-
DEVELO
PMENT
4815RESEARCH ARTICLE
INTRODUCTIONIn the vertebrate retina, the six major neuronal
cell types and onetype of Müller glia are arranged in a
well-defined laminar structure.Whereas photoreceptors (rod and
cones) are located in the outernuclear layer (ONL), the three types
of interneurons (bipolar,horizontal and amacrine cells) and the
Müller glial cells comprisethe inner nuclear layer (INL). The
ganglion cell layer (GCL) is madeof retinal ganglion cells (RGCs)
and displaced amacrine cells. Basedon their anatomical and
functional disparities, retinal neurons arefurther divided into a
number of subtypes (Masland, 2001a;Masland, 2001b). For example,
there is one rod bipolar (RB)subtype and nine cone bipolar (CB)
subtypes in mouse retina basedon their synaptic inputs from rod or
cone photoreceptors and theirsub-laminar localization (Ghosh et
al., 2004). Bipolar cells can befurther classified as ON and OFF
subtypes according to theirresponses to visual stimuli. RB cells
are all of the ON subtype andrelay visual information to RGCs
indirectly through the AII subtypeof amacrine cells. CB cells
synapse directly with RGCs and conveyboth rod- and cone-generated
visual information to RGCs. CertainOFF-CB cells also contact rod
photoreceptors directly (DeVries andBaylor, 1995; Hack et al.,
1999). Amacrine cells are mainlylocalized to the INL and the GCL,
and make synapses with bipolarcells and RGCs. They constitute the
most diverse cell type within theretina where they function to
modulate synaptic activity betweenbipolar and ganglion cells
(Masland, 2001a; Masland, 2001b).Similarly, amacrine cells are
classified into subtypes based oncriteria such as sub-laminar
localization [the inner plexiform layer(IPL), the GCL, and the
innermost layer of the INL known as the
amacrine cell layer (ACL)], morphology (e.g. starburst, parasol
andmidget) and neurotransmitter type (e.g. GABAergic,
dopaminergicand cholinergic).
Transcription factors play important roles in retinal cell
fatedetermination and genetic manipulation of these factors often
leadsto a loss or an alteration of one or more cell fates in the
retina(Cepko, 1999; Marquardt, 2003; Marquardt and Gruss,
2002).Bipolar cell fate specification is dependent on the combined
actionof the homeodomain protein Chx10 and the bHLH factors
Mash1and Math3 (Ascl1 and Neurod4, respectively – both Mouse
GenomeInformatics) (Burmeister et al., 1996; Hatakeyama et al.,
2001).Chx10-null or Mash1-Math3 double-null mice exhibit a
completeloss of bipolar cells (Burmeister et al., 1996; Hatakeyama
et al.,2001). Studies of Mash1-Math3 double-null retinas also show
thatMash1 and Math3 function redundantly to regulate bipolar
cellgenesis and that loss of the two genes leads to an absence
ofbipolar cells (Hatakeyama et al., 2001). Gain-of-function
studiesdemonstrate that misexpression of Chx10, Mash1 or Math3
alonedoes not promote bipolar cell genesis. By contrast,
ectopicexpression of Mash1 or Math3 together with Chx10 increases
thepopulation of mature bipolar cells. Thus, it is hypothesized
thatwhereas Chx10 provides the INL-specific identity, Mash1
andMath3 determine bipolar cell fate (Hatakeyama et al.,
2001).Recently, targeted deletion studies have revealed that Vsx1,
a paired-like homeodomain factor, and Bhlhb4, an Olig family bHLH
factor,are required for the development of the CB and RB
subtypes,respectively. Mice lacking Vsx1 are able to generate CB
cells.However, the terminal differentiation of CB cells in
Vsx1-null retinais incomplete, indicating that Vsx1 is required
solely for the latedifferentiation of OFF-CB cells (Chow et al.,
2004; Ohtoshi et al.,2004). Similarly, Bhlhb4 plays no role in the
initial generation ofbipolar cells, and loss of Bhlhb4 prevents
nascent RB cells frommaturating and eventually results in their
apoptosis (Bramblett et al.,2004).
The expression of Math3, NeuroD (Neurod1 – Mouse
GenomeInformatics) and Pax6 is associated with differentiating
amacrinecells as well as other retinal cells (Jones et al., 1998;
Morrow et al.,
Requirement for Bhlhb5 in the specification of amacrine andcone
bipolar subtypes in mouse retinaLiang Feng1, Xiaoling Xie1, Pushkar
S. Joshi1, Zhiyong Yang1, Koji Shibasaki1, Robert L. Chow2 andLin
Gan1,3,4,*
The mammalian retina comprises six major neuronal cell types and
one glial type that are further classified into multiple
subtypesbased on their anatomical and functional differences.
Nevertheless, how these subtypes arise remains largely unknown at
themolecular level. Here, we demonstrate that the expression of
Bhlhb5, a bHLH transcription factor of the Olig family, is
tightlyassociated with the generation of selective GABAergic
amacrine and Type 2 OFF-cone bipolar subtypes throughout
retinogenesis.Targeted deletion of Bhlhb5 results in a significant
reduction in the generation of these selective bipolar and amacrine
subtypes.Furthermore, although a Bhlhb5-null mutation has no effect
on the expression of bHLH-class retinogenic genes, Bhlhb5
expressionoverlaps with that of the pan-amacrine factor NeuroD and
the expression of Bhlhb5 and NeuroD is negatively regulated
byganglion cell-competence factor Math5. Our results reveal that a
bHLH transcription factor cascade is involved in regulating
retinalcell differentiation and imply that Bhlhb5 functions
downstream of retinogenic factors to specify bipolar and amacrine
subtypes.
KEY WORDS: Bhlhb5 (Beta3), bHLH, Math5 (Atoh7), NeuroD
(Neurod1), Math3 (Neurod4), Amacrine cell, Bipolar cell, Retina,
Neurogenesis,Transcription factors, Mouse
Development 133, 4815-4825 (2006) doi:10.1242/dev.02664
1Center for Aging and Developmental Biology, University of
Rochester, Rochester,NY 14642, USA. 2Department of Biology,
University of Victoria, Victoria, BC V8W3N5, Canada. 3Department of
Ophthalmology and 4Department of Neurobiologyand Anatomy,
University of Rochester, Rochester, NY 14642, USA.
*Author for correspondence (e-mail:
[email protected])
Accepted 28 September 2006
-
DEVELO
PMENT
4816
1999; Nishina et al., 1999). Mutation of NeuroD, Pax6 or
Math3alone does not impair the differentiation of amacrine
cells.Conditional deletion of Pax6 in retina results in the
formation of onlyamacrine cells (Marquardt et al., 2001). Although
the developmentof amacrine cells is unaffected in mice lacking
either NeuroD orMath3, mice deficient for both Math3 and NeuroD are
devoid ofamacrine cells (Inoue et al., 2002). Cell lineage analysis
studies havedemonstrated that retinal progenitors of the Math3 and
NeuroDdouble-null mice fail to differentiate into amacrine cells.
Instead ofundergoing programmed cell death, these progenitors
trans-differentiate into ganglion and Müller glial cells (Inoue et
al., 2002).Misexpression of Math3 or NeuroD alone in developing
retina doesnot promote the differentiation of amacrine cells, but
results in theformation of rod cells (Inoue et al., 2002).
