Regulation of retinal cone bipolar cell differentiation and photopic vision by the CVC homeobox gene Vsx1

Cone bipolar cells of the vertebrate retina connect photoreceptors with ganglion cells to mediate photopic vision. Despite this important role, the mechanisms that regulate cone bipolar cell differentiation are poorly understood. VSX1 is a CVC domain homeoprotein specifically expressed in cone bipolar cells. To determine the function of VSX1, we generated Vsx1 mutant mice and found that Vsx1 mutant retinal cells form but do not differentiate a mature cone bipolar cell phenotype. Electrophysiological studies demonstrated that Vsx1 mutant mice have defects in their cone visual pathway, whereas the rod visual pathway was unaffected. Thus, Vsx1 is required for cone bipolar cell differentiation and regulates photopic vision perception.     

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.     

The Yeast a1 and α2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding

In diploid cells of the yeast Saccharomyces cerevisiae, the α2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the α2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-α2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-α2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the α2 or a1 protein. Interestingly, an α2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of α2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the α2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-α2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.     

Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.     

金鱼Vsx1基因结构及其内含子多态性分析

Vsx1是第一个在金鱼中发现的编码含有同源异型框(homeodomain)和CVC结构域蛋白的基因。该基因在胚胎发育的不同阶段在胚胎的不同区域和不同组织中表达,并已经证明它在视网膜视锥双极细胞的分化和正常功能的维持中具有重要作用。为了进一步研究该基因在不同发育阶段组织特异性表达的调节机制,实验用PCR方法分析了金鱼Vsx1的基因组结构和内含子多态性。结果表明:金鱼Vsx1基因由5个外显子和4个内含子组成,与斑马鱼、人、小鼠的Vsx1基因结构相同。4个内含子中,第一内含子有两种序列差异明显的类型,但第二、三、四内含子无明显差异。第一内含子的一种类型比另外一种类型多39个碱基,这39个碱基中包括了真核生物增强子的核心序列。这一观测结果提示金鱼Vsx1第一内含子可能与该基因的发育阶段特异性和组织特异性表达调节有关。同时,第一内含子序列的明显差异也为分析Vsx1不同等位基因或基因拷贝的表达活性,以及组织特异性表达调节方式提供了合适的探针序列。     

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous,Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.     

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.)

Two Fix^? mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix^? (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix^? in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix^? line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.