Biomedical subjects

D S Sepich

Publications and source records attributed to D S Sepich.

12 recordsLinked to original sources

PubMed2001

The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension.

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

Amino Acid Sequence↗

PubMed2000

Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension.

Convergence and extension are gastrulation movements that participate in the establishment of the vertebrate body plan. Using new methods for quantifying convergence and extension movements of cell groups, we demonstrate that in wild-type embryos, dorsal convergence of lateral cells is initially slow, but speeds up between the end of the gastrula period and early segmentation. Convergence and extension movements of lateral cells in trilobite mutants are normal during the gastrula period but reduced by early segmentation. Morphometric studies revealed that during epiboly wild-type gastrulae become ovoid, whereas trilobite embryos remain rounder. By segmentation, trilobite embryos exhibit shorter, broader embryonic axes. The timing of these morphological defects correlates well with impaired cell movements, suggesting reduced convergence and extension are the main defects underlying the trilobite phenotype. Our gene expression, genetic, and fate mapping analyses show the trilobite mutation affects movements without altering dorsoventral patterning or cell fates. We propose that trilobite function is required for cell properties that promote increased speed of converging cells and extension movements in the dorsal regions of the zebrafish gastrula.

Animals↗

PubMed1998

An altered intron inhibits synthesis of the acetylcholine receptor alpha-subunit in the paralyzed zebrafish mutant nic1.

The acetylcholine receptor (AChR), an oligomeric protein composed of five subunits, is a component of the postsynaptic membrane at the vertebrate neuromuscular junction that plays a central role in synaptic transmission. The zebrafish mutation nic1 blocks the expression of functional and clustered nicotinic muscle AChRs. To understand the mechanisms underlying this lack of AChRs, we characterized the molecular defect in nic1 mutants. Our results suggest that the mutation affects the gene coding for the alpha-subunit of the AChR. Southern blot hybridization and DNA sequence analyses showed that the nic1 AChR alpha-subunit gene lacks part of intron 6 where the splicing branchpoint normally forms. Several lines of evidence suggest that this deletion blocks normal splicing; most nic1 alpha-subunit mRNAs retain intron 6 and are larger and less abundant than wild-type, some nic1 alpha-subunit mRNAs are internally deleted, and wild-type alpha-subunit mRNA rescues nic1 mutant cells. The nic1 mutation reduces the size of an intron, which prevents efficient splicing of the pre-mRNA, thus blocking synthesis of the alpha-subunit and assembly of AChRs. By this route, the nic1 mutation leads to paralysis.

Amino Acid Sequence↗

PubMed1994

Autonomous expression of the nic1 acetylcholine receptor mutation in zebrafish muscle cells.

The nic1b107 (nic1) mutation blocks expression of both functional and clustered acetylcholine receptors (AChRs) in zebrafish muscle. Normally, signaling between motoneurons and muscles regulates AChR clustering. To learn if signaling is affected and to identify the primary cellular target of the nic1 mutation, we made mosaic embryos by transplanting motoneurons and muscle precursors from wild-type to mutant embryos. Genotypically mutant muscle cells fail to cluster AChRs even when contacted by wild-type motoneurons, whereas genotypically mutant motoneurons induce AChR clustering on wild-type muscle cells. Moreover, mutant muscle cells fail to cluster AChRs under culture conditions that induce AChR clustering on wild-type cells. We conclude that the nic1 mutation acts autonomously in muscle cells rather than by affecting signaling between motoneurons and muscle. The wild-type nic1 gene is necessary in muscle for expression and clustering of AChRs.

Animals↗

PubMed1988

Development of segmentation in zebrafish.

