with a Nikon DXM1200F digital camera.
Cell counts and statistical analysis
For apoptosis analysis, five pairs of matched retina sections of
Isl1-null and littermate controls were collected at regularly spaced
intervals to completely survey each retina. After anti-activated caspase 3
immunolabeling, images were taken and the immunoreactive cells were counted
with Image J program (NIH). Results from five sections were averaged to obtain
the apoptotic cell number for each eye. For analyses of BRN3A+ or BRN3B+
cells, whole-mount retinas were used. In these cases, three pictures were
obtained from the central region of the retinas. The immunoreactive cells were
counted and averaged. To compare the optic nerve size, three pairs of matched
cross-sections of null and control optic nerves were collected and processed
for H&E staining. The boundary of each optic nerve was outlined using
Adobe Photoshop. The size of optic nerve was determined by measuring the
number of pixels contained in the outlined area.
Lipophilic dye tracing
For anterograde labeling of the optic pathway, mouse heads at E13.5 and
E15.5 were fixed overnight in 4% paraformaldehyde in PBS. After enucleation of
the right eye, DiI crystals (Molecular Probes) were implanted unilaterally in
the optic disc. After incubation in PBS containing 0.1% sodium azide at
37°C for 1-2 weeks, the brains were dissected to expose the optic chiasm
and visualized under a Nikon SMZ1500 fluorescent stereomicroscope.
Chromatin immunoprecipitation (ChIP)
Chromatin from retinas at indicated stages was collected according to the
protocol supplied with the ChIP assay kit (Upstate Biotechnologies). Mouse
anti-ISL1 and goat anti-BRN3B were used for immunoprecipitation. Promoter
regions with BRN3-binding consensus sequences were detected in the
precipitated material by PCR (primer set details can be provided on request).
Brn3b ORF was used as a negative control.
Luciferase activity assay
CV1 epithelial cells were cultured in 24-well plates in DMEM with 10% FBS.
Transfections were carried out with Lipofectamine 2000 (Invitrogen) when cells
reached 70% confluence. Brn3b expression plasmid and
Brn3a-luciferase reporter construct were generous gifts from Dr Eric
Turner (Trieu et al., 2003).
Isl1 expression plasmid was generated by inserting Isl1 cDNA
into pcDNA expression vector (Invitrogen). For each transfection, 100 ng of
Isl1 and/or Brn3b expression plasmid, 200 ng of
Brn3a-luciferase reporter construct and 5 ng of PRL
(Promega) Renilla luciferase control plasmid were used. The total
amount of DNA was balanced by adding empty pcDNA vector. Cells were harvested
36 hours after transfection and luciferase activity was measured with the
Dual-Luciferase Reporter Assay System (Promega). The firefly luciferase
activity was normalized by renilla luciferase activity.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Elshatory et al., 2007b;
Galli-Resta et al., 1997).
Anti-ISL1 is raised against the C-terminal of ISL1 homeodomain and reacts to
ISL1 specifically but also recognizes ISL2 with a lower affinity
(Tanabe et al., 1998). In
mouse, weak ISL2 expression is first detected in a very few RGCs in the
central GCL at E13.5 and in about 30% RGCs postnatally (see Fig. S1A-D in the
supplementary material) (Brown et al.,
2000; Pak et al.,
2004). Targeted deletion of Isl2 reveals that
Isl2 is not necessary for the generation and survival of RGCs
(Pak et al., 2004). To confirm
that the above anti-ISL1 labeling reveals the true Isl1 expression pattern, we
compared the ISL1-immunostaining in wild-type (WT) and Isl2-null
retinas at E13.5 to E15.5 and found no difference in the labeling patterns
(Fig. 1D-F, data not shown; see
Fig. S1E-J in the supplementary material). Thus, at E11.5 to E15.5, all cells
labeled with anti-ISL1 in the retina represent ISL1-expressing cells.
|
The early expression of ISL1 in nascent RGCs and its co-localization with
BRN3B suggested that ISL1 could function in parallel to BRN3B or immediately
upstream or downstream of BRN3B during RGC development. To distinguish these
possibilities, we analyzed the expression of ISL1 in Math5-null and
Brn3b-null mice. In Math5-null retinas at E13.5, the
expression of ISL1 and BRN3B was dramatically decreased
(Fig. 1M,N,P,Q), consistent
with our previous finding that Brn3b and Isl1 are downstream
genes of Math5 (Wang et al.,
2001; Yang et al.,
2003). By contrast, we observed no discernible changes in
Isl1 expression in Brn3b-null retinas at E14.5
(Fig. 1O,R), a stage before the
onset of RGC death caused by the absence of Brn3b
(Gan et al., 1999). Therefore,
Isl1 probably functions upstream of or in parallel to Brn3b
during RGC development.