Recently, it has beenshown that the winged helix/forkhead
transcription factor Foxn4plays an essential role in the formation
of amacrine and horizontalcells (Li et al., 2004). Loss of Foxn4
function leads to the completeabsence of horizontal cells and to a
reduction in amacrine cells.Interestingly, the expression of NeuroD
and Math3 are reduced inFoxn4-null retina, suggesting that Foxn4
acts upstream of NeuroDand Math3 in the amacrine differentiation
pathway. The abovegenetic studies reveal the roles of these
transcription factors as pan-amacrine-determining factors. However,
the transcription factorsresponsible for retinal subtype
specification remain elusive.
Bhlhb5 is a member of the Olig subfamily of bHLH
transcriptionfactors. It has previously been shown to be expressed
in a restrictedmanner in the developing central nervous system
(CNS), sensoryorgans, kidney and hair follicles (Bramblett et al.,
2002; Brunelli etal., 2003; Kim et al., 2002; McLellan et al.,
2002; Peyton et al.,1996). Due to its inability to bind DNA alone
or in combination withother bHLH proteins, Bhlhb5 is thought to
function as a negativeregulator of other bHLH proteins (Peyton et
al., 1996). In vitroevidence has shown that Bhlhb5 represses Pax6
promoter activitythrough a non-DNA-binding mechanism (Xu et al.,
2002). However,the role of Bhlhb5 in embryonic development remains
unknown. Inthis report, we demonstrate that Bhlhb5 is predominantly
expressedin post-mitotic cells in the developing mouse retina.
Co-localizationof Bhlhb5 and retinal cell type-specific markers
reveals that Bhlhb5expression is restricted to selective GABAergic
amacrine and Type2 OFF-CB cells. Targeted deletion of Bhlhb5 leads
to a loss of Type2 OFF-CB and selective GABAergic amacrine cells,
implying thatBhlhb5 is indispensable for the formation of these
cells. Expressionstudies of early embryonic retinas have
demonstrated that Bhlhb5 ismostly expressed in cells of the NeuroD+
and Math5+ lineage, andthat the increased formation of displaced
amacrine cells in Math5-null retina is associated with a
substantial increase in cellsexpressing Bhlhb5 and NeuroD.
Furthermore, whereas loss ofBhlhb5 has no effect on the expression
of NeuroD, and Math5,Mash1 and Ngn1 (Atoh7, Ascl1 and Neurog2,
respectively – allMouse Genome Informatics), Math3 or Chx10, it
does lead to thereduced generation of GABAergic amacrine and OFF-CB
subtypes.These studies strongly argue for the crucial role of
Bhlhb5 as a factordownstream of the bHLH-class of retinogenic
factors in thespecification of amacrine and bipolar subtypes.
MATERIALS AND METHODSAnimalsThe Math5lacZ and Math5GFP knock-in
mice were generated previously(Wang et al., 2001). To generate the
Bhlhb5lacZ allele, genomic sequenceswere isolated from a mouse
129S6 (formally 129SvEvTac) BAC library(CHORI) using Bhlhb5 coding
sequences as a probe. The Bhlhb5lacZ
targeting construct was generated by inserting 4.0 kb of
5�-flankingsequences that ends immediately upstream of the
translational initiation
codon ATG and 3.6 kb of 3�-flanking sequences into the HindIII
and theXbaI sites of the pKI-lacZ vector, respectively (see Fig. S2
in thesupplementary material). The knock-in construct removed the
entire Bhlhb5coding sequences and placed the lacZ reporter gene
under the control ofBhlhb5 regulatory sequences. To generate
Bhlhb5lacZ knock-in mice, anAscI-linearized Bhlhb5lacZ targeting
construct was electroporated into AB1embryonic stem (ES) cells (a
gift from Dr A. Bradley, Baylor College ofMedicine, Houston, USA).
Three targeted ES clones were obtained from atotal of 192 G418- and
FIAU-resistant ES clones. Genotypes of targetedclones were
confirmed by Southern blotting. Two targeted ES clones wereinjected
into C57BL/6J blastocysts to generate mouse chimeras
andheterozygous Bhlhb5lacZ/+ mice were generated in a 129S6 and
C57BL/6Jmixed background as previously described (Gan et al., 1999;
Gan et al.,1996). PCR methods were used to genotype mice from
subsequent breedingof Bhlhb5lacZ/+ heterozygotes. The PCR primers
used to identify the wild-type Bhlhb5 mice were Bhlhb5-2F
5�-GACAGCGACGGCCGCT-3� andBhlhb5-2R 5�-GTGCACTGTTTGCAG-3�, and the
lacZ knock-in allele5�-AGGGCCGCAAGAAAACTATCC-3� and
5�-ACTTCGGCACCTTA-CGCTTCTTCT-3�. Embryos were designated as E0.5 at
noon on the day atwhich vaginal plugs were observed. All animal
procedures in this study wereapproved by the University Committee
of Animal Resources (UCAR) at theUniversity of Rochester.
Histochemistry, immunohistochemistry, in situ hybridization,
BrdUlabeling and X-Gal stainingStaged mouse embryos were dissected
and fixed in 4%paraformaldehyde/PBS (PFA) at 4°C for 1-2 hours. The
samples were thenembedded and frozen in OCT medium (Tissue-Tek) and
sectioned at 14 �m.BrdU pulse-labeling and Hematoxylin and Eosin
staining were performedas described (Pan et al., 2005).
�-Galactosidase activity was determined byX-Gal staining as
previously described (Gan et al., 1999). For in situhybridization
experiments, 20 �m cryosections were used as previouslydescribed
(Li and Joyner, 2001). The working dilutions and sources
ofantibodies used in this study were: goat anti-Bhlhb5 (1:1000,
Santa Cruz),mouse anti-bromodeoxyuridine (BrdU) (1:50,
Developmental StudiesHybridoma Bank), mouse anti-Brn3a (1:100,
Santa Cruz), goat anti-Brn3b(1:2000, Santa Cruz), mouse
anti-calbindin (1:5000, Sigma), rabbit anti-calretinin (1:2000,
Oncogene), rabbit anti-activated caspase-3 (1:200, BDPharmingen),
anti-ChAT (choline acetyltransferase, 1:200, Chemicon),sheep
anti-Chx10 (1:200, Exalpha), rabbit anti-cyclin D3 (1:200,
SantaCruz), mouse anti-GAD65 (glutamate decarboxylase 65 kD, 1:200,
BDBiosciences), mouse anti-�-galactosidase (LacZ) (1:500, DSHB),
rabbitanti-LacZ (1:500, Chemicon), mouse anti-Isl1/2 (1:400, DSHB),
goat anti-NeuroD (1:500, E-17, Santa Cruz), rabbit anti-NK3R
(1:200) (Chow et al.,2001), rabbit anti-parvalbulmin (1:200,
Sigma), rabbit anti-phosphorylatedhistone H3 (1:400, Santa Cruz),
and mouse anti-Pax6 (1:200, DSHB), rabbitanti-PKC� (1:5000, Sigma),
rabbit anti-Prox1 (1:1000, Covance), rabbitanti-recoverin (1:200)
(Chow et al., 2001), mouse anti-syntaxin (1:5000,Sigma), rabbit
anti-TH (tyrosine hydroxylase, 1:200, Chemicon), rabbit anti-Vsx1
(1:100) (Chow et al., 2001). Alexa-conjugated secondary
antibodies(Molecular Probes) were used at a dilution of 1:1000.
Images were capturedwith a Zeiss Axioplan microscope. Whole-Mount
immunostaining wasperformed as described (Wang et al., 2001), and
was scored using a confocalmicroscope. To quantify the number of
different cell type-specific markerson sections of retina, three or
more age-matched retinas were analyzed foreach cell type. All data
are presented as the mean±s.d. Statistical analysiswas performed
using two-sample Student’s t test for unequal variances.
