1 Center for Aging and Developmental Biology, University of Rochester,
Rochester, NY 14642, USA
2 Department of Ophthalmology, 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 8 December 2004
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SUMMARY |
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Key words: Brn3, POU-domain, Retina, Neurogenesis, Retinal ganglion cells, Transcription factors, Pou4f
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Introduction |
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To investigate the roles of Brn3 genes, we and others have created targeted
mutations in Brn3 genes and have shown that individual null mutations lead to
distinct defects in the development of sensory neurons
(Xiang et al., 1997a). Mice
carrying Brn3a-null mutation suffer from a selective loss of neurons
in somatosensory ganglia and in selective brainstem nuclei, display
uncoordinated limb movement and impaired suckling, and die shortly after birth
(McEvilly et al., 1996
;
Xiang et al., 1996
). A
detailed examination of the role of Brn3a in neurogenesis has demonstrated the
axonal growth and pathfinding defects in trigeminal and dorsal root neurons
prior to their programmed cell death (Eng
et al., 2001
). Last, Brn3a is required for axon
pathfinding and target field innervation of spiral and vestibular ganglion
neurons in the inner ear (Huang et al.,
2001
).
In contrast to the studies using Brn3a mutants,
Brn3b-null mutants display a loss of 70% of RGCs in adult
retinal ganglion cell layer (GCL) (Erkman
et al., 1996
; Gan et al.,
1996
). Further studies by using Brn3b-lacZ and
Brn3b-AP knock-in mice have shown that Brn3b is not required
for cell fate specification and migration of RGCs. Rather, Brn3b
expression is essential for axon growth, pathfinding, fasciculation and
survival of RGCs (Gan et al.,
1999
; Wang et al.,
2000
). Analysis of RGC axons in postnatal Brn3b-null mice
also reveals that Brn3b is involved in the pathfinding of RGC axons
at multiple points along their pathways and in the establishment of
topographic order in the superior colliculus
(Erkman et al., 2000
).
In the third group of Brn3 mutants, Brn3c-null mice lack
vestibular and auditory hair cells in the inner ear, are deaf and have
impaired balancing abilities (Erkman et
al., 1996; Xiang et al.,
1997a
). Recently, Brn3c was shown to be essential for the
differentiation of a small number of RGCs
(Wang et al., 2002
). This
finding is exacerbated in Brn3b and Brn3c double knockout
mice, where significantly more RGCs are lost than in either Brn3b or
Brn3c single mutants (Wang et
al., 2002
).
The unique phenotypes associated with Brn3 knockouts illustrate that each Brn3 gene has a great degree of functional specificity. One explanation of their functional uniqueness is that the unique protein sequences of Brn3 factors could render each characteristic biochemical properties and thus, distinctive transcriptional activities. Conversely, based on their highly conserved POU-domains and identical DNA-binding properties, all Brn3 factors could also be functionally equivalent in transcriptional activities. The distinctive neuronal defects related to the loss of each Brn3 gene could simply reflect its characteristic spatiotemporal expression patterns. To test these hypotheses, we perform in vivo gene-replacement experiments to investigate whether knocking-in one Brn3 gene to replace the other can functionally rescue the neuronal defects associated with the loss of the other Brn3 gene. We demonstrate that targeted replacement of Brn3b with Brn3a corrects the retinal defects identified in Brn3b-null mice. The RGCs expressing Brn3aki in the absence of Brn3b are able to form fasciculated axons, to generate proper axon projection, and to avoid the fate of programmed cell death. Furthermore, Brn3aki expression from the Brn3b locus restores the early developmental expression profiles of Brn3b downstream target genes. Our results strongly argue for the functional equivalence of Brn3 transcription factors and imply a shared Brn3 regulatory pathway in the development of various sensory neurons.
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Materials and methods |
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Histochemistry
To compare retinas and optic nerves in Brn3blacZ/+,
Brn3bAP/lacZ and Brn3b3a/lacZ mice, eye
cups and brain tissues containing optic nerves, optic chiasms and optic tracks
from weight- and gender-matched mice at 6 weeks age were paraffin-embedded and
sectioned at 10 µm. Optic nerves were cut at 1 mm anterior to the
optic chiasms. Sections were de-waxed and stained with Hematoxylin and Eosin.