Targeted disruption of Isl1 in retina
Conventional Isl1 knockout mice do not survive beyond E11.5,
probably owing to the failure in vascular development
(Pfaff et al., 1996). To
assess the role of ISL1 in RGC development that occurs during mid- to late
gestation stages, we generated an Isl1 conditional knockout allele
(Isl1loxP) by flanking exon 2, which encodes the first LIM
domain, with loxP sequences (Fig.
2A). Cre recombinase-mediated deletion of the loxP-flanked exon 2
resulted in a null mutation via a reading frame shift. Additionally, a
lacZ knock-in allele, Isl1lacZ, was created by
replacing the exon 2 with a nuclear lacZ reporter gene
(Fig. 2B). The genotypes of
Isl1 mutant mice were confirmed by Southern blotting and PCR
(Fig. 2C). The expression of
β-galactosidase in Isl1lacZ/+ mice faithfully
recapitulated the pattern of endogenous Isl1 as shown by in situ
hybridization and immunostaining (see Fig. S2 in the supplementary material)
and, thus, served as an excellent marker of Isl1-expressing
cells.
Retina-specific removal of Isl1 was achieved by breeding
heterozygous Isl1lacZ/+ or Isl1loxP/+
with Six3-cre mice and subsequently crossing with
Isl1loxP/loxP mice. Six3-cre mice express Cre
recombinase in the eye field and the ventral forebrain from E9 to E9.5
(Furuta et al., 2000), and
have been used successfully as an effective retina-specific deleter
(Mu et al., 2005b). In our
experiments, we observed consistently a greater than 90% deletion of Isl1
in Isl1loxP/lacZ; Six3-cre and Isl1loxP/loxP;
Six3-cre retinas at E13.5 (Fig.
2D; data not shown). In the following experiments,
Isl1loxP/lacZ; Six3-cre and Isl1loxP/loxP;
Six3-cre mice were used interchangeably as Isl1 nulls.
Isl1loxP/+, Isl1loxP/loxP and wild-type mice
were phenotypically indistinguishable and were designated as controls
hereafter. The Isl1 nulls were born at Mendelian frequencies (26%,
n=96) with no overt morphological defects, but exhibited moderate
growth retardation postnatally. Their body weights were about 91% of their
littermate controls at P10 (n=7 pairs) and about 80% at 6 weeks
(n=5 pairs).
Major loss of RGCs in the absence of Isl1
To assess the importance of ISL1 during RGC development, we analyzed BRN3B
expression in Isl1-null retinas at different embryonic stages
(Fig. 3A-H). At E13.5 and
E15.5, the peak period of RGC generation, there was no substantial difference
in the number and distribution of BRN3B+ RGCs in Isl1-null retinas
compared with controls (Fig.
3A,B,E,F), indicating that Isl1 is dispensable for the
generation and migration of RGCs and for the onset of BRN3B expression.
However, BRN3B+ RGCs were drastically reduced in the absence of Isl1
at E17.5 (Fig. 3C,G). This
reduction progressed in a central-to-peripheral wave and was most evident at
E18.5 (Fig. 3D,H).
|
;
Perry et al., 1983).
Consistently, we detected few apoptotic cells in wild-type retinas throughout
embryogenesis. In Isl1-null retinas, although there was no
significant change in the number of apoptotic cells during the peak of RGC
genesis at E13.5 to E15.5, a significant increase in the number of apoptotic
cells was detected from E16.5 (2.8-fold of the wild-type level at E16.5,
t-test P<0.01; 5.3-fold at E17.5, P<0.01; and
2.6 fold at E18.5, P<0.05, Fig.
3K). The apoptotic cells were primarily detected in the GCL
(Fig. 3I,J) and the timing of
increased apoptosis corresponded to the massive loss of BRN3B+ RGCs.
To quantify RGC loss as a result of Isl1-null mutation, we
performed whole-mount immunostaining of adult retinas using anti-BRN3A and
BRN3B antibodies (Fig. 4A-E).