RESULTSExpression of Bhlhb5 during retinal developmentThe onset
of Bhlhb5 expression was detected at E11.5 within theneuroblast
layer (NBL) of the central retina (Fig. 1A). Asretinogenesis
progressed from central to peripheral retina from E12to E15.5,
Bhlhb5 expression expanded to the entire retina with themajority of
Bhlhb5+ cells being detected in the proliferating NBL(Fig.
1B-D,J-M). From E17.5 to P0, Bhlhb5 expression becamerestricted to
the GCL and to the inner boundary of the NBL,
RESEARCH ARTICLE Development 133 (24)
-
DEVELO
PMENT
presumably the newly formed ACL (Fig. 1E,F,N,O). In
situhybridization and immunostaining experiments revealed
identicalexpression profiles for Bhlhb5 mRNA and protein,
indicating thatBhlhb5 expression is mostly regulated at the
transcriptional level. AtP7, Bhlhb5 expression became localized to
three distinctive rows:two in the INL and one in the GCL, and this
expression pattern wasmaintained in the adult retina (Fig. 1G-I,
arrowheads). In the INL,Bhlhb5+ cells were divided into two groups
based on theirexpression levels and sub-laminar locations: a higher
expressionlevel in cells at the inner boundary (presumptive
amacrine cells) anda lower level in cells at the outer boundary
(presumptive bipolarand/or horizontal cells).
To test whether Bhlhb5 is expressed in progenitors or in
nascentneurons, co-localization of Bhlhb5 with bromodeoxyuridine
(BrdU),an S-phase marker, and with phosphorylated histone 3
(Ser-10), anM-phase marker, was performed in E12.5 and E13.5
retinas. Bhlhb5expression was absent in the vast majority of
proliferating cells in S-and M-phase (Fig. 1P-U). The expression of
Bhlhb5 in post-mitoticcells in the NBL, and its expression in
selective groups of cells of theINL and the GCL at later stages,
suggest that Bhlhb5 could play arole in the differentiation of
specific retinal subtypes.
Restricted expression of Bhlhb5 in GABAergicamacrine and OFF-CB
subtypesWe further determined the identities of Bhlhb5+ cells by
co-immunolabeling adult mouse retinas with anti-Bhlhb5 and cell
type-specific markers. Strongly anti-Bhlhb5-labeled cells were
mostlydetected in the ACL and the GCL, and somewhat weakly
labeledcells within the outer boundary of the INL (Fig. 2A).
Co-labeling ofBhlhb5 with the pan-amacrine cell marker Pax6
demonstrated thatall Bhlhb5+ cells in the ACL and in the GCL were
Pax6+ (Fig. 2A-C), implying their amacrine identity. The absence of
Brn3a (Pou4f1– Mouse Genome Informatics) and cyclin D3 expression
in Bhllhb5+
cells further excluded their identity as ganglion and Müller
cells (seeFig. S1A-A�,I-I� in the supplementary material). To
further definethe subtypes of Bhlhb5+ amacrine cells, we
double-labeled retinaswith anti-Bhlhb5 and amacrine
subtype-specific markers. Whereasnone of the Bhlhb5+ amacrine cells
expressed markers forcholinergic amacrine (Isl1 and ChAT) (see Fig.
S1B-B�,C-C� in thesupplementary material), dopaminergic amacrine
(TH) (see Fig.S1D-D� in the supplementary material), calretinin
(Fig. S1E-E� inthe supplementary material), or AII amacrine
(parvalbumin) (seeFig. S1F-F� in the supplementary material)
subtypes, a majority ofBhlhb5+ cells expressed the GABAergic marker
GAD65 (Gad2 –Mouse Genome Informatics), indicating that the Bhlhb5+
amacrinecells were mostly of the GABAergic subtype (Fig. 2D-F).
Inaddition, the Prox1+ displaced amacrine cells in the GCL
wereBhlhb5+ (Fig. 2G-I, arrows). The co-localization of Prox1+
andBhlhb5+ cells in the outer boundary of the INL implied
theiridentities as bipolar or horizontal cells (Fig. 2G-I,
arrowheads).
4817RESEARCH ARTICLEBhlhb5 in retinal subtype specification
Fig. 1. Expression profile of Bhlhb5 in retinogenesis.
Retinalsections from the indicated developmental stages were
immunolabeledwith anti-Bhlhb5 (green) and the nuclei
counter-stained with PropidiumIodide (PI, red) (A-I) or probed with
a Bhlhb5 in situ probe (J-O). Theonset of Bhlhb5 expression starts
at E11.5 in the central retina (A). AtE12.5 to E15.5, Bhlhb5
expression expands toward the peripheral retinaand is mostly
detected in cells in the NBL (B-D,J-M). At E17.5 to P0,Bhlhb5
expression becomes localized in the inner boundary of the NBLand
the GCL (E,F,N,O). Inserts in E and F show the enlarged view of
thecorresponding boxed regions. At P7 to P28, two rows of
Bhlhb5expression are seen in the INL and one in the GCL
(arrowheads, G-I).(P-U) Bhlhb5 expression is mostly observed in
post-mitotic cells of thedeveloping retina. Anti-Bhlhb5 (green)
labeling of E12.5 retina showsBhlhb5 in nuclei of cells in the NBL
(P) and anti-BrdU (red) labels thenuclei of proliferating cells at
S-phase (Q). (R) Overlay image of P and Q.(S-U) Anti-Bhlhb5 (S,
red) and anti-phosphorylated histone H3 (PH3) (T,green) show that
Bhlhb5+ cells are mostly negative for PH3 labeling.Abbreviations
for this and other figures: L, lens; NBL, neuroblast layer;GCL,
ganglion cell layer; IPL, inner plexiform layer; INL, inner
nuclearlayer; OPL, outer plexiform layer; ONL, outer nuclear layer.
Scale bars:100 �m.
-
DEVELO
PMENT
4818
Nevertheless, Bhlhb5 was not co-expressed with the horizontal
cellmarker calbindin-28K (see Fig. S1H-H� in the
supplementarymaterial). Co-immunolabeling with anti-Bhlhb5 and an
antibody toChx10, a pan-bipolar cell marker, demonstrated that
Bhlhb5 wasexpressed in bipolar cells (Fig. 2J-L, arrowheads).
Carefulexamination of the double-labeled bipolar cells revealed
that Bhlhb5was expressed in those bipolar cells with a lower level
of Chx10 butnot those with a higher level of Chx10 (Fig. 2J-L
inserts, asterisks).Additional labeling experiments showed that
Bhlhb5+ cells did notexpress the RB cell-specific marker, PKC� (see
Fig. S1G-G� in thesupplementary material). Rather, all of these
Bhlhb5+ bipolar cellswere labeled with the CB-specific marker Vsx1
and representedapproximately one third (37%) of the Vsx1+ CB cell
population (Fig.2M-O, arrowheads). As Vsx1 is expressed in 60-70%
of all CB cells(Chow et al., 2004), Bhlhb5 is expressed in
approximately 21-25%of CB cells. Furthermore, whereas not all
recoverin+ Type 2 OFF-CB cells (red arrowheads) expressed Bhlhb5
(white arrowheads), allBhlhb5+ CB cells were recoverin+ (Fig.
2P-R), confirming theseBhlhb5+ bipolar cells as Type 2 OFF-CB
cells. In conclusion,Bhlhb5 is expressed selectively in GABAergic
amacrine and Type 2OFF-CB cells in the adult retina.