Areas of the optic nerves were photographed and the sizes of optic nerves were
computed and compared by Scion Image.
Immunohistochemistry, in situ hybridization, BrdU labeling and X-gal staining
Staged embryo and tissue samples were dissected and immediately fixed in 4%
paraformaldehyde. The samples were then embedded and frozen in OCT medium
(Tissue-Tek). For immunolabeling, cryosections were cut at 15 µm. The
working dilutions and sources of antibodies used in this study were: mouse
anti-bromodeoxyuridine (BrdU) (1:200, Becton Dickson), mouse anti-Brn3a
(1:100, Santa Cruz), goat anti-Brn3b (1:2,000, Santa Cruz), rabbit
anti-phospho-histone 3 (1:400, Santa Cruz) and anti-nonphosphorylated
neurofilament H (SMI-32) (1:2,000, Sternberger Monoclonals). Alexa-conjugated
secondary antibodies (Molecular Probes) were used at a concentration of 1:400.
For in situ hybridization experiments, 20 µm cryosections were used as
previously described (Li and Joyner,
2001).
Detection of ß-galactosidase activity was determined by X-gal staining
(Gan et al., 1999). Briefly,
retinal tissues were fixed in 4% paraformaldehyde in PBS at 4°C for 30
minutes. Whole retinas or 16 µm frozen sections were stained overnight at
room temperature with 0.1% X-gal, 5 mM potassium ferricyanide, 5 mM potassium
ferrocyanide, 2 mM MgCl2 in PBS. For bromodeoxyuridine (BrdU)
(Sigma) pulse-labeling experiments, pregnant females were injected
intraperitoneally with 100 µg BrdU/gram body weight 1 hour before they were
sacrificed. Embryo processing and BrdU labeling were performed as previously
described (Mishina et al.,
1995
).
Lipophilic dye tracing
For optic nerve labeling, E17.5 embryonic mouse heads were fixed overnight
in 4% paraformaldehyde in PBS. After the right eyes were enucleated, crystals
of DiI (Molecular Probes) were implanted unilaterally into the optic discs.
After incubation at 37°C in PBS containing 0.1% sodium azide for 2 weeks,
the brains were dissected. The labeled optic nerves were exposed and
visualized with a Nikon fluorescence dissecting microscope.
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Results |
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Previously, studies of adult retinas have shown that the
Brn3b-null mutation leads to the reduced retinal expression of
Brn3a (Gan et al.,
1996). To test if the loss of Brn3a expression is caused
by Brn3b-null mutation rather than the apoptosis of RGCs, we asked
whether the downregulation of Brn3a occurs prior to the onset of RGC
apoptosis in developing Brn3b-null retinas. In situ hybridization of
wild-type retinas showed the onset of Brn3a and Brn3b
expression at E12.5 and E11.5, respectively
(Fig. 2A,B). In
Brn3b-null retinas, Brn3a expression was greatly reduced
starting at E12.5 (Fig. 2C). As
the apoptosis of 70% Brn3b-null RGCs does not start until E14.5 in
Brn3b-null mice (Gan et al.,
1999
; Xiang,
1998
), downregulation of Brn3a is unlikely to be due to
the loss of RGCs. Rather, the initiation of Brn3a expression in
normal retina depends on Brn3b expression and the loss of
Brn3b directly causes the decreased Brn3a expression.
|
Absence of retinal defects of in adult Brn3b3a knock-in mice
To detect the effect of Brn3aki on retinal development
in Brn3b-null mice, we examined the potential defects in retinal
structures and in RGC number and properties. Hematoxylin and eosin staining of
retinal structure and X-Gal staining of nuclear Brn3b-lacZ activity
(as the specific marker for all RGCs) showed a loss of 70% RGCs in the
GCL of Brn3bAP/lacZ retina in mice 6 weeks of age
(Fig. 3B,E). Interestingly, the
RGC number and the overall structure were indistinguishable in
Brn3b3a/lacZ and wild-type or
Brn3blacZ/+ retinas (compare
Fig. 3A with 3C, and 3D with
3F). As another measure to detect the changes in RGCs, we
immunolabeled retinas with mouse monoclonal antibody SMI-32, which
predominantly labels the axons of large RGCs
(Nixon et al., 1989
).