In adult retinas, BRN3A and BRN3B are each expressed in 
70% of RGCs in a
partially overlapping pattern and their combined expression reveals nearly the
entire RGC population (Xiang et al.,
1995). In Isl1-null retinas, both BRN3A+ and BRN3B+ cells
were reduced to about 33% of those in wild type. To rule out the possibility
that this reduction was due to the downregulation of BRN3A and BRN3B, rather
than the loss of RGCs, we also analyzed RGC axons as another parameter of RGC
number. SMI32 antibody recognizes a non-phosphorylated epitope on the
neurofilament H-chain and specifically labels large RGCs and their nerve
fibers (Nixon et al., 1989).
In Isl1-null retinas, the number of SMI32+ axon bundles was
significantly reduced and the remaining ones appeared to be less fasciculated
(Fig. 4F,G). Consistently, the
ventral view of mouse brains revealed that Isl1 nulls have much
thinner optic nerves, optic chiasms and optic tracts
(Fig. 4H,I). Quantification
analysis showed a 69% reduction in the cross-section area of optic nerves in
Isl1 nulls. These data indicate that ISL1 is required for RGC
survival but not for the generation and migration of RGCs or for the
initiation of BRN3B expression in retinas.
ISL1 and BRN3B regulate common downstream target genes
The co-expression of ISL1 and BRN3B raises the possibility that they
regulate a common set of downstream genes in differentiating RGCs. To test
this possibility, we examined retinas at E14.5, a time point at which neither
the reduction of BRN3B expression nor the progressive RGC apoptosis has
occurred. BRN3A, ISL2, IRX4, and OLF1 (EBF1 - Mouse Genome Informatics) are
TFs whose expression immediately follows that of BRN3B during retinal
development. BRN3A is expressed in the post-migrated RGCs starting from E12.5
and is thought to play a redundant role with BRN3B
(Pan et al., 2005). ISL2 is
selectively expressed in one-third of contralaterally projecting RGCs and
represses the ipsilateral targeting program
(Pak et al., 2004). IRX4 is
involved in intra-retina pathfinding of RGCs
(Jin et al., 2003). OLF1, with
an early onset of retinal expression from E12.5
(Xiang, 1998), has no
identified function in RGC differentiation. ABLIM1, GAP43 and L1CAM all play
crucial roles in RGC axon growth and pathfinding by mediating cytoskeleton
changes or cell-cell interactions
(Demyanenko and Maness, 2003;
Erkman et al., 2000;
Suh et al., 2004;
Zhang et al., 2000). SHH is
secreted by differentiated RGCs and negatively regulates the differentiation
of retinal progenitors into RGCs (Wang et
al., 2005; Wang et al.,
2002b; Zhang and Yang,
2001). When compared with the controls, the expression of the
above genes in the GCL was reduced in mice lacking either Isl1 or
Brn3b (Fig. 5; see
Table S1 in the supplementary material). Interestingly, we also observed that
the reduction in the expression of Brn3a, Olf1 and Ablim1
was less severe in Isl1 null than in Brn3b null retinas
(Fig. 5A,C,E). By contrast, a
more significant decrease in Isl2 expression was seen in
Isl1-null retinas (Fig.
5B). The expression analysis presented here not only supports a
direct role of ISL1 in RGC differentiation but also suggests that ISL1 and
BRN3B probably function cooperatively through regulating the common downstream
target genes.
|
|
), our
analysis revealed that in the wild-type embryos at E13.5, a large proportion
of RGC axons already passed the midline of the optic chiasm and extended in
the contralateral optic tract. At the same time, a sizeable proportion of
axons turned away from the midline and proceeded in the ipsilateral optic
tract (Fig. 6A). However, in
Isl1 nulls and Brn3b nulls
(Fig. 6B,C), most axons failed
to reach the midline at E13.5. At E15.5, the optic chiasm structure in
wild-type mice was well defined and resembled its adult-like morphology
(Fig. 6D). In both
Isl1 nulls and Brn3b nulls, though RGC axons projected into
the contra- and ipsilateral optic tracts, several pathfinding defects were
apparent (Fig. 6E,F). First,
the optic nerves were smaller, indicating that a substantial fraction of RGCs
failed to send out axons or that the axons did not exit the optic disc.
Second, the axons in the optic tracts were less fasciculated and appeared to
be grouped into two bundles (Fig.