Retinal defects in Bhlhb5-null miceTo investigate the role of
Bhlhb5 in retinogenesis in vivo, wegenerated a targeted deletion
allele of Bhlhb5 by removing the entireBhlhb5 ORF (see Fig. S2 in
the supplementary material). Theresulting heterozygous Bhlhb5lacZ/+
mice were normal and showedno discernible defects. The homozygous
Bhlhb5lacZ/lacZ mutantswere born indistinguishable from the
wild-type or heterozygous
littermates at birth. Examination of the offsprings from
heterozygousintercrosses revealed that the null mutants were
fertile and were bornin a normal Mendelian ratio with 46 wild type
(19.1%), 130Bhlhb5lacZ/+ (53.9%) and 65 Bhlhb5lacZ/lacZ (27.0%).
However,Bhlhb5-null mice displayed signs of slower weight gain than
thewild-type or heterozygous littermates at approximately 3 weeks
ofage, and developed skin lesions between 1-2 months of age (data
notshown).
To determine the role of Bhlhb5 in retinogenesis, we
firstexamined the retinas by Hematoxylin and Eosin staining.
Whereasno noticeable change in the thickness and laminar
organization wasfound in Bhlhb5-null retinas during embryogenesis
(Fig. 3A-F,K),there was a significant reduction in the thickness of
the INLpostnatally (Fig. 3G-K). Given the restriction of Bhlhb5
expressionto selective amacrine and CB subtypes, the decrease in
the INL ofBhlhb5-null mice indicated a loss of these interneuron
subtypes.Therefore, we analyzed the changes in specific retinal
subtypesusing subtype-specific markers at P21, a time when all
retinal cellsare generated and mature in mice. Anti-Pax6 labeling
revealed thatthe total number of amacrine cells was reduced in the
INL and theGCL (Fig. 4A,G). Immunolabeling studies demonstrated a
lossof 44.2±6% GABAergic amacrine cells, a significant reductionof
TH+ amacrine cells, and the absence of Prox1+ displacedamacrine
cells, respectively (Fig. 4B-D,H-J and see Fig. S3in the
supplementary material). Although the anti-ChATimmunolabeling was
somewhat weak in Bhlhb5-null retina (Fig.4E,K), the number of ChAT+
cells was unchanged (Fig. 4M,S andsee Fig. S3 in the supplementary
material). Similarly, no overtchanges in the number of AII (Prox1+)
and calretinin+ amacrine
RESEARCH ARTICLE Development 133 (24)
Fig. 2. Expression of Bhlhb5 in GABAergic amacrine and OFF-cone
bipolar subtypes. Sections from P28 mouse retinas were
double-immunolabeled with anti-Bhlhb5 (red) and subtype-specific
markers (green) as indicated. (A-C) The Bhlhb5+ cells in the GCL
and the ACL are Pax6+
amacrine cells. (D-F) Bhlhb5 is expressed in GAD65+ GABAergic
amacrine cells. (G-I) Bhlhb5 is co-expressed with Prox1 in
displaced amacrine cells(arrows) in the GCL and bipolar cells
(arrowheads) in the INL. (J-L) Bhlhb5 is co-expressed with Chx10 in
bipolar cells (arrowheads). Inserts show thedouble-labeling of
bipolar cells (asterisks) at high magnification. (M-O) All Bhlhb5+
bipolar cells are Vsx1+ cone bipolar cells (white arrowheads).(P-R)
All Bhlhb5+ bipolar cells (white arrowheads) are recoverin+ Type 2
OFF-bipolar cells; red arrowheads indicate the OFF-bipolar
cellsimmunoreactive to recoverin only. Scale bar: 50 �m.
-
DEVELO
PMENT
subtypes were observed (Fig. 4D,F,J,L, and see Fig. S3 in
thesupplementary material). Among the bipolar subtypes, the
Vsx1+
CB subtype was reduced by 36.3±7% (Fig. 4N,T and see Fig. S3in
the supplementary material) whereas the PKC�+ and Go�+
bipolar cells (Fig. 4O,P,U,V) and the Brn3b+ and Brn3a+ RGCs
were unaffected (Fig. 4Q,R,W,X). Altogether, our
resultsdemonstrated that targeted deletion of Bhlhb5 resulted in
thereduction of specific retinal subtypes, particularly the
CB,GABAergic and displaced amacrine subtypes that normallyexpress
Bhlhb5.
4819RESEARCH ARTICLEBhlhb5 in retinal subtype specification
Fig. 3. Developmental abnormality of Bhlhb5-null retinas. (A-J)
Retinal sections from Bhlhb5–/– and wild-type control retinas at
indicateddevelopmental stages were stained with Haemotoxylin and
Eosin. Compared with the control retina (A,C,E), no overt change in
retinal thicknessand laminar organization is seen in the mutant
(B,D,F) from E15.5-P0. At P14 to P28, the INL of Bhlhb5-null
retinas is thinner and the number ofcells in the INL is reduced by
approximately 40% (G-J). (K) Quantitation of cells in the INL and
the GCL per 250 �m length of retinal section atE17.5 to P28. Each
bar represents the mean±s.d. for three or more retinas. Scale bars:
50 �m.
Fig. 4. Selective loss of retinal cell subtypes in Bhlhb5-null
retinas. (A-X) Sections from P21 mouse retinas were immunolabeled
with subtype-specific markers (green) and nuclear-counterstained
with PI (red). Loss of Bhlhb5 leads to a severe loss of amacrine
cells immunoreactive for Pax6(A,G) and GAD65 (B,H) and to an
absence of TH+ (C,I) and Prox1+ (D,J) amacrine cells. There is no
overt change in the number of amacrine cellsimmunoreactive to ChAT
(E,K), calretinin (F,L) and Isl1 (M,S). A significant loss of Vsx1+
CB cells was observed in Bhlhb5-null retina (N,T). However,no
discernible change is seen in the number of PKC�+ RB (O,U) and Go�+
ON-bipolar (P,V) cells, and Brn3b+ (Q,W) and Brn3a+ (R,X) ganglion
cells.Scale bar: 100 �m.
-
DEVELO
PMENT
4820
Impaired genesis of GABAergic amacrine and CBcells in the
absence of Bhlhb5The close association of Bhlhb5 expression with
the genesis ofamacrine and bipolar cells suggested a role for
Bhlhb5 in regulatingretinal cell differentiation. We then tested
whether the formation ofamacrine and bipolar subtypes was impaired
in the absence ofBhlhb5. In mice, most bipolar cells are generated
postnatally; Vsx1expression is first detected in presumptive CB
cells from P5 to P6(Chow et al., 2001), thus serving as a suitable
early marker for CBcells. Whereas no overt change in Chx10
expression was observedin bipolar cells and progenitors in
Bhlhb5-null retinas (Fig. 5D,H),loss of Bhlhb5 resulted in a
reduction of approximately 35% inVsx1+ CB cells (Fig. 5A,E), a
value comparable to the reduction inP21 retinas (Fig. 4N,T). A
comparable loss of cells labeled withantibody to recoverin, a Type
2 OFF-bipolar cell marker, andantibody to NK3R (Tacr3 – Mouse
Genome Informatics), a Type 1and 2 OFF-bipolar cell marker (Chow et
al., 2001), was also seen inBhlhb5-null retinas (Fig. 5B,C,F,G).
Similarly, immunolabeling ofretinal sections at P0 and P6 revealed
the agenesis of Prox1+
displaced amacrine cells in the GCL of Bhlhb5-null mice,
whereasthe generation of Prox1+ horizontal, bipolar and amacrine
cells inthe INL was not affected (Fig. 5I,J,M,N). Additionally, the
numberof amacrine cells immunoreactive for Pax6 and GAD65 were
greatlyreduced in P6 and P7 retinas (Fig. 5K,L,O,P), implying a
reducedgeneration of selective GABAergic subtypes. We also tested
thepossibility that GABAergic amacrine and OFF-CB subtypes
wereinitially generated but later died of apoptosis in Bhlhb5-null
retinas.Anti-activated-caspase-3 immunolabeling revealed no
increase in
apoptotic cells in Bhlhb5-null retinas from E15.5 to P10 (see
Fig.S4A-D in the supplementary material and data not shown).