Brn3bAP/lacZ retinas showed significantly fewer and less
fasciculated axon bundles projecting into the optic disc (OD)
(Fig. 3H). Staining of
Brn3b3a/lacZ (Fig.
3I) and wild-type or Brn3blacZ/+
(Fig. 3G) retinas revealed
similarly dense and fasciculated RGC axons. RGCs are the only output retinal
neurons whose axons exit the eye to form the optic nerve with each RGC
contributing a single axon to the optic nerve. We examined the optic nerves of
wild-type, Brn3blacZ/AP and
Brn3b3a/lacZ mice as an alternative measurement of RGC
numbers. When compared with those of wild-type or
Brn3blacZ/+ (Fig.
3J,M), the optic nerves of Brn3blacZ/AP
(Fig. 3K,N) were greatly
reduced in the cross-sectional area (12.7±4.6% of wild-type,
n=10). Conversely, the expression of a single copy of
Brn3aki (Fig.
3L,O) was sufficient to restore normal optic nerve size to a level
comparable with those of wild-type mice (85.2±6.6% of wild-type,
n=9). These data indicate that the knock-in of Brn3a into
Brn3b locus is capable of rescuing the defects in retinal structure
and RGC number of Brn3b-null retinas.
|
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For animals with binocular vision, the RGC axons from different retinal
regions segregate at the optic chiasm to form the contralateral and
ipsilateral visual pathways (Fig.
5A). In mice, the ipsilaterally projected axons arise from the
ventrotemporal retina and amount to 3% of total RGC axons. As shown in
Fig. 5B, anterograde DiI
labeling of wild-type mice at E17.5, when the segregation of RGC axons is
nearly completed, revealed that a majority of RGC axons projected
contralaterally with a small percentage of axons projecting into the
ipsilateral pathway. In Brn3bAP/lacZ mice, although a
large proportion of RGC axons formed the contralateral optic tract, an
abnormally large proportion of axons exhibited severe pathfinding defects at
the optic chiasm and projected into ipsilateral optic track or into the optic
nerve of the opposite eye (Fig.
5C). Conversely, no apparent pathfinding defects were identified
at the optic chiasm of Brn3b3a/3a mice (n=4)
(Fig. 5D), indicating that
Brn3aki fully substitutes Brn3b in regulating the pathfinding
decision of RGC axons.
|
To test whether Brn3a and Brn3b share the identical transcription activities, as well as to determine whether Brn3aki rescues the RGC defects by restoring the RGC expression of Brn3b downstream target genes, we compared the expression of these genes in E14.5 retinal sections of wild-type, Brn3blacZ/AP and Brn3b3a/3a embryos. The control Brn3b probe confirmed the absence of Brn3b expression in Brn3blacZ/AP and Brn3b3a/3a retinas (Fig. 6A). Brn3a ORF probe detected the reduced expression of endogenous Brn3a in Brn3blacZ/AP retina and Brn3b-like expression pattern of Brn3aki in Brn3b3a/3a retina (Fig. 6B). Compared with wild-type controls, loss of Brn3b in Brn3blacZ/AP mice resulted in the downregulation of Brn3a, Irx2, Irx6, Ablim, Gfi1, Gli1, Isl2, Olf1, L1, Gap43, Shh and Hermes (Rbpms Mouse Genome Informatics) (Fig. 6C-D, G-P) and the upregulation of Dlx1 and Dlx2 expression (Fig. 6E,F). When retinas from Brn3b3a/3a knock-in mice were examined, the expression levels of all of above genes were restored to those observed in wild-type retinas (Fig. 6C-P). The expression analyses demonstrate the identical ability for Brn3a and Brn3b to activate or suppress in RGCs the expression of Brn3b downstream genes and support the notion that rescue of Brn3b-null phenotypes by Brn3aki is achieved by restoring the expression of Brn3b downstream genes. Furthermore, Brn3aki expression in Brn3b3a/3a retina lead to the activation of endogenous Brn3a expression (Fig. 6C), implying the presence of positive feedback regulation of Brn3a expression in the developing retina.