6E,F, arrows). These observations are consistent with the
recognized function of BRN3B in RGC axon growth and pathfinding
(Erkman et al., 2000;
Gan et al., 1999), and the
similar roles of ISL1 in the projections of spinal motoneuron and sensory
neuron axons (Kania and Jessell,
2003; Segawa et al.,
2001; Thor and Thomas,
1997). Taken together, our data suggest that the axon growth
defects occur prior to the onset of RGC apoptosis and may contribute to RGC
death in both mutants.
|
|
; Lin et al.,
2004; Wang et al.,
2001). However, unlike Math5-null mutation, loss of both
Isl1 and Brn3b did not affect the genesis of RGCs. X-Gal
staining of E13.5 retina sections revealed the comparable number of
Brn3b-lacZ-labeled nascent RGCs in the NBL and GCL of
control, Brn3b-null and Isl1/Brn3b-null mice
(Fig. 7M-O). Taken together,
these data imply that Isl1 and Brn3b function together
downstream of Math5 to control the terminal differentiation but not
the genesis of almost all RGCs.
Simultaneous binding of ISL1 and BRN3B to RGC-specific promoters
To investigate whether the cooperative function of ISL1 and BRN3B during
RGC development was mediated by their direct regulation of RGC-specific genes,
we explored the co-occupancy of these two factors on RGC-specific promoters in
vivo with ChIP assays. The BRN3-binding site, ATNA(A/T)T(T/A)AT
(Gruber et al., 1997;
Trieu et al., 2003), was found
in many genes expressed in RGCs. We examined four of them, Brn3b, Shh,
Brn3a and Isl2, to determine whether they are directly regulated
by both ISL1 and BRN3B. Besides Brn3b, these genes were chosen
because their expression in RGCs immediately follows that of ISL1 and BRN3B
(Quina et al., 2005;
Wang et al., 2005;
Xiang, 1998) and depends on
ISL1 and BRN3B (Fig. 5). We
immunoprecipitated the chromatin from wild-type retinas at E13.5-14.5 and
PCR-amplified the promoter regions containing BRN3-binding consensus sequence.
Both anti-ISL1 and anti-BRN3B antibodies co-precipitated with the promoter
sequences of Brn3b, Shh, Brn3a and Isl2
(Fig. 8A and Fig. S3 in the
supplementary material). Neither of these antibodies precipitated with
Brn3b-coding sequences in the controls
(Fig. 8A). To further sustain
the specificity of our assays, we also incorporate several negative controls,
including IgG-precipitation, anti-BRN3B precipitation of chromatin derived
from Brn3b-null retinas and anti-ISL1 precipitation with cerebellum
tissues where ISL1 was not expressed. (Fig.
8A; see Fig. S3 in the supplementary material). Moreover,
anti-ISL1 was not able to co-precipitate with these promoters in
Brn3b-null retinas, implying that the binding of ISL1 to these
promoters depends on BRN3B.
|
) and
co-transfected CV1 cells with Brn3a-luciferase reporter and
Isl1 and/or Brn3b expression plasmids. Although BRN3B alone
activated Brn3a-luciferase expression to 2.34-fold of the control
level (t-test P<0.001) and ISL1 had no effect on
Brn3a-luciferase expression (1.01-fold, P>0.05),
co-expression of ISL1 and BRN3B activated Brn3a-luciferase expression
by 5.07 fold (P<0.001, Fig.
8B). Thus, ISL1 and BRN3B simultaneously bind to and
synergistically regulate the expression of their target genes. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lee and Pfaff,
2003; Shirasaki and Pfaff,
2002). Among these TFs, the POU-HD and LIM-HD TFs are often
co-expressed in the same differentiating neurons and play essential roles
during late stage of the neuronal differentiation in both invertebrates and
vertebrates. In this study, we use neural retina to explore the functional
mechanism of LIM-HD and POU-HD TFs during neurogenesis. Using retina-specific
knockout of Isl1, we show that similar to those in
Brn3b-null retinas, a majority of RGCs in Isl1-nulls are
defective in axon growth and pathfinding, and die prenatally. Moreover, a loss
of greater than 95% RGCs in Isl1/Brn3b nulls, the necessity of both
ISL1 and BRN3B for the expression of common downstream genes, and their
simultaneous binding to and synergistic regulation of RGC-specific genes
demonstrate the parallel and cooperative nature of ISL1 and BRN3B function in
RGC development.
Expression and function of ISL1 in RGC development
The onset of ISL1 expression in post-mitotic cells at E11.5 and its
complete co-localization with BRN3B in nascent RGCs suggest a role for ISL1 in
RGC development. We found that targeted disruption of Isl1 does not
affect the genesis of RGCs. Rather, it results in the apoptosis of a
significant number of RGCs (Fig.