Takentogether, targeted deletion of Bhlhb5 specifically diminished
thegeneration of selective OFF-CB and GABAergic amacrine
subtypes.
Upregulation of Bhlhb5 and NeuroD in math5-nullretinasPrevious
studies have indicated that NeuroD and Math3 playredundant roles in
the differentiation of amacrine cells (Inoue et al.,2002). To
determine the genetic relationship of Bhlhb5 and NeuroDin the
amacrine differentiation pathway, we investigated whetherBhlhb5
co-expressed with NeuroD in developing retinas.Immunolabeling
experiments demonstrated that although bothNeuroD- and
Bhlhb5-expressing cells were similarly distributedthroughout the
NBL of E13.5 retina, NeuroD was detected in agreater number of
cells than Bhlhb5 (Fig. 6A-D), and virtually allBhlhb5+ cells
expressed NeuroD (Fig. 6E,F). We then tested whetherthe absence of
Bhlhb5 could affect the expression of NeuroD andMath3. As shown by
anti-NeuroD labeling and in situ hybridizationfor Math3, the
expression of both NeuroD and Math3 was detectedmostly in the NBL
of developing retinas and was unaltered inBhlhb5-null retinas (Fig.
6G,H,L,M). Similarly, the expression ofother retinogenic factors
such as Math5, Ngn2 and Mash1 wasunaffected by the targeted
deletion of Bhlhb5 (Fig. 6I,K,N-P).Therefore, it is unlikely that
Bhlhb5 functions upstream of theseretinogenic factors during
retinal neurogenesis. Rather, it could actdownstream of these
factors to control the differentiation of retinalsubtypes.
RESEARCH ARTICLE Development 133 (24)
Fig. 5. Decreased genesis of Type2 cone bipolar, GABAergic
anddisplaced amacrine cells.(A-H) Immunolabeling of retinalsections
at P6 with bipolar subtype-specific markers reveals a dramaticdrop
in the genesis of CB cellsimmunoreactive for Vsx1 (A,E),recoverin
(B,F, white arrowheads)and NK3R (C,G, white arrowheads)in
Bhlhb5-null mice, whereas thetotal number of bipolar cells
labeledby Chx10 is unchanged (D,H).(I,J,M,N) Anti-Prox1 labeling
ofretinal sections at P0 (I,M) and P6(J,N) demonstrates the absence
ofdisplaced amacrine genesis in theGCL (white arrowheads) in
Bhlhb5-null mice, whereas the Prox1+
horizontal cells (red arrowheads) inBhlhb5-null mice are
formednormally. (K,L,O,P) Anti-Pax6 (K,O)and anti-GAD65 (L,P)
labeling alsodemonstrate a significant decreasein the genesis of
amacrine cells inthe INL. Scale bar: 100 �m.
-
DEVELO
PMENT
Previously published studies have shown that the loss of
RGC-determining factor Math5 in mice, or of lakritz in zebrafish,
resultsin an increase in displaced amacrine cells, suggesting that
theexpression of Math5 or lakritz suppresses the amacrine cell
fateduring normal retinogenesis and that null mutations in these
genescause a cell fate conversion from RGC to displaced amacrine
cellfates (Kay et al., 2001; Wang et al., 2001; Yang et al., 2003).
Bhlhb5expression in displaced amacrine cell lineages provided us
with atool to examine the molecular basis of this cell fate change.
Whole-mount immunolabeling of normal and Math5-null retinas at
P21showed that although the Bhlhb5+ CB cells in the INL
weredramatically reduced in Math5-null retinas, the number of
Bhlhb5+
displaced amacrine cells was increased sevenfold (Fig. 7A-H).
Thisincreased number of Bhlhb5+ cells in Math5-null retina was
detectedthroughout embryogenesis (see Fig. S5 in the
supplementarymaterial). We further examined whether Math5 could
suppress thegeneration of amacrine cells by negatively regulating
the expressionof Bhlhb5 and NeuroD within the Math5+ cell lineage.
Theexpression of Math5 mRNA is transiently detected in progenitors
ofthe NBL and is not suitable to trace Math5+ lineage (Brown et
al.,1998; Yang et al., 2003). We have previously shown that the
nuclearMath5-LacZ knock-in reporter protein is relatively stable,
servingas a suitable marker to trace Math5+ cells in the NBL and in
nascentRGCs (Wang et al., 2001). Co-localization of Bhlhb5 and
LacZrevealed that loss of Math5 resulted in a significant increase
in thenumber of cells expressing Bhlhb5 in E13 retina and that
themajority of these Bhlhb5+ cells expressed LacZ (Fig. 7I-P).
Asimilar increase in retinal cells expressing LacZ and NeuroD
was
observed in E13.5 Math5-null retina (Fig. 7Q-X). Therefore,
theincreased generation of displaced amacrine cells in
Math5-nullretinas corresponded with the premature expression of
NeuroD andBhlhb5 in the Math5+ cell lineage.
DISCUSSIONThe expression of specific retinogenic bHLH factors
plays crucialroles in the cell fate selection of retinal
progenitors. In this report,we have identified that a crucial
aspect of retinogenesis, namely theformation of specific amacrine
and bipolar subtypes, depends on theactivity of Bhlhb5. Targeted
disruption of Bhlhb5 causes theselective loss of GABAergic amacrine
and Type 2 OFF-cone bipolarcells. Although loss of Bhlhb5 has no
impact on the expression ofretinogenic bHLH factors, Bhlhb5
expression co-localizes with thatof NeuroD. Moreover, Math5
negatively regulates the expression ofBhlhb5 and NeuroD. Therefore,
our findings establish a key step inthe molecular mechanism of
retinogenesis and define a bHLHcascade that connects the regulation
of pan-neuronal typedetermination by retinogenic factors with the
subsequent formationof retinal neuronal subtypes.
Bhlhb5 expression as an early, specific marker forGABAergic
amacrine and OFF-CB subtypesRetinal neurons are born in the NBL and
migrate to their definedlaminar layers within the retina soon after
their birth. The early onsetand the spatiotemporal profile of
Bhlhb5 expression duringembryogenesis and early postnatal
development coincide with thegenesis of amacrine and bipolar cells,
respectively (Fig. 1). Our
4821RESEARCH ARTICLEBhlhb5 in retinal subtype specification
Fig. 6. Normal expression of retinogenic bHLH factors in
Bhlhb5-null retinas. (A-F) Immunolabeling shows a largely
overlapping expressionof Bhlhb5 (green) and NeuroD (red) in E13
wild-type retina. B, D and F show the enlarged view of the
corresponding boxed regions in A, C and E,respectively. (G,L)
Anti-NeuroD labeling reveals no change in NeuroD expression in
Bhlhb5-null retinas at E13. (H-K,M-P) Similarly, the expression
ofMath3 (H,M), Ngn2 (I,N), Math5 (J,O) and Mash1 (K,P) is
unaffected in Bhlhb5-null retina at E14.5 as assessed by in situ
hybridization. Scale bars:100 �m.
