Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.
ABSTRACT: Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.
Project description:Diffuse esophageal leiomyomatosis (DL), a benign smooth-muscle-cell tumor, is characterized by abnormal cell proliferation. DL is sometimes associated with X-linked Alport syndrome (AS), an inherited nephropathy caused by COL4A5 gene mutations. COL4A5 is tightly linked, in a head-to-head fashion, to the functionally related and coordinately regulated COL4A6 gene. No X-linked AS cases are due to COL4A6 mutations, but all DL/AS cases are always associated with deletions spanning the 5' regions of the COL4A5/COL4A6 cluster. Unlike the COL4A5 breakpoints, those of COL4A6 are clustered within intron 2 of the gene. We identified a DL/AS deletion and the first characterization of the breakpoint sequences. We show that a deletion eliminates the first coding exon of COL4A5 and the first two coding exons of COL4A6. The breakpoints share the same sequence, which, in turn, is closely homologous to the consensus sequences of topoisomerases I and II. Additional DNA evidence suggested that the male patient is a somatic mosaic for the mutation. Immunohistochemical analysis using alpha-chain-specific monoclonal antibodies supported this conclusion, since it revealed the absence of the alpha5(IV) and alpha6(IV) collagen chains in most but not all of the basement membranes of the smooth-muscle-cell tumor. We also documented a similar segmental staining pattern in the glomerular basement membranes of the patient's kidney. This study is particularly relevant to the understanding of DL pathogenesis and its etiology.
Project description:Hereditary hearing loss is the most common human sensorineural disorder. Genetic causes are highly heterogeneous, with mutations detected in >40 genes associated with nonsyndromic hearing loss, to date. Whereas autosomal recessive and autosomal dominant inheritance is prevalent, X-linked forms of nonsyndromic hearing impairment are extremely rare. Here, we present a Hungarian three-generation family with X-linked nonsyndromic congenital hearing loss and the underlying genetic defect. Next-generation sequencing and subsequent segregation analysis detected a missense mutation (c.1771G>A, p.Gly591Ser) in the type IV collagen gene COL4A6 in all affected family members. Bioinformatic analysis and expression studies support this substitution as being causative. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes, which forms a heterotrimer with two alpha-5 chains encoded by COL4A5. Whereas mutations in COL4A5 and contiguous X-chromosomal deletions involving COL4A5 and COL4A6 are associated with X-linked Alport syndrome, a nephropathy associated with deafness and cataract, mutations in COL4A6 alone have not been related to any hereditary disease so far. Moreover, our index patient and other affected family members show normal renal and ocular function, which is not consistent with Alport syndrome, but with a nonsyndromic type of hearing loss. In situ hybridization and immunostaining demonstrated expression of the COL4A6 homologs in the otic vesicle of the zebrafish and in the murine inner ear, supporting its role in normal ear development and function. In conclusion, our results suggest COL4A6 as being the fourth gene associated with X-linked nonsyndromic hearing loss.
Project description:Deletions encompassing the 5' termini of the paired type IV collagen genes COL4A5 and COL4A6 on chromosome Xq22 give rise to Alport syndrome (AS) and associated diffuse leiomyomatosis (DL), a syndrome of disseminated smooth-muscle tumors involving the esophagus, large airways, and female reproductive tract. In this study, we report isolation and characterization of two deletion junctions. The first, in a patient described elsewhere, arose by a nonhomologous recombination event fusing a LINE-1 (L1) repetitive element in intron 1 of COL4A5 to intron 2 of COL4A6, resulting in a 13.4-kb deletion. The second, in a previously undescribed family, arose by unequal homologous recombination between the same L1 and a colinear L1 element in intron 2 of COL4A6, resulting in a>40-kb deletion. L1 elements have contributed to the emergence of this locus as a site of frequent recombinations by diverse mechanisms. These give rise to AS-DL by disruption of type IV collagen and perhaps other as yet unidentified genes, evidenced by deletions as small as 13.4 kb.
Project description:X-linked Alport syndrome is a progressive nephropathy associated with mutations in the COL4A5 gene. The kidney usually lacks the alpha3-alpha6 chains of collagen type IV, although each is coded by a separate gene. The molecular basis for this loss remains unclear. In canine X-linked hereditary nephritis, a model for X-linked Alport syndrome, a COL4A5 mutation results in reduced mRNA levels for the alpha3, alpha4, and alpha5 chains in the kidney, implying a mechanism coordinating the production of these 3 chains. To examine whether production of alpha6 chain is under the same control, we studied smooth muscle cells from this animal model. We determined the canine COL4A5 and COL4A6 genes are separated by 435 bp, with two first exons for COL4A6 separated by 978 bp. These two regions are >/= 78% identical to the human sequences that have promoter activity. Despite this potential basis for coordinated transcription of the COL4A5 and COL4A6 genes, the alpha6 mRNA level remained normal in affected male dog smooth muscle while the alpha5 mRNA level was markedly reduced. However, both alpha5 and alpha6 chains were absent at the protein level. Our results suggest that production of the alpha6 chain is under a control mechanism separate from that coordinating the alpha3-alpha5 chains and that the lack of the alpha6 chain in Alport syndrome is related to a failure at the protein assembly level, raising the possibility that the alpha5 and alpha6 chains are present in the same network. The lack of the alpha6 chain does not obviously result in disease, in particular leiomyomatosis, as is seen in Alport patients with deletions involving the COL4A5 and COL4A6 genes.