Recent findings on the nature and origin of segmentation in zebrafish, Brachydanio rerio, are reviewed. Segmented peripheral tissues include the trunk and tail myotomes, that are derived from somitic mesoderm, and the pharyngeal arches that are derived from head mesoderm in addition to other sources. Two major regions of the central nervous system, the spinal cord and hindbrain, are also segmentally organized, as deduced from the distribution of identified neurones in both regions and by formation of neuromeres in the hindbrain that contain single sets of these neurones. Neural and mesodermal segments in the same body region can be related to one another by their patterns of motor innervation. This relationship is simple for the spinal/myotomal segments and complex for the hindbrain/pharyngeal arch segments. Development of the segments is also complex. Mesodermal and ectodermal progenitors have separate embryonic origins and indeterminate cell lineages, and the embryonic cells migrate extensively before reaching their definitive segmental positions. Results of heat-shock experiments suggest that development of myotomal and spinal segments are regulated coordinately in postgastrula embryos. Segmental patterning may be a relatively late feature of zebrafish embryonic development.

Animals↗

PubMed1987

Constitutive expression of Slp genes in mouse strain B10.WR directed by C4 regulatory sequences.

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.

Animals↗

PubMed1987

cDNA sequence of a novel sex-limited protein (Slp) from mice constitutive for Slp expression. Sequence comparisons suggest that Slp has no functional role.

Murine sex-limited protein (Slp) is a serum protein that shares 95% sequence identity with murine complement component C4 but does not have C4 activity. Mouse strain B10.WR, which carries the H-2w7 haplotype, has up to 4 Slp genes and is unusual in that both males and females express Slp. Here we report the sequence of a complete pro-Slp cDNA from this strain that we designate Slpw7.2. We find that the Slpw7.2 sequence differs at multiple dispersed sites from three previously reported Slp sequences: two complete pro-Slp sequences, Slpw7.1 and SlpFM, from the B10.WR and FM strains, respectively, and a partial sequence from the B10.WR strain that is distinct from Slpw7.1 as well. A detailed comparison of the complete Slpw7.1, Slpw7.2, and SlpFM sequences reveals that nucleotide changes that alter the amino acid sequence (replacement substitutions) are accumulating at the same relative rate as changes that do not affect the amino acid sequence (silent substitutions); in addition, the amino acid changes themselves tend to be nonconservative. Our results suggest that at least three Slp genes are transcriptionally active in B10.WR mice; that the protein product of the Slpw7.2 transcript predominates in B10.WR serum; and that the Slp protein probably has no function. The Slp system may be particularly suitable for the study of the evolution in the absence of selective pressures of a gene that encodes a stable protein.

Amino Acid Sequence↗

PubMed1985

Complete cDNA sequence of the fourth component of murine complement.

We have used the method of Okayama and Berg to construct cDNA clones that span the entire length of the fourth component of murine complement (C4) from mouse strain B10.WR. The cDNA sequence spans 5372 nucleotides and encodes a pre-pro-C4 molecule 1738 amino acids in length; it includes 56 and 99 untranslated bases at the 5' and 3' ends, respectively, of the C4 mRNA. The deduced pre-pro-C4 molecule includes a 19 amino acid signal peptide and two highly basic interchain regions that presumably are excised during maturation of the protein. The nucleotide sequence shows 79% identity with the human C4 cDNA sequence over the length of the protein-coding region. A comparison of the B10.WR C4 sequence with those of C4 and sex-limited protein (Slp) from mouse strain FM (i) suggests that the difference in hemolytic activity between the C4 proteins from B10.WR and FM is due to structural changes distant from both the C1 cleavage site and the internal thioester site and (ii) raises the possibility that the C4 gene from B10.WR has undergone genetic exchange with its adjoining Slp genes.

Amino Acid Sequence↗

PubMed1985

Murine sex-limited protein: complete cDNA sequence and comparison with murine fourth complement component.

