Takahisa Furukawa

Crx, a Novel otx-like Homeobox Gene, Shows Photoreceptor-Specific Expression and Regulates Photoreceptor Differentiation

We have isolated a novel otx-like homeobox gene, Crx, from the mouse retina. Crx expression is restricted to developing and mature photoreceptor cells. CRX bound and transactivated the sequence TAATCC/A, which is found upstream of several photoreceptor-specific genes, including the opsin genes from many species. Overexpression of Crx using a retroviral vector increased the frequency of clones containing exclusively rod photoreceptors and reduced the frequency of clones containing amacrine interneurons and Müller glial cells. In addition, presumptive photoreceptor cells expressing a dominant-negative form of CRX failed to form proper photoreceptor outer segments and terminals. Crx is a novel photoreceptor-specific transcription factor and plays a crucial role in the differentiation of photoreceptor cells.

Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor

Genes associated with inherited retinal degeneration have been found to encode proteins required for phototransduction, metabolism, or structural support of photoreceptors. Here we show that mutations in a novel photoreceptor-specific homeodomain transcription factor gene (CRX) cause an autosomal dominant form of cone-rod dystrophy (adCRD) at the CORD2 locus on chromosome 19q13. In affected members of a CORD2-linked family, the highly conserved glutamic acid at the first position of the recognition helix is replaced by alanine (E80A). In another CRD family, a 1 bp deletion (E168 [delta1 bp]) within a novel sequence, the WSP motif, predicts truncation of the C-terminal 132 residues of CRX. Mutations in the CRX gene cause adCRD either by haploinsufficiency or by a dominant negative effect and demonstrate that CRX is essential for the maintenance of mammalian photoreceptors.

rax, Hes1, and notch1 promote the formation of Müller glia by postnatal retinal progenitor cells.

We are interested in the mechanisms of glial cell development in the vertebrate central nervous system. We have identified genes that can direct the formation of glia in the retina. rax, a homeobox gene, Hes1, a basic helix-loop-helix gene, and notch1, a transmembrane receptor gene, are expressed in retinal progenitor cells, downregulated in differentiated neurons, and expressed in Müller glia. Retroviral transduction of any of these genes resulted in expression of glial markers. In contrast, misexpression of a dominant-negative Hes1 gene reduced the number of glia. Cotransfection of rax with reporter constructs containing the Hes1 or notch1 regulatory regions led to the upregulation of reporter transcription. These data suggest a regulatory heirarchy that controls the formation of glia at the expense of neurons.

Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development

Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Here, using conditional gene ablation in mice, we show that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converted differentiating photoreceptor cells to amacrine-like neurons and led to a total lack of pinealocytes in the pineal gland. We also found that Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. Our results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development.

Comprehensive Analysis of Photoreceptor Gene Expression and the Identification of Candidate Retinal Disease Genes

To identify the full set of genes expressed by mammalian rods, we conducted serial analysis of gene expression (SAGE) by using libraries generated from mature and developing mouse retina. We identified 264 uncharacterized genes that were specific to or highly enriched in rods. Nearly half of all cloned human retinal disease genes are selectively expressed in rod photoreceptors. In silico mapping of the human orthologs of genes identified in our screen revealed that 86 map within intervals containing uncloned retinal disease genes, representing 37 different loci. We expect these data will allow identification of many disease genes, and that this approach may be useful for cloning genes involved in classes of disease where cell type-specific expression of disease genes is observed.

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix–like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

Ptf1a determines horizontal and amacrine cell fates during mouse retinal development

The vertebrate neural retina comprises six classes of neurons and one class of glial cells, all derived from a population of multipotent progenitors. There is little information on the molecular mechanisms governing the specification of cell type identity from multipotent progenitors in the developing retina. We report that Ptf1a, a basic-helix-loop-helix (bHLH) transcription factor, is transiently expressed by post-mitotic precursors in the developing mouse retina. Recombination-based lineage tracing analysis in vivo revealed that Ptf1a expression marks retinal precursors with competence to exclusively produce horizontal and amacrine neurons. Inactivation of Ptf1a leads to a fate-switch in these precursors that causes them to adopt a ganglion cell fate. This mis-specification of neurons results in a complete loss of horizontal cells, a profound decrease of amacrine cells and an increase in ganglion cells. Furthermore, we identify Ptf1a as a primary downstream target for Foxn4, a forkhead transcription factor involved in the genesis of horizontal and amacrine neurons. These data, together with the previous findings on Foxn4, provide a model in which the Foxn4-Ptf1a pathway plays a central role in directing the differentiation of retinal progenitors towards horizontal and amacrine cell fates.

elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.

Misexpression of the Emx-related homeobox genes cVax and mVax2 ventralizes the retina and perturbs the retinotectal map.

The mechanisms that establish the dorsal-ventral (D-V) axis of the eye are poorly understood. We isolated two homeobox genes from mouse and chicken, mVax2 and cVax, whose expression during early eye development is restricted to the ventral retina. In chick, ectopic expression of either Vax leads to ventralization of the early retina, as assayed by expression of the transcription factors Pax2 and Tbx5, and the Eph family members EphB2, EphB3, ephrinB1, and ephrinB2, all of which are normally dorsally or ventrally restricted. Moreover, the projections of dorsal but not ventral ganglion cell axons onto the optic tectum showed profound targeting errors following cVax misexpression. mVax2/cVax thus specify positional identity along the D-V axis of the retina and influence retinotectal mapping.