Project description:The genes for the alpha 5(IV) and alpha 6(IV) chains of human basement membrane collagen type IV have been found together on chromosome X at segment q22 and have been reported to be arranged in a head-to-head fashion. Here we report the 5' flanking sequences of COL4A5 and COL4A6 and that COL4A6 is transcribed from two alternative promoters in a tissue-specific fashion. Analysis of the sequence immediately upstream of the transcription start sites revealed some features of housekeeping genes--i.e., the lack of a TATA motif and the presence of CCAAT and CTC boxes. Further analysis revealed that COL4A6 contains two alternative promoters that control the generation of two different transcripts. One transcription start site (from exon 1') is 442 bp away from the transcription start site of COL4A5, while an alternative transcription start site (from exon 1) is located 1050 bp from the first one and drives the expression of a second transcript that encodes an alpha 6(IV) chain with a different signal peptide. Reverse transcription-PCR experiments revealed that the transcript from exon 1' is abundant in placenta, whereas the transcript from exon 1 is more frequently found in kidney and lung. These results provide additional clues to answering the general question of what mechanisms are used to generate unique basement membrane structures in different tissues.
Project description:Type IV collagen is a predominant component of basement membranes, and glomeruli of a kidney filter approximately 70-90 liters of plasma every day through a specialized glomerular basement membrane (GBM). In Alport syndrome, a progressive disease primarily affecting kidneys, mutations in GBM-associated type IV collagen genes (COL4A3, COL4A4, or COL4A5) lead to basement membrane structural defects, proteinuria, renal failure, and an absence of all three GBM collagen triple helical chains because of obligatory posttranslational assembly requirements. Here, we demonstrate that transplantation of wild-type bone marrow (BM) into irradiated COL4A3(-/-) mice results in a possible recruitment of BM-derived progenitor cells as epithelial cells (podocytes) and mesangial cells within the damaged glomerulus, leading to a partial restoration of expression of the type IV collagen alpha3 chain with concomitant emergence of alpha4 and alpha5 chain expression, improved glomerular architecture associated with a significant reduction in proteinuria, and improvement in overall kidney histology compared with untreated COL4A3(-/-) mice or irradiated COL4A3(-/-) mice with BM from adult COL4A3(-/-) mice. The alpha3(IV) collagen produced by BM-derived podocytes integrates into the GBM and associates with other alpha-chains to form type IV collagen triple helical networks. This study demonstrates that BM-derived stem cells can offer a viable strategy for repairing basement membrane defects and conferring therapeutic benefit for patients with Alport syndrome.
Project description:A genome-wide association study (GWAS) was conducted to identify expression quantitative trait loci (eQTLs) for the genes involved in phosphatidylinositol-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway.Data on mRNA expression of 341 genes in lymphoblastoid cell lines of 373 Europeans recruited by the 1000 Genomes Project using Illumina HiSeq2000 were utilized. We used their genotypes at 5,941,815 nucleotide variants obtained by Genome Analyzer II and SOLiD.The association analysis revealed 4166 nucleotide variants associated with expression of 85 genes (P?<?5?×?10). A total of 73 eQTLs were identified as association signals for the expression of multiple genes. They included 9 eQTLs for both of the genes encoding collagen type I alpha 1 (COL1A1) and integrin alpha 11 (ITGA11), which synthesize a major complex of plasma membrane. They also included eQTLs for type IV collagen molecules; 13 eQTLs for both collagen type IV alpha 1 (COL4A1) and collagen type IV alpha 2 (COL4A2) and 18 eQTLs for both collagen type IV alpha 5 (COL4A5) and collagen type IV alpha 6 (COL4A6). Some genes expressed by the eQTLs might induce expression of the genes encoding type IV collagen. One eQTL (rs16871986) was located in the promoter of palladin (PALLD) gene which might synthesize collagen by activating fibroblasts through the PI3K/AKT pathway. Another eQTL (rs34845474) was located in an enhancer of cadherin related family member 3 (CDHR3) gene which can mediate cell adhesion.This study showed a profile of eQTLs for the genes involved in the PI3K/AKT pathway using a healthy population, revealing 73 eQTLs associated with expression of multiple genes. They might be candidates of common variants in predicting genetic susceptibility to cancer and in targeting cancer therapy. Further studies are required to examine their underlying mechanisms for regulating expression of the genes.
Project description:Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen ?1(IV) and ?2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen ?5(IV) and ?6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform ?6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of ?6(IV) chain in epithelial keratinization. These findings indicate that ?112:?556 (IV) network, which is the only network that includes the ?6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.
Project description:To examine the role of germinal centers (GCs) in the generation and selection of high affinity antibody-forming cells (AFCs), we have analyzed the average affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific AFCs and serum antibodies both during and after the GC phase of the immune response. In addition, the genetics of NP-binding AFCs were followed to monitor the generation and selection of high affinity AFCs at the clonal level. NP-binding AFCs gradually accumulate in bone marrow (BM) after immunization and BM becomes the predominant locale of specific AFCs in the late primary response. Although the average affinity of NP-specific BM AFCs rapidly increased while GCs were present (GC phase), the affinity of both BM AFCs and serum antibodies continued to increase even after GCs waned (post-GC phase). Affinity maturation in the post-GC phase was also reflected in a shift in the distribution of somatic mutations as well as in the CDR3 sequences of BM AFC antibody heavy chain genes. Disruption of GCs by injection of antibody specific for CD154 (CD40 ligand) decreased the average affinity of subsequent BM AFCs, suggesting that GCs generate the precursors of high affinity BM AFCs; inhibition of CD154-dependent cellular interactions after the GC reaction was complete had no effect on high affinity BM AFCs. Interestingly, limited affinity maturation in the BM AFC compartment still occurs during the late primary response even after treatment with anti-CD154 antibody. Thus, GCs are necessary for the generation of high affinity AFC precursors but are not the only sites for the affinity-driven clonal selection responsible for the maturation of humoral immune responses.
Project description:The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.