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Discussion |
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Functional equivalency of Brn3 factors
The highly conserved Brn3 factors share a significantly high sequence
homology, especially in the functionally important POU-domains and bind to the
identical DNA sequences. Though the targeted deletion of each Brn3 gene
results in unique defects in neuronal development, there is a tight
correlation of Brn3 spatiotemporal expression patterns and their knockout
phenotypes. In the developing retina, though the expression of Brn3a
and Brn3c in RGCs mostly overlaps with that of Brn3b, a
comparison of temporal expression profiles of Brn3 genes shows that
Brn3b is expressed in retina 1 day earlier than Brn3a and
Brn3c. Deletion of Brn3b causes the loss of RGCs and the
downregulation of Brn3a and Brn3c expression in RGCs
(Gan et al., 1996). The
downregulation of Brn3a is detected at E12.5 and before the onset of
RGCs apoptosis after E14.5 in Brn3b-null mice, suggesting that in a
majority of RGCs, Brn3b acts upstream to activate the expression of
Brn3a. Thus, it is probable that the loss of these RGCs in
Brn3b knockout mice is due to the requirement for early
Brn3b expression during RGC development.
Similarly, the specific neuronal defects observed in Brn3a-null
mice are closely linked to its distinctive expression in these neurons. In
developing dorsal root ganglia, Brn3a expression starts at E9.5 and
precedes those of Brn3b and Brn3c
(McEvilly et al., 1996).
Brn3a is also the only Brn3 family member to express in red nuclei
and knockout of Brn3a specifically leads to apoptosis of neurons in
both dorsal root ganglia and red nuclei
(Xiang et al., 1996
). In
addition, in the caudal region of the inferior olivary nucleus, Brn3a
and Brn3b are co-expressed in neurons at the ventral boundaries and
Brn3a expression is further extended more dorsally. Removal of
Brn3a in the knockout mice results in the loss of
Brn3a-expressing neurons in regions dorsal to the ventral boundaries
but not at the ventral boundaries where the expression of Brn3a and
Brn3b overlaps (Xiang et al.,
1996
).
In inner ear, although both Brn3a and Brn3b are
co-expressed in vestibular and cochlear ganglion cells of the developing inner
ear, Brn3a expression starts at E9 and persists throughout
development, and Brn3b expression does not start until E12.5. In
agreement with their expression patterns, loss of Brn3a results in
the degeneration of spiral and vestibular ganglion cells and the absence of
Brn3b expression in these cells
(Huang et al., 2001).
Likewise, Brn3c is the only Brn3 gene to express in inner ear hair
cells and the loss of hair cells in Brn3c knockout mice also reflects
its unique spatial expression pattern
(Xiang et al., 1997a
).
In our present study, we express Brn3a in all
Brn3b-expressing cells at a physiological level comparable with that
of endogenous Brn3b by choosing a knock-in approach to replace
Brn3b-coding regions with Brn3a cDNA. We have shown that
Brn3aki can fully replace Brn3b and rescue the retinal
defects associated with Brn3b-null mutation. Our in vivo studies
demonstrate that Brn3 genes are functionally equivalent and that their
distinctive roles in development are determined by their unique expression
profiles. Consistent with our results, gain-of-function studies in chicken
retinas have shown that all Brn3 proteins play a similar role to promote the
development of RGCs (Liu et al.,
2000). However, our findings appear different from other published
in vitro studies. Those studies as such have shown that both Brn3a and Brn3b
activate an iNOS promoter in BHK-21 fibroblast cells
(Gay et al., 1998
). However,
the two genes exhibit antagonistic effects on the transcriptional regulation
of human papilloma virus (HPV) type 16 and 18 E6 and E7 genes in cell lines of
cervical origin (Ndisdang et al.,
1998
) and on in vitro differentiation of ND7 cells
(Smith et al., 1997
). All of
these in vitro studies are performed in culture of different cell lines and,
sometimes, in lines derived from cells with no known Brn3 expression or
function. In addition, these transactivation experiments have used strong
viral promoters that express Brn3 factors at levels significantly higher than
normal physiological concentrations. Thus, it is rather difficult to properly
compare and to decipher the precise in vivo role of each Brn3 factor under
such in vitro conditions.
