Center for Advanced Biotechnology and Medicine and Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: xiang{at}cabm.rutgers.edu)
Accepted 7 January 2004
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SUMMARY |
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Key words: Barhl2, Homeobox gene, Retina, Retinogenesis, Glycinergic amacrine cell
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Introduction |
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The amacrine cells are important interneurons that serve to integrate and
modulate visual signals presented to ganglion cells, the output neurons of the
retina. In the mouse retina, at least 26 morphological types of amacrine cells
have been identified (MacNeil and Masland,
1998). The majority of them contain either glycine or GABA
inhibitory neurotransmitters and hence amacrine cells can be divided into two
major groups glycinergic and GABAergic
(Vaney, 1990
). Glycinergic
amacrine cells, which in the mouse comprise
35% of the amacrine cell
population (Marquardt et al.,
2001
), are usually small-field amacrine cells with diffuse
dendritic trees (Menger et al.,
1998
; Pourcho and Goebel,
1985
). AII cells are thus far the best characterized glycinergic
amacrine cells with a bistratified morphology. They are the major amacrine
cells that participate in the rod pathway circuitry
(Famiglietti and Kolb, 1975
;
Kolb and Famiglietti, 1974
;
Strettoi et al., 1992
;
Vaney, 1985
). GABAergic
amacrine cells generally have wider dendritic fields than those of glycinergic
cells and they comprise
40% of all amacrine cells in the mouse retina
(Marquardt et al., 2001
;
Pourcho and Goebel, 1983
;
Vaney, 1990
). Many GABAergic
amacrine cells contain other neurotransmitters such as acetylcholine and
dopamine in addition to GABA (Vaney,
1990
; Wassle and Boycott,
1991
), as exemplified by the direction-selective starburst
amacrine cells which are not only GABAergic but cholinergic as well
(Brecha et al., 1988
;
Fried et al., 2002
;
Kosaka et al., 1988
;
O'Malley and Masland, 1989
;
O'Malley et al., 1992
;
Vaney and Young, 1988
).
During retinogenesis, the seven classes of retinal cells are produced from
multipotent progenitor cells following a loose temporal order, with amacrine
cells being generated in a period spanning from embryonic day 11 (E11) to
postnatal day 4 (P4) (Young,
1985). It has been postulated that, in response to changes of
intrinsic and extrinsic cues, retinal progenitors undergo a series of changes
in competence to give rise to the various retinal cell types
(Cepko, 1999
;
Harris, 1997
;
Livesey and Cepko, 2001
).
Recent advances by molecular genetic approaches have begun to unravel the
molecular bases underlying the determination and differentiation of different
retinal cell types, including amacrine cells. It has been shown by gene
targeting that the homeobox gene Pax6 is required to maintain the
multipotency of retinal progenitors to generate all retinal cell types but
amacrine cells (Marquardt et al.,
2001
; Marquardt and Gruss,
2002
). Overexpression experiments have demonstrated that the basic
helix-loop-helix (bHLH) factor Neurod1 alone or in combination with Pax6 is
capable of promoting amacrine cell differentiation
(Inoue et al., 2002
;
Morrow et al., 1999
).
Similarly, another bHLH factor Math3 (Neurod4 Mouse Genome
Informatics) together with Pax6 can promote amacrine cell differentiation but
Math3 alone lacks this activity (Inoue et
al., 2002
). In compound knockout mice deficient for both
Neurod1 and Math3, no amacrine cells are produced in the
retina (Inoue et al., 2002
).
However, the formation of amacrine cells is essentially normal in single
mutants null for either Neurod1 or Math3
(Inoue et al., 2002
;
Morrow et al., 1999
),
suggesting that Neurod1 and Math3 are redundantly required
for fate determination of amacrine cells. The absence of Pax6 does
not alter Neurod1 expression
(Marquardt et al., 2001
),
thereby allowing the differentiation of amacrine cells to occur.
Despite our current knowledge of amacrine cell development, it remains
unclear what factors are involved in conferring retinal progenitors with the
potential to generate amacrine cells, or in specifying their subtype identity.
Barhl1 and Barhl2/MBH1, two mammalian homeobox genes,
represent homologues of the Drosophila BarH genes that are required
for normal development of the compound eye and external sensory organs
(Bulfone et al., 2000;
Higashijima et al., 1992a
;
Higashijima et al., 1992b
;
Kojima et al., 1991
;
Li et al., 2002
;
Saito et al., 1998
). In the
mouse, Barhl1 is expressed in the developing inner ear hair cells and
central nervous system (CNS), and has been shown by gene-targeting to be
essential for the long-term maintenance of cochlear hair cells
(Li et al., 2002
). The
expression pattern of Barhl2 has not yet been well characterized
during mouse development. One area of Barhl2 expression is in the
interneurons of the spinal cord and ectopic Barhl2 expression has
been reported to promote the differentiation and migration of these neurons
(Saba et al., 2003
).