-
DEVELO
PMENT
4822
expression studies demonstrate that the expression of Bhlhb5
isunambiguously restricted to selective GABAergic amacrine andType
2 OFF-CB subtypes (Fig. 2). To the best of our knowledge,Bhlhb5 is
the earliest transcription factor to be specifically expressedin
these amacrine and bipolar subtypes during early
retinogenesis.Thus, Bhlhb5 expression serves as the earliest
subtype-specificcellular marker for these cells. Additionally,
because not allrecoverin+ Type 2 OFF-CB cells express Bhlhb5 or are
affected inBhlhb5-null retinas, Type 2 bipolar cells can be further
divided intotwo subtypes based on Bhlhb5 expression.
Requirement for Bhlhb5 in the generation of Type2 OFF-CB and
selective GABAergic amacrinesubtypesPrevious studies have shown
that Math3 and Mash1, along withChx10, play an essential role in
regulating the generation of allbipolar cells, but not in defining
bipolar subtypes (Burmeister etal., 1996; Hatakeyama et al., 2001).
Targeted mutagenesis studieshave also revealed that though not
playing a role in the initial
bipolar genesis, Vsx1 and Bhlhb4 are required for the
terminaldifferentiation and maturation of CB and RB subtypes,
respectively(Bramblett et al., 2002; Cheng et al., 2005; Chow et
al., 2004;Ohtoshi et al., 2004). A recent study shows that the null
mutation ofthe Iroquois homeobox gene Irx5 in mice causes a partial
loss ofType 2 and Type 3 OFF CB cells. However, it is unclear
whetherthese defects result from a failure in their specification
ordifferentiation (Cheng et al., 2005). In this study, we
demonstratethat Bhlhb5 expression in the INL is closely associated
with theperiod of bipolar generation in the first postnatal week
and that itsexpression is tightly restricted in Type 2 OFF-CB
cells. In Bhlhb5-null retinas, there is a significant reduction in
Vsx1+ and recoverin+
Type 2 CB cells (Figs 4, 5), indicating that Bhlhb5 is indeed
requiredfor the genesis of a majority of Type 2 OFF-CB cells and
that it actsupstream of Vsx1 during CB development. Although the
geneticrelationship between Bhlhb5 and Mash1 or Math3 needs to
befurther examined in mice lacking Mash1 and Math3, the
unalteredexpression of Mash1 and Math3 in Bhlhb5-null retinas
suggests thatBhlhb5 is unlikely to function upstream of Mash1 and
Math3 (Fig.
RESEARCH ARTICLE Development 133 (24)
Fig. 7. Upregulation of Bhlhb5 and NeuroD expression in the
Math5-LacZ cell lineage in Math5-null retina. (A-H) Retinal
sectionsimmunolabeled with anti-Bhlhb5 (green) and nuclei
counterstained with PI (red) show that loss of Math5 results in a
large increase in Bhlhb5+
displaced amacrine cells and a significant decrease in Bhlhb5+
bipolar cells. (A,E) Low magnification of retinas at P21. (B,F)
Enlarged view of thecorresponding boxed regions in A and E,
respectively. (C,G) Confocal sections of Bhlhb5+ displaced amacrine
cells in the GCL. (D,H) Confocalsections of Bhlhb5+ cells in the
INL. (I-P) Co-immunolabeling of Bhlhb5 (red) and LacZ (green)
indicates that loss of Math5 results in a significantincrease in
the number of cells expressing Bhlhb5 in E13 retina and that a
majority of these Bhlhb5+ cells express Math5-LacZ. The boxed areas
in Kand O are shown at high magnification in L and P. (Q-X)
Similarly, an increase in retinal cells expressing Math5-LacZ and
NeuroD is observed inE13.5 Math5-null mice. The boxed areas of S
and W are shown at high magnification in T and X. The Math5-GFP
fluorescence is undetectableunder the fixation and detection
conditions used and all green signals are derived from either LacZ
or anti-Bhlhb5 staining. Scale bars: in A, 200 �mfor A and E; in
all other panels, 100 �m.
-
DEVELO
PMENT
6). Instead, given the requirement for Mash1 and Math3
inpan-bipolar development, Bhlhb5 can conceivably play a
roledownstream of Mash1 and Math3 to regulate the formation
ofbipolar subtypes.
Similarly, whereas NeuroD and Math3 are essential for
thegeneration of amacrine cells, our studies have shown that
Bhlhb5is expressed only in selective GABAergic and displaced
amacrinesubtypes, and that targeted deletion of Bhlhb5 leads to a
specificreduction in these amacrine subtypes (Fig. 4 and see Fig.
S3 in thesupplementary material). During early retinogenesis,
Bhlhb5expression in the NBL is mostly confined to cells that
expressNeuroD and loss of Bhlhb5 does not alter the retinal
expressionof NeuroD and Math3 (Fig. 6), suggesting that Bhlhb5
isunlikely to function upstream of NeuroD and Math3
inretinogenesis. Although further studies are needed to testwhether
Bhlhb5 is downstream of NeuroD and Math3 and, inparticular, whether
its expression in retina is reduced in micedeficient for NeuroD and
Math3, it is plausible that Bhlhb5 isexpressed in a subset of
NeuroD+ cells after they acquire a pan-amacrine identity to render
these cells a GABAergic amacrinesubtype identity.
During retinal development, all retinal cell types are
generatedfrom the same pool of multipotent progenitors. The
bHLH-classretinogenic factors have been shown to play crucial roles
inregulating the cell fate choices of progenitors and loss of
thesefactors frequently results in cell fate phenotypes. Math3
andNeuroD double-null retinas lack amacrine cells but gain moreRGCs
and Müller cells (Inoue et al., 2002). Loss of both Mash1and Math3
leads to a cell fate switch from bipolar to Müller cells(Tomita et
al., 2000). In this study, the loss of Type 2 OFF-CB andselective
GABAergic amacrine cells in Bhlhb5-null retinas was notaccompanied
by an overt increase in other retinal cell types (Fig.4). One
explanation for the lack of a readily identifiable cell fateswitch
is that the amacrine and CB subtypes affected in Bhlhb5-null
retinas only represent a small population of retinal cells andthat
any consequent cell fate change would be less obvious.Additionally,
analysis of cell proliferation during retinogenesisrevealed a
slight decrease in the number of proliferating cells inpostnatal
Bhlhb5-null retinas (see Fig. S4E-J in the supplementarymaterial).
It is possible that the reduced cell proliferation couldalso
contribute to the loss of late-born cells in the INL of Bhlhb5-null
retinas. To detect a possible cell fate change, Cre
recombinasecould be used to replace the Bhlhb5 allele. Cell lineage
analysisusing Bhlhb5Cre knock-in and lineage-reporter mice could be
usedto trace the fates of Bhlhb5-expressing cells. Comparison of
theretinal cell fates of Bhlhb5+ lineage in the presence and
absence ofBhlhb5 would accurately reveal whether Bhlhb5 is
exclusivelyexpressed in selective OFF-CB and GABAergic amacrine
lineages,and whether Bhlhb5-expressing cells switch fates in the
absenceof Bhlhb5. Our results have also shown that the
Bhlhb5-nullmutation leads to a reduction in TH+ dopaminergic
amacrine cells(Fig. 4). It is possible that such a reduction
results, as a non-cell-autonomous mechanism of Bhlhb5, from the
loss of otheramacrine and bipolar subtypes. Alternatively, the loss
could bethrough a cell-autonomous mechanism as Bhlhb5 could
betransiently expressed in the dopaminergic amacrine cell
lineageand be essential for their development. Due to the lack
ofembryonic and early postnatal markers for dopaminergic
amacrinecells, we are unable to distinguish these two possibilities
in thisstudy. Future cell lineage analysis with Bhlhb5Cre
knock-inmice could effectively show whether Bhlhb5 is expressed
indopaminergic amacrine lineage.