Murine sex-limited protein (Slp) is a structural homologue of the murine fourth complement component (C4) that lacks C4 activity and has no known function. The genes for C4 and Slp lie closely linked in the S region of the murine major histocompatibility complex. We have sequenced a cDNA clone that spans the entire protein-coding region of Slp from the mouse strain B10.WR. The sequence contains a 1735 amino acid-long open reading frame encoding a putative prepro-Slp flanked by 51 and 103 untranslated nucleotides at the 5' and 3' ends respectively; it shows 96% nucleotide and 94% amino acid identity with our previously reported complete sequence of murine C4 from the same mouse strain. The present complete Slp sequence differs slightly from our previously reported partial sequence from the same mouse strain; this suggests that at least two distinct Slp genes are transcribed in B10.WR mice. We suggest, by analogy with procaryotic DNA-binding proteins, that a three amino acid deletion in Slp, close to the Cls cleavage site, makes that site resistant to proteolysis; this renders Slp inactive. We also speculate on the possibility that Slp might be a gene in evolutionary transition; one that is midway in the evolution of a completely silent pseudogene or a new gene with a novel function.

Amino Acid Sequence↗

PubMed1985

Mice constitutive for sex-limited protein (SLP) expression contain multiple Slp gene sequences.

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes map to the S region of H-2. Serum C4 levels vary as much as 20-fold between C4 high (C4H) and C4 low (C4L) strains, and Slp expression can be null (SlpO), limited to male mice of a subset of C4H strains (Slp+), or "constitutive" (SlpC), in which female as well as male mice express Slp. In this study, we compare, by genomic Southern blot analysis, the C4 and Slp genes from eight congenic inbred mouse strains representative of three distinct phenotypes: C4H, Slp+ (two strains), C4H, SlpO (two strains), C4H, SlpC (three strains), and C4L, SlpO (one strain). By using cDNA probes that recognize both C4 and Slp genes, and are derived from the extreme 5' and 3' ends of the mRNA as well as internal coding sequences, we find no evidence to suggest that strain-specific variations in the expression of C4 and Slp are due to gross deletions of major portions of the structural genes. In most cases, two distinct C4/Slp genes are detected; hybridization with C4- and Slp-specific probes indicate that one of these is C4 and the other is Slp. The three SlpC strains are exceptional: they carry at least four C4/Slp genes; one of these hybridizes to the C4-specific probe whereas the remaining genes hybridize to the Slp-specific probe. Hence, multiple duplication of a gene containing Slp sequences has occurred in certain strains of mice, and this is accompanied by constitutive expression of the Slp protein.

Animals↗

PubMed1984

Genes for murine fourth complement component (C4) and sex-limited protein (Slp) identified by hybridization to C4- and Slp-specific cDNA.

Murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins whose structural genes lie in the S region of the murine H-2 complex. We have cloned two very similar (95% nucleotide sequence identity) cDNAs; they encode amino acid sequences that are distinct but that correspond equally well with the limited amino acid sequence available for murine and human C4. We have identified one of these as C4 cDNA and the other as Slp cDNA by comparing, in a RNA blot hybridization experiment, the extent of hybridization of the two cDNAs to liver mRNAs from inbred mouse strains expressing varying amounts of C4 and Slp proteins in their plasma. The C4 and Slp cDNAs were used in a Southern blot experiment to identify the C4 and Slp genes in the molecular map of the S region.

Amino Acid Sequence↗

PubMed1983

cDNA clone spanning the alpha-gamma subunit junction in the precursor of the murine fourth complement component (C4).

cDNA clones carrying parts of murine fourth complement component (C4, serum substance protein) mRNA sequences have been identified by differential hybridization to mRNA from a high C4-producing strain, B10.WR, and a congeneic low C4 strain, B10.BR, followed by hybrid-selected translation and DNA sequence analysis. One clone, pMLC4/w7-2, encodes an open amino acid reading frame that includes four tandem arginine residues immediately preceding a sequence 85% homologous with the NH2-terminal sequence of the human C4 gamma-chain. The amino acid composition of the predicted sequence upstream of the tandem arginines matches quite closely with the composition of a similar sized peptide at the COOH terminus of the human C4 alpha chain. The latter result raises questions regarding the nature and extent of plasma-mediated postsynthetic processing of the C4 alpha-chain COOH terminus. The results also demonstrate that strain differences in plasma C4 levels (low C4 vs. high C4) reflect differences in steady-state levels of liver C4 mRNA in these strains.

Amino Acid Sequence↗