3). The observed RGC defect in Isl1-null retinas is
consistent with the spatiotemporal expression of ISL1 in nascent RGCs and
supports its role in the late stages of RGC development. In addition to RGCs,
ISL1 is also expressed in developing cholinergic amacrine and ON-bipolar cells
in mouse retina (Elshatory et al.,
2007a). In a separate study, we have examined the effect of the
Isl1-null mutation on these other two types of retinal neurons and
found the postnatal loss of nearly all these cells in the absence of
Isl1 (Elshatory et al.,
2007b), further supporting its essential role in the late stages
of retinal neurogenesis.
In normal retina development, about half of RGCs die during the first
postnatal week owing to the deprivation of target-derived neurotrophins as the
result of axon mis-projection (Farah and
Easter, 2005; Perry et al.,
1983). It is possible that the massive RGC death in
Isl1-null or Brn3b-null retinas could also result from a
similar mechanism, based on their recognized roles in axon guidance. In
Drosophila, Islet is required for proper axon trajectory of spinal
motoneurons (Thor and Thomas,
1997). Mis-expression of Isl1 in chicken also causes axon
targeting errors of the spinal motoneurons
(Kania and Jessell, 2003).
Moreover, mis-guidance of RGC axons at multiple decision-making points has
been reported in Brn3b-null mutants
(Erkman et al., 2000). In this
study, we show that the defect in RGC axons arises prior to RGC apoptosis in
Isl1 nulls or Brn3b nulls, suggesting that the defect in
axon outgrowth and/or targeting contribute to the excessive apoptosis of RGCs.
Interestingly, in Isl1- null or Brn3b-null retinas, there is
a significant downregulation of genes implicated in axon growth
(Gap43), fasciculation (L1cam) and guidance (Isl2,
Ablim1) (Fig. 5).
Furthermore, DiI anterograde tracing experiments reveal that at E13.5, a
significant amount of pioneer RGC axons fail to reach the midline in
Isl1 nulls or Brn3b nulls
(Fig. 6). At E15.5, although
the optic chiasm does form in these null mutants, it is always associated with
axon growth and fasciculation defects.
|
). Taken
together, these data suggest that ISL1 and BRN3B co-bind to Brn3b
promoter directly and maintain Brn3b expression in retina. The
continuous expression of Brn3b then maintains the survival of
developing RGCs. Alternatively, ISL1 could directly control the terminal differentiation and survival of RGCs by regulating the expression of genes essential for these processes. Supporting this hypothesis are our findings that genes with expression in differentiating RGCs are significantly downregulated in Isl1-null retinas before the reduction of BRN3B expression and the initiation of RGC apoptosis (Fig. 5). Moreover, inconsistent with the reduced Shh expression and its role as a retinal progenitor mitogen, there is a slight decrease of M-phase retinal progenitors in Isl1-null retinas (see Fig. S4 in the supplementary material). The expression of these RGC genes is also diminished in Brn3b nulls, suggesting that these two TFs regulate a common set of downstream target genes. Interestingly, we have also noticed that the extent of downregulation of certain RGC genes differs in Isl1 nulls than in Brn3b nulls. For example, the expression of Brn3a, Olf1 and Ablim1 is reduced more severely in Brn3b nulls, while Isl2 expression is more significantly downregulated in Isl1 nulls (Fig. 5), suggesting the expression of these genes could have different dependency on ISL1 than on BRN3B. BRN3B alone is probably sufficient to maintain the expression of Brn3a, Olf1 and Ablim1 in certain RGCs, whereas ISL1 is essential for the expression of Isl2. The resolution of this different dependency awaits the future transcriptional regulation analysis of these genes.
ISL1 and BRN3B co-regulate the RGC differentiation program
Our data indicate that ISL1 and BRN3B simultaneously bind to the promoter
regions of RGC-specific genes and synergistically regulate their expression
during RGC differentiation. This finding is consistent with prior studies of
the cooperative function of POU-HD and LIM-HD factors. The POU-HD factor PIT1
(POU1F1 - Mouse Genome Informatics) and the LIM-HD factor P-LIM (LHX3 - Mouse
Genome Informatics) are co-expressed during pituitary development. PIT1 and
P-LIM interact with each other and both bind to promoter sequences containing
PIT1-binding sites (Bach et al.,
1995). The LIM-domains of P-LIM are not required for DNA binding
but are crucial for the synergistic interaction with PIT1 on distal target
genes, including Pit1. In C. elegans, UNC-86 dimerizes with
MEC-3 to play an essential role in regulating the terminal differentiation of
touch sensory cells (Duggan et al.,
1998; Lichtsteiner and Tjian,
1995; Xue et al.,
1992; Xue et al.,
1993). However, in contrast to the interaction between PIT1 and
P-LIM, the coupling of UNC-86 and MEC-3 does not require the LIM-domains and
MEC-3 alone binds poorly to the promoters. In retina, we demonstrate that
anti-ISL1 does not co-precipitate with promoters tested in the absence of
BRN3B (see Fig. S3A in the supplementary material), suggesting the binding of
ISL1 to these promoters depends on BRN3B. Moreover, ISL1 alone is not
sufficient to activate Brn3a-luciferase reporter, but is essential
for the synergetic activation of Brn3a when co-expressed with BRN3B
(Fig. 8B).