Genetic cascade of bHLH transcription factors inthe
determination of retinal cell typesWe have previously shown that
during normal retinal development,Math5+ cell lineage contributes
to retinal cell types includingganglion, cone, horizontal and
amacrine cells and that targeteddeletion of Math5 leads to a cell
fate conversion from RGC toamacrine cells (Wang et al., 2001; Yang
et al., 2003). We havehypothesized that in addition to promoting
RGC differentiation,Math5 negatively regulates amacrine
differentiation pathways bysuppressing the expression of key
transcription factors, particularlythe bHLH-class factors. The
upregulation of amacrine factors
4823RESEARCH ARTICLEBhlhb5 in retinal subtype specification
Progenitors
RGC competent precurcors (Math5+)
GABAergic amacrine
Other amacrine
Ganglion
Horizontal
Rod
Cone
Cone and amacrine precursors (Math5-)
RGC precursors (Math5+)
Horizontal precursors (Math5-/Math3+/ NeuroD+/Ngn2+)
Amacrine precursors (Math3+/NeuroD+)
Bhlhb5
Bhlhb5
M ller precursors
Rod precursors (Math3+/NeuroD+)
Bipolar precursors (Math3+/Mash1+)
M ller Other bipolar
Type 2 OFF- cone bipolar
Cone precursors
Rod, bipolar and M ller precursors (Math5-)
Fig. 8. A model for the role of Bhlhb5 in the generation
ofGABAergic amacrine and Type 2 CB cells. The retinal
progenitorsexit the cell cycle and are divided into the Math5+ and
Math5–
precursor pools based on the expression of Math5. Precursors
with thetransient activation of Math5 are RGC-competent. Some of
theseprecursors choose the RGC differentiation pathway and generate
nearlyall RGCs. The remaining precursors lose RGC-competence when
Math5expression ceases, express other retinogenic bHLH factors and
generatehorizontal cells or, along with precursors from the Math5–
pool,produce amacrine and cone cells. Together with NeuroD and
Math3,Bhlhb5 determines the genesis GABAergic amacrine cells. The
rod,bipolar and Müller cells are derived from the Math5– pool of
precursorsand Bhlhb5 expression provides the precursors with
competence todifferentiate into Type 2 bipolar cells.
-
DEVELO
PMENT
4824
Bhlhb5 and NeuroD in the absence of Math5 (Fig. 7)
providesexperimental evidence to support our hypothesis. Moreover,
wehave demonstrated that removal of Math5 leads to alleviation
ofinhibition of NeuroD and Bhlhb5 expression in the cells of
Math5+
lineage (Fig. 7). Therefore, the suppression of the
amacrinedifferentiation pathway by Math5 could be mediated through
itsnegative regulation of NeuroD and Bhlhb5 expression. Mu et
al.have also recently reported that NeuroD expression is
upregulatedin Math5-null retina (Mu et al., 2005). Furthermore, our
expressionstudies show that a group of cells exists that express
NeuroD andBhlhb5 but not Math5-LacZ in normal and Math5-null
retinas. It isnot clear whether these cells expressing NeuroD or
Bhlhb5 alonearise from Math5+ lineage as Math5-LacZ expression
onlytransiently labels the cells of Math5+ lineage (Wang et al.,
2001;Yang et al., 2003). By Math5-Cre-mediated lineage tracing,
wehave demonstrated that although Math5+ cell lineage
producesnearly all RGCs and horizontal cells and a limited number
ofphotoreceptor and amacrine cells, none of the bipolar cells
arederived from Math5+ cell lineage. Thus, these Math5– and
NeuroD+
or Bhlhb5+ cells are probably derived from a separate,
Math5-independent progenitor pool. Taken together, our expression
andtargeted deletion analyses suggest the following model
ofretinogenesis (Fig. 8). As a selective pool of retinal
progenitors exitthe cell cycle, the transient expression of Math5
in these post-mitotic precursors endows them with a short period of
RGCcompetence. During this competence period, Math5 activates
anetwork of transcription factors including Brn3b and Isl1 to
initiatethe RGC differentiation program (Yang et al., 2003).
Additionally,Math5 suppresses the non-RGC differentiation pathways
bynegatively regulating the non-RGC-specifying factors such
asNeuroD and Bhlhb5. As Math5 expression diminishes in
theprecursors uncommitted to RGC fate, these precursors lose
RGCcompetence, start to express non-RGC-specifying factors
andbecome competent to adopt amacrine, horizontal and cone
cellfates. Whereas Math5+ lineage is mostly limited to
selectiveamacrine, horizontal and cone cells, and no bipolar, rod
or Müllercells are derived from Math5+ lineage (Yang et al., 2003)
(data notshown), there must exist a separate pool of Math5-
precursors thatnever express Math5 but express other retinal cell
fate-specifyingfactors. The highly restricted expression of Bhlhb5
in amacrine andCB subtypes suggests that Bhlhb5 could play a role,
downstream ofretinogenic bHLH factors, in allowing amacrine- and
bipolar-competent precursors to adopt subtype identities by
activating theexpression of subtype-specific genes such as those
encodingGAD65 in GABAergic amacrine cells, and Vsx1, NK3R
andrecoverin in Type 2 OFF-CB cells. How Bhlhb5 functions at
thetranscriptional level remains unknown. It is conceivable that
Bhlhb5could dimerize with retinogenic or other bHLH factors to
fine-tunetheir roles in cell differentiation and to allow for
subtype distinction.It remains unknown what factors make selective
Math5+ cells adoptRGC or non-RGC fates and whether retinal cells
generated fromMath5+ lineages belong to specific retinal
subtypes.
We thank the members of the L.G.’s laboratory for helpful
discussions andtechnical assistance. This work was supported by NIH
grants EY013426 andEY015551 to L.G., a Rochester Eye Bank research
grant to L.F., the Research toPrevent Blindness unrestricted grant
to the Department of Ophthalmology atthe University of Rochester,
and funding from the Canada Research ChairsProgram and the New
Investigator Award from the Foundation FightingBlindness, Canada to
R.L.C.
Supplementary materialSupplementary material for this article is
available
athttp://dev.biologists.org/cgi/content/full/133/24/4815/DC1
ReferencesBramblett, D. E., Copeland, N. G., Jenkins, N. A. and
Tsai, M. J. (2002).
BHLHB4 is a bHLH transcriptional regulator in pancreas and brain
that marks thedimesencephalic boundary. Genomics 79, 402-412.
Bramblett, D. E., Pennesi, M. E., Wu, S. M. and Tsai, M. J.
(2004). Thetranscription factor Bhlhb4 is required for rod bipolar
cell maturation. Neuron43, 779-793.
Brown, N. L., Kanekar, S., Vetter, M. L., Tucker, P. K., Gemza,
D. L. and Glaser,T. (1998). Math5 encodes a murine basic
helix-loop-helix transcription factorexpressed during early stages
of retinal neurogenesis. Development 125, 4821-4833.
Brunelli, S., Innocenzi, A. and Cossu, G. (2003). Bhlhb5 is
expressed in the CNSand sensory organs during mouse embryonic
development. Gene Expr. Patterns3, 755-759.
Burmeister, M., Novak, J., Liang, M. Y., Basu, S., Ploder, L.,
Hawes, N. L.,Vidgen, D., Hoover, F., Goldman, D., Kalnins, V. I. et
al. (1996). Ocularretardation mouse caused by Chx10 homeobox null
allele: impaired retinalprogenitor proliferation and bipolar cell
differentiation. Nat. Genet. 12, 376-384.