Barhl2 expression has also been found in the GCL of the developing
retina (Saito et al., 1998
);
however, it is not yet known whether Barhl2 has a role during retinal
development. In this study, we aimed to: (1) systemically examine the spatial
and temporal expression pattern of Barhl2 during mouse development;
(2) identify the types of retinal cells that express Barhl2; and (3)
investigate whether Barhl2 plays a role during retinogenesis. We find
that Barhl2 displays an expression pattern in the CNS that is
distinct from that of Barhl1. In the retina, Barhl2 is
expressed by developing amacrine, horizontal and ganglion cells. Forced
Barhl2 expression promotes the formation of glycinergic amacrine
cells but has no effect on GABAergic neurons, suggesting that Barhl2
is involved in the specification of subtype identity of amacrine cells.
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Materials and methods |
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Generation of an anti-Barhl2 antibody
DNA fragment corresponding to amino acids 3-132 of the mouse Barhl2 protein
was amplified by PCR and inserted into pGEMEX (Promega) and pMAL-cR1 (New
England Biolabs) vectors to express fusion proteins with the bacteriophage T7
gene 10 protein and bacterial maltose-binding protein, respectively. Antibody
production and affinity purification were performed as described previously by
Xiang et al. (Xiang et al.,
1995; Xiang et al.,
1993
).
Plasmid construction and virus preparation and injection
The control-GFP plasmid used is pLZRS-IRES-EGFP
(Kim et al., 2002
), a
replication-incompetent murine retroviral vector derived from LZRSpBMN-Z
(Kinsella and Nolan, 1996
). To
construct the Barhl2-GFP plasmid, the above-mentioned cDNA fragment containing
the entire Barhl2-coding region was subcloned into the XhoI site of
the control-GFP vector. For construction of the Barhl2-EnR-GFP plasmid, DNA
segment corresponding to amino acids 208-314 of Barhl2 containing the
homeodomain was PCR amplified and inserted into the NcoI and
EcoRI sites of the EnR-Slax13 shuttle vector
(Morgan and Fekete, 1996
). The
fused sequence was then released, blunt-ended and transferred into the
control-GFP vector. Control-GFP, Barhl2-GFP and Barhl2-EnR-GFP viruses were
produced in Phoenix Eco retroviral packaging cells (ATCC) by transfection of
relevant plasmids using the Lipofectamine reagent (Gibco-BRL). Ten to 12 days
after transfection, viruses were collected and concentrated as described
(Morgan and Fekete, 1996
).
In vivo infection of P0 mouse retinas by retroviruses was performed as
described (Turner et al.,
1990). Infected retinas were harvested from P30 mice for analysis.
Retinal explant culture and viral infection were carried out also as
previously described (Tomita et al.,
1996
).
Immunostaining and antibodies
To prepare retinal sections, eyeballs were enucleated and immersion-fixed
with 4% paraformaldehyde in PBS for 1 hour at 4°C. Following fixation the
retinas were dissected out, treated with 30% sucrose in PBS for 1-2 hours at
4°C with gentle shaking, embedded in OCT compound and cryosectioned at
12-16 µm. Immunoperoxidase staining of mouse retinal sections was performed
as described (Xiang et al.,
1995; Xiang et al.,
1993
). For double immunofluorescence, retinal sections were
treated with methanol for 2 minutes at 4°C, rinsed with PBS and then
stained with 0.02 µg/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS
for 5 minutes at room temperature. After washes in PBS, they were blocked with
5% normal serum, incubated overnight with a mixture of primary antibodies at
4°C, rinsed with PBS and then incubated with a mixture of
fluorophore-conjugated secondary antibodies for 1 hour at room tempeature.
Images were captured by a digital camera mounted on a Nikon Eclipse E800
microscope.
