requirement for bhlhb5 in the speciﬁcation of amacrine and cone bipolar … · subtype and nine...

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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

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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,

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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.

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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


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.

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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.


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.

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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.


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.

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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


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.

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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.


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.

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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


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.

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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

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