Cepko, C. L. (1999). The roles of intrinsic and extrinsic cues
and bHLH genes in thedetermination of retinal cell fates. Curr.
Opin. Neurobiol. 9, 37-46.
Cheng, C. W., Chow, R. L., Lebel, M., Sakuma, R., Cheung, H.
O.,Thanabalasingham, V., Zhang, X., Bruneau, B. G., Birch, D. G.,
Hui, C. C. etal. (2005). The Iroquois homeobox gene, Irx5, is
required for retinal cone bipolarcell development. Dev. Biol. 287,
48-60.
Chow, R. L., Snow, B., Novak, J., Looser, J., Freund, C.,
Vidgen, D., Ploder, L.and McInnes, R. R. (2001). Vsx1, a rapidly
evolving paired-like homeobox geneexpressed in cone bipolar cells.
Mech. Dev. 109, 315-322.
Chow, R. L., Volgyi, B., Szilard, R. K., Ng, D., McKerlie, C.,
Bloomfield, S. A.,Birch, D. G. and McInnes, R. R. (2004). Control
of late off-center cone bipolarcell differentiation and visual
signaling by the homeobox gene Vsx1. Proc. Natl.Acad. Sci. USA 101,
1754-1759.
DeVries, S. H. and Baylor, D. A. (1995). An alternative pathway
for signal flowfrom rod photoreceptors to ganglion cells in
mammalian retina. Proc. Natl.Acad. Sci. USA 92, 10658-10662.
Gan, L., Xiang, M., Zhou, L., Wagner, D. S., Klein, W. H. and
Nathans, J.(1996). POU domain factor Brn-3b is required for the
development of a large setof retinal ganglion cells. Proc. Natl.
Acad. Sci. USA 93, 3920-3925.
Gan, L., Wang, S. W., Huang, Z. and Klein, W. H. (1999). POU
domain factorBrn-3b is essential for retinal ganglion cell
differentiation and survival but not forinitial cell fate
specification. Dev. Biol. 210, 469-480.
Ghosh, K. K., Bujan, S., Haverkamp, S., Feigenspan, A. and
Wassle, H.(2004). Types of bipolar cells in the mouse retina. J.
Comp. Neurol. 469, 70-82.
Hack, I., Peichl, L. and Brandstatter, J. H. (1999). An
alternative pathway for rodsignals in the rodent retina: rod
photoreceptors, cone bipolar cells, and thelocalization of
glutamate receptors. Proc. Natl. Acad. Sci. USA 96,
14130-14135.
Hatakeyama, J., Tomita, K., Inoue, T. and Kageyama, R. (2001).
Roles ofhomeobox and bHLH genes in specification of a retinal cell
type. Development128, 1313-1322.
Inoue, T., Hojo, M., Bessho, Y., Tano, Y., Lee, J. E. and
Kageyama, R. (2002).Math3 and NeuroD regulate amacrine cell fate
specification in the retina.Development 129, 831-842.
Jones, S. E., Jomary, C., Grist, J., Thomas, M. R. and Neal, M.
J. (1998).Expression of Pax-6 mRNA in the retinal degeneration (rd)
mouse. Biochem.Biophys. Res. Commun. 252, 236-240.
Kay, J. N., Finger-Baier, K. C., Roeser, T., Staub, W. and
Baier, H. (2001).Retinal ganglion cell genesis requires lakritz, a
zebrafish atonal Homolog.Neuron 30, 725-736.
Kim, M. H., Gunnersen, J., Augustine, C. and Tan, S. S. (2002).
Region-specificexpression of the helix-loop-helix gene BETA3 in
developing and adult brains.Mech. Dev. 114, 125-128.
Li, J. Y. and Joyner, A. L. (2001). Otx2 and Gbx2 are required
for refinement andnot induction of mid-hindbrain gene expression.
Development 128, 4979-4991.
Li, S., Mo, Z., Yang, X., Price, S. M., Shen, M. M. and Xiang,
M. (2004). Foxn4controls the genesis of amacrine and horizontal
cells by retinal progenitors.Neuron 43, 795-807.
Marquardt, T. (2003). Transcriptional control of neuronal
diversification in theretina. Prog. Retin. Eye Res. 22,
567-577.
Marquardt, T. and Gruss, P. (2002). Generating neuronal
diversity in the retina:one for nearly all. Trends Neurosci. 25,
32-38.
Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R.,
Guillemot, F.and Gruss, P. (2001). Pax6 is required for the
multipotent state of retinalprogenitor cells. Cell 105, 43-55.
Masland, R. H. (2001a). The fundamental plan of the retina. Nat.
Neurosci. 4,877-886.
Masland, R. H. (2001b). Neuronal diversity in the retina. Curr.
Opin. Neurobiol.11, 431-436.
McLellan, A. S., Langlands, K. and Kealey, T. (2002). Exhaustive
identification ofhuman class II basic helix-loop-helix proteins by
virtual library screening. GeneExpr. Patterns 2, 329-335.
Morrow, E. M., Furukawa, T., Lee, J. E. and Cepko, C. L. (1999).
NeuroD
RESEARCH ARTICLE Development 133 (24)
-
DEVELO
PMENT
regulates multiple functions in the developing neural retina in
rodent.Development 126, 23-36.
Mu, X., Fu, X., Sun, H., Beremand, P. D., Thomas, T. L. and
Klein, W. H.(2005). A gene network downstream of transcription
factor Math5 regulatesretinal progenitor cell competence and
ganglion cell fate. Dev. Biol. 280, 467-481.
Nishina, S., Kohsaka, S., Yamaguchi, Y., Handa, H., Kawakami,
A., Fujisawa,H. and Azuma, N. (1999). PAX6 expression in the
developing human eye. Br. J.Ophthalmol. 83, 723-727.
Ohtoshi, A., Wang, S. W., Maeda, H., Saszik, S. M., Frishman, L.
J., Klein, W.H. and Behringer, R. R. (2004). Regulation of retinal
cone bipolar celldifferentiation and photopic vision by the CVC
homeobox gene Vsx1. Curr. Biol.14, 530-536.
Pan, L., Yang, Z., Feng, L. and Gan, L. (2005). Functional
equivalence of Brn3POU-domain transcription factors in mouse
retinal neurogenesis. Development132, 703-712.
Peyton, M., Stellrecht, C. M., Naya, F. J., Huang, H. P.,
Samora, P. J. andTsai, M. J. (1996). BETA3, a novel
helix-loop-helix protein, can act as anegative regulator of BETA2
and MyoD-responsive genes. Mol. Cell. Biol. 16,626-633.
Tomita, K., Moriyoshi, K., Nakanishi, S., Guillemot, F. and
Kageyama, R.(2000). Mammalian achaete-scute and atonal homologs
regulate neuronalversus glial fate determination in the central
nervous system. EMBO J. 19, 5460-5472.
Wang, S. W., Kim, B. S., Ding, K., Wang, H., Sun, D., Johnson,
R. L., Klein, W.H. and Gan, L. (2001). Requirement for math5 in the
development of retinalganglion cells. Genes Dev. 15, 24-29.
Xu, Z. P., Dutra, A., Stellrecht, C. M., Wu, C., Piatigorsky, J.
and Saunders, G.F. (2002). Functional and structural
characterization of the human gene BHLHB5,encoding a basic
helix-loop-helix transcription factor. Genomics 80, 311-318.
Yang, Z., Ding, K., Pan, L., Deng, M. and Gan, L. (2003). Math5
determines thecompetence state of retinal ganglion cell
progenitors. Dev. Biol. 264, 240-254.
4825RESEARCH ARTICLEBhlhb5 in retinal subtype specification