The following primary antibodies were used: anti-syntaxin (Sigma);
anti-protein kinase C (PKC
) (Amersham); anti-glutamine synthase
(GS) (Chemicon); anti-Pax6 (Developmental Studies Hybridoma Bank); anti-Brn3a
(Chemicon); anti-choline acetyltransferase (ChAT) (Chemicon); anti-green
fluorescent protein (GFP) (mouse monoclonal, Chemicon; rabbit polyclonal, MBL
International Corporation); anti-calbindin-D28K (Sigma); anti-calretinin
(Chemicon); anti-GABAB (Sigma); anti-GABA (Sigma); anti-GABA
transporter-1 (GAT-1) (Chemicon); anti-tyrosine hydroxylase (TH) (Chemicon);
anti-recoverin (Dizhoor et al.,
1991
); anti-Barhl2 (this work); and anti-glycine transporter 1
(GLYT1) (Chemicon).
Quantification
To quantify GFP+ cells and GFP+ cells colocalized
with cell type-specific markers on sections of retinas infected with
control-GFP, Barhl2-GFP or Barhl2-EnR-GFP viruses, 500-3500 GFP+
cells were usually scored for each retina and at least three retinas were
analyzed for each type. For retinas labeled with the anti-TH antibody;
however, more than 10000 GFP+ cells were counted in each retina
owing to the paucity of TH-immunoreactive cells. All data were tested for
significance using two sample Student's t-test with unequal
variances.
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Results |
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Expression of Barhl2 in amacrine, horizontal and ganglion cells but not in mitotic progenitors during retinogenesis
As revealed by RNA in situ hybridization, during retinogenesis,
Barhl2 transcripts were found primarily in the inner neuroblastic
layer at E14.5 and then in the INL and GCL at late embryonic and postnatal
stages (Fig. 2A,B).
Barhl1 transcripts, by contrast, were not detectable in embryonic and
postnatal retinas (Fig. 2G). We
further made use of a lacZ reporter knocked in the Barhl1
locus to examine whether Barhl1 was expressed in the retina
(Li et al., 2002). No
ß-galactosidase activity was ever seen in retinas of
Barhl1lacZ/+ mice at any developmental stages
(Fig. 2H), demonstrating that
of the two Barhl genes, Barhl2 is uniquely expressed in the
developing retina.
|
By immunostaining retinal sections from various developmental stages, we used the anti-Barhl2 antibody to examine retinal expression patterns of the protein. Consistent with it being a homeodomain transcription factor, Barhl2 is nuclear (Fig. 2C-F). Similar to its transcripts detected by in situ hybridization, Barhl2 is first seen in cells within the inner neuroblastic layer at E13.5. By E17.5, it is distributed largely in cells of the INL and GCL (Fig. 2C). Interestingly, however, anti-Barhl2 immunolabeled additional cells scattered within the outer neuroblastic layer at E13.5-17.5 (Fig. 2C). At P1, anti-Barhl2 also additionally labeled a layer of evenly spaced cells in the outer neuroblastic layer (Fig. 2D), which at P14 became a layer of cells lining at the border between the outer plexiform layer and INL (Fig. 2E), a position where horizontal cells are normally situated. At P14 and in the adult, the large majority of Barhl2-immunoreactive cells are located within the inner half of the INL as well as in the GCL, where amacrine and ganglion cells reside (Fig. 2E,F).
The cells expressing Barhl2 in the outer neuroblastic layer of embryonic retinas could be either mitotic progenitors or newly generated cells en route to the INL and GCL or both. To distinguish these possibilities, S-phase cells within E17.5 retinas were pulse labeled by the thymidine analog BrdU, followed by double-immunostaining using anti-Barhl2 and anti-BrdU antibodies (Fig. 3A). In the outer neuroblastic layer, we found that none of the small number of scattered cells immunoreactive for Barhl2 colocalized with any of the large number of proliferative cells labeled by anti-BrdU (Fig. 3A). Thus, Barhl2 appears to be expressed exclusively by postmitotic cells during retinal development.
|
Forced Barhl2 expression promotes the formation of glycinergic amacrine cells
As a first step to understand the role of Barhl2 during retinal
development, we investigated the effect of forced Barhl2 expression in retinal
progenitors. Overexpression of Barhl2 in the mouse retina was achieved by a
replication-incompetent murine retroviral vector derived from LZRSpBMN-Z
(Fig. 4A)
(Kim et al., 2002;
Kinsella and Nolan, 1996
).
Retinas were infected at P0 by subretinal injection of Barhl2-GFP or
control-GFP viruses and analyzed at P30. The laminar position and morphology
of GFP-positive cells were monitored in retinal sections. Although most
virus-infected GFP+ cells became rod cells located within the ONL
in retinas infected with either control-GFP or Barhl2-GFP viruses, there
appeared to be a significant increase in the number of GFP+ cells
residing in the inner region of the INL in retinas infected with Barhl2-GFP
viruses (Fig. 4B,C). To more
accurately assess cell distribution, we quantified the number of
GFP+ cells located in different retinal layers. In retinas infected
with Barhl2-GFP viruses, we found that the percentage of GFP+ cells
located within the inner half of the INL increased from 8.6% in the control to
16.9% (Fig. 4D). By contrast,
the proportion of GFP+ cells distributed within the outer half of
the INL dropped sharply from 9.4% in the control to only 1.5% in retinas
infected with Barhl2-GFP viruses (Fig.