As Brn3 factors are often expressed in the same cells, if Brn3 factors
indeed possess unique biochemical properties and function antagonistically,
disruption of the proper equilibriums of Brn3 factors within the same cells
could have a detrimental effect on their development and survival. However,
previously published studies have failed to show any developmental and
survival defects in mice heterozygous for any of the three Brn3 mutations
(Erkman et al., 1996;
Gan et al., 1999
;
Gan et al., 1996
;
McEvilly et al., 1996
;
Xiang et al., 1997b
;
Xiang et al., 1996
). Recent
studies have shown that the autoregulation of Brn3a can compensate
for the loss of one allele by increasing transcription from the remaining
allele in trigeminal and dorsal root ganglia
(Trieu et al., 2003
). Such
dose compensation mechanisms in retina have not been reported yet. Additional
in vivo gene replacement experiments of Brn3 are needed to demonstrate whether
the biochemical roles of all Brn3 factors are identical. Nonetheless, our
results clearly provide the first in vivo evidence to support this theory.
Brn3b regulatory pathway in the development and survival of retinal ganglion cells
The postmitotic expression of Brn3b in retina is consistent with our
previous findings that Brn3b acts downstream of bHLH-class transcription
factor Math5 to regulate the terminal differentiation and survival of RGCs
(Gan et al., 1999;
Wang et al., 2001
;
Yang et al., 2003
). In
Brn3b-null mice, RGCs fail to project axons properly and die of
programmed cell death. Brn3b probably controls these complicated processes by
modulating the expression of genes essential for RGC differentiation and
survival. Recently, a cDNA microarray analysis of gene expression profiles in
wild-type and Brn3b-null retinas demonstrated that Brn3b regulates
the expression of discrete sets of genes, including genes encoding
transcription factors, secreted signaling molecules, and proteins for neuron
integrity and function (Mu et al.,
2004
). Similarly, we have shown in this study that the expression
of additional genes with known roles in axon growth and pathfinding are indeed
altered in Brn3b-null retina.
It remains unclear whether the Brn3b downstream targets are
primarily regulated by Brn3b or Brn3a in retina. Brn3b-null mutation
leads to the diminished expression of Brn3a
(Gan et al., 1996). A
comparable phenomenon is observed in Brn3a-null mice, where
Brn3b expression is drastically reduced in trigeminal, dorsal root,
spiral and vestibular ganglia (Huang et
al., 2001
; McEvilly et al.,
1996
; Xiang et al.,
1996
). The lack of overt retinal phenotypes in Brn3a-null
mice (McEvilly et al., 1996
;
Xiang et al., 1996
) and the
restoration of Brn3b downstream target gene expression by
Brn3aki suggest that both Brn3a and Brn3b play equivalent
roles to regulate these genes. Interestingly, ectopic expression of
Brn3aki in Brn3b-defecient retina also activates the
expression of endogenous Brn3a, suggesting a mutual, positive
feedback regulation of Brn3a and Brn3b genes. Activation of
Brn3b probably leads to the activation and maintenance of
Brn3a and Brn3b expression in RGCs. Vice versa, initial
activation of Brn3a could result in the activation and maintenance of
Brn3a and Brn3b expression in other sensory neurons. The
exact role of such feedback control in Brn3 gene expression is unclear.
Nevertheless, as Brn3 factors function downstream of the initial
differentiation events initiated by bHLH transcription factors, such a
positive feedback mechanism would allow the irreversible activation of
terminal differentiation programs of sensory neurons.
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ACKNOWLEDGMENTS |
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Footnotes |
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