Previous studies have shown by gel-shift assays that BRN3B binds to the
BRN3-binding site (SBRN3) in the first intron of Shh and activates
the expression of a reporter gene containing SBRN3 in cultured HEK293 cells
(Mu et al., 2004). Using ChIP
assay, we demonstrate that BRN3B binds to this SBRN3-containing region in vivo
in the developing retinas. Additionally, we reveal the simultaneously binding
of ISL1 to the same region in the first intron of Shh. Intriguingly,
ISL1 has also been reported as an upstream regulator of Shh during
cardiac morphogenesis (Lin et al.,
2006). It would be interesting to test whether ISL1 controls
Shh expression through its first intron in developing heart.
Taken together, our expression and targeted deletion analysis suggests a
Math5-Isl1/Brn3b pathway of RGC development
(Fig. 8C). The expression of
MATH5 endows the post-mitotic precursors with RGC competence and activates the
expression of Isl1 and Brn3b to initiate the RGC
differentiation program. Math5 also suppresses the non-RGC differentiation
pathways by negatively regulating the non-RGC specifying factors such as NGN2,
NEUROD, MATH3 (NEUROG2, NEUROD1 and NEUROD4, respectively - Mouse Genome
Informatics) and BHLHB5 (Feng et al.,
2006; Mu et al.,
2005a). As the loss of either Isl1 or Brn3b does
not affect the initial expression of the other, their expression is regulated
in parallel by upstream TFs such as MATH5. The joint action of ISL1 and BRN3B
leads to the expression of RGC-specific genes including Brn3a, Shh,
Olf1 and Ablim1, as well as the autofeedback regulation of
Brn3b. Although our data imply that the majority of RGCs require both
factors to activate their terminal differentiation, the presence of a few RGCs
in Isl1/Brn3b-nulls suggests the existence of a pathway independent
of BRN3B or ISL1. Published studies show that other TFs, such as DLX1 and
DLX2, also participate in regulating the terminal differentiation and survival
of 33% RGCs (de Melo et al.,
2005). Interestingly, the expression of Dlx1 and
Dlx2 is upregulated in Brn3b-null retinas, suggesting BRN3B
normally represses Dlx1/2 expression
(Mu et al., 2004;
Pan et al., 2005). Thus, it is
possible that BRN3B and DLX1/2 are required for the development of
complementary sets of RGCs (de Melo et
al., 2005). Though the remaining RGCs in Brn3b-null
retinas represent most or all RGC subtypes
(Lin et al., 2004), it remains
to be tested whether any specific RGC subtype is selectively lost in
Isl1-null or Dlx1/2-null mice.
In addition to retina, both ISL1 and BRN3 TFs are co-expressed in the
developing dorsal root ganglia, trigeminal ganglia, and the spiral and
vestibular ganglia of the inner ear
(Artinger et al., 1998;
Avivi and Goldstein, 1999;
Huang et al., 2001;
Radde-Gallwitz et al., 2004;
Sohal et al., 1996). Our
findings that BRN3B and ISL1 cooperate in RGC differentiation strongly argue
for a common functional mechanism of these two classes of TFs in neurogenesis
in these other areas of the developing nervous systems. It remains unknown
whether these two factors directly couple with each other in transcriptional
complexes. Interactions of LIM-HD proteins are generally mediated by LDB
family co-factors with the direct interaction between UNC-86 and MEC-3 as the
only exception (Hobert and Westphal,
2000; Lichtsteiner and Tjian,
1995). In combination with immunoprecipitation, future
experiments, such as in vitro pull down, in vitro DNA binding and yeast
two-hybrid assays, can be used to resolve the biochemistry nature of this
interaction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/1981/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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