4D). There was no difference in the number of GFP+
cells distributed in the ONL and GCL in retinas infected with either
control-GFP or Barhl2-GFP viruses (Fig.
4D). Thus, forced Barhl2 expression significantly increases the
formation of cells located within the inner half of the INL at the expense of
cells located within the outer half of the INL.
|
|
|
The effect of forced Barhl2 expression on bipolar and Müller cells could result from increased death of virus-transduced cells. To test this possibility, we measured the number of apoptotic GFP+ cells by assaying for the active caspase 3 immunoreactivity in P4 and P10 retinas infected with control-GFP or Barhl2-GFP viruses. No significant difference was observed in the percentage of apoptotic GFP+ cells between control retinas and retinas infected with Barhl2-GFP viruses at P4 (Barhl2 mean±s.d., 0.29±0.10%, n=4; control, 0.22±0.06%, n=3) or P10 (Barhl2, 0.12±0.06%, n=3; control, 0.15±0.03%, n=3). As Barhl2 is expressed only by postmitotic cells and hence is unlikely to affect cell proliferation, it most probably exerts its retinal function by influencing specification and/or differentiation of retinal cell types.
As retinal progenitors change their competent states during development, we
investigated whether overexpressed Barhl2 had different effects at embryonic
stages, in particular, on the earlier-born horizontal and ganglion cells. In
these experiments, retinal explants from E13.5, E17.5 or P0 mice were infected
with Barhl2-GFP or control-GFP viruses and cultured in vitro for 14 days
before analysis. Compared with P0 retinas infected in vivo, we found that very
similar results could be obtained from these explants
(Fig. 6). For example, with
E17.5 retinal explants, overexpressed Barhl2 increased amacrine cells positive
for syntaxin, Pax6, GLYT1 or calbindin, reduced cells immunoreactive for
PKC or GS, but did not alter the number of cells positive for GAT-1
(Fig. 6B). Similarly, it had no
effect on calbindin+ horizontal cells or Brn3a+ ganglion
cells (Fig. 6B).
Overexpression of a dominant-negative form of Barhl2 inhibits the formation of glycinergic amacrine cells
To test whether Barhl2 acts as a transcriptional activator for the
specification of glycinergic amacrine cells, we constructed a
dominant-negative viral plasmid, Barhl2-EnR-GFP, by fusing the repressor
domain of the Drosophila engrailed protein to the Barhl2 homeodomain
region (Fig. 7A). Compared with
retinas infected with control-GFP viruses, we found that the fraction of
GFP+ cells distributed in the INL was reduced from 19.7% to 10.4%
in retinas infected with Barhl2-EnR-GFP viruses
(Fig. 7D,E). This decrease
manifested a proportional reduction of GFP+ cells within both the
inner and outer halves of the INL and was accompanied by an 10% increase
in the number of GFP+ cells located in the ONL
(Fig. 7B).
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Discussion |
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Barhl2 is expressed in postmitotic amacrine, horizontal and ganglion cells during retinogenesis
The expression of Barhl2 in the retina has provided an excellent
opportunity to study its function in the CNS using retina as a model system
because of its easy accessibility and well-characterized cell classes. We
therefore focused our efforts to investigate the cellular localization of
Barhl2 factor and its function during retinogenesis. RNA in situ hybridization
reveals the presence of Barhl2 transcripts within the inner
neuroblastic layer at E14.5 and in the INL and GCL in postnatal retinas. Owing
to the presence of several cell classes in these layers, however, it is
impossible to unequivocally identify the types of cells that express
Barhl2 by in situ hybridization. We thus produced a specific
anti-Barhl2 antibody and unambiguously identified by immunostaining all
classes of cells that express Barhl2, which include amacrine, horizontal and
ganglion cells (Fig. 3). In
addition, BrdU labeling experiment indicated that Barhl2 was expressed only by
postmitotic cells.
During mouse retinogenesis, birthdating studies have demonstrated that
amacrine, horizontal and ganglion cells begin to exit the cell cycle at E11
(Young, 1985). Our analysis
has revealed the onset of Barhl2 expression at E13.5, indicating that
Barhl2 is unlikely to be involved in the fate commitment and initial
generation of amacrine, horizontal and ganglion cells. The scattered cells
immunoreactive for Barhl2 in the outer neuroblastic layer resemble those of
Brn3b+ migrating ganglion cells or Lim1+ migrating
horizontal cells (Liu et al.,
2000
; Xiang,
1998
). They most probably represent newly generated neurons that
are differentiating and migrating toward the inner retina to become amacrine,
horizontal and ganglion cells. Thus, given its expression only in
differentiating and mature neurons, Barhl2 may play a role in the
differentiation and maintenance of amacrine, horizontal and ganglion
cells.
Barhl2 promotes the differentiation of glycinergic amacrine cells at the expense of bipolar and Müller cells
The amacrine cells come as many as more than 26 morphological types to fine
tune the physiological output of the retina
(MacNeil and Masland, 1998).
For example, the starburst amacrine cells play a central role in the
establishment of retinal direction-selectivity
(Fried et al., 2002
;
Vaney and Taylor, 2002
).
Amacrine cells can also be divided into two major nonoverlapping groups
according to the types of neurotransmitters they contain. These are
glycinergic and GABAergic amacrine cells. To date, it remains essentially
unknown what factors are involved in the differentiation of these different
amacrine cell subtypes. Our work identifies Barhl2 as a key regulator that
confers the identity of glycinergic amacrine cells. This effect appears to be
specific as overexpressed Barhl2 does not cause even a slight change in the
number of GABAergic cells (Fig.
6). Similarly, the Barhl2 homeodomain, when fused with the
engrailed repressor, suppresses glycinergic amacrine cells but has no effect
on GABAergic neurons. Interestingly, Pax6, apart from its essential
role in early retinal development, is expressed in differentiating amacrine
cells and has been shown to positively regulate the formation of glycinergic
amacrine cells as well (Marquardt et al.,
2001
).
Despite the requirement of Pax6 for retinal progenitors to acquire
multipotency, the absence of Pax6 in mice permits the formation of
amacrine cells (Marquardt et al.,
2001), implicating that another unknown factor(s) must be required
to make the progenitors competent for the generation of amacrine cells.
Furthermore, gene targeting has demonstrated that Math3 and
Neurod1 are redundantly required to specify amacrine cells from
progenitors (Inoue et al.,
2002
; Morrow et al.,
1999
). These data and our present study have prompted us to
propose a genetic pathway governing the determination and differentiation of
amacrine cells (Fig. 8). In
this model, Math3 and Neurod1 together specify amacrine cells from retinal
progenitors rendered competent by an unknown regulator(s). Barhl2, probably
together with Pax6, then promotes the differentiation of glycinergic amacrine
cells from fate-restricted amacrine progenitors. Such fate-restricted amacrine
progenitors have been identified by fate tracing experiments in early rat and
Xenopus retinas (Alexiades and
Cepko, 1997
; Huang and Moody,
1997
; Moody et al.,
2000
). However, it remains to be determined what factors are
involved in the differentiation of GABAergic amacrine cells. Apart from the
absolute requirement of Pax6 for retinal progenitors to produce all retinal
cells other than amacrine neurons
(Marquardt et al., 2001
), the
possibility cannot be ruled out that Pax6 also plays a redundant role to
confer progenitors with the capacity of amacrine cell generation. Consistent
with this notion, forced Pax6 expression can promote an amacrine cell fate in
combination with Math3 or Neurod1 (Inoue
et al., 2002
). Thus, there is a possibility that Pax6+
progenitor cells may also be specified into amacrine cells by Math3 and
Neurod1 (Fig. 8).
|
Many of the transcription factors involved in retinal development have been
shown to act as both positive and negative regulators depending on different
cell types. For example, Rax1 and Hes1 are able to promote a Müller glial
cell fate as well as inhibit neuron differentiation
(Furukawa et al., 2000). By
contrast, Xath5 promotes a ganglion cell fate but blocks the formation of
Müller and bipolar cells (Kanekar et
al., 1997
). Similarly, we show here that Barhl2 can function as a
positive factor to specify glycinergic amacrine cells while negatively
regulating the formation of bipolar and Müller cells. As Barhl2 is not
expressed in mitotic retinal progenitors, conceivably, it may prevent newly
selected amacrine cells from differentiating into bipolar or Müller
cells. This bifunctional property of retinal developmental regulators may
serve to control the production of proper numbers of different cell types and
to minimize developmental errors during retinogenesis.
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ACKNOWLEDGMENTS |
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