CAZIP,a novel protein expressed in the developing heart and nervous system

Recently, we have performed a whole genome micro‐array analysis on human embryonic stem cells differentiating toward cardiomyocytes, which resulted in the identification of novel genes that were highly up‐regulated during differentiation. Here, we describe one of these novel genes annotated as KIAA0774. The predicted protein contains a leucine‐zipper domain at the C‐terminus and has at least two isoforms (358 and 1354 amino acids). Whole‐mount in situ hybridization confirmed that the mRNA of both the mouse and chicken orthologs of KIAA0774 is expressed during early cardiac development. Hence, we named this protein CAZIP (ca rdiac zi pper p rotein). Later during embryonic development, Cazip was also expressed in parts of the nervous system. Northern blot and real‐time polymerase chain reaction analysis showed that Cazip is expressed in heart and brain in adult mice. These results suggest a role for CAZIP in development and function of the heart and nervous system in vertebrates. Developmental Dynamics 238:2903–2911, 2009. © 2009 Wiley‐Liss, Inc.     

PLEXIN-D1, a novel plexin family member, is expressed in vascular endothelium and the central nervous system during mouse embryogenesis.

The genetic defect in M?bius syndrome 2 (MBS2, MIM 601471), a dominantly inherited disorder characterised by paralysis of the facial nerve, is situated at chromosome 3q21-q22. We characterised the cDNA and predicted protein, and examined the expression pattern during mouse embryogenesis of a positional candidate gene, PLEXIN-D1 (PLXND1). The cDNA for PLXND1 is 7095 base pairs in length, coding for a predicted protein of 1925 amino acids. The protein features all known domains of plexin family members, with the exception of the third Met-related sequence. Northern analysis revealed a very low expression of PLXND1 in adult mouse and adult human tissues. To investigate the expression of PlxnD1 during embryogenesis, RNA in situ hybridisation was performed on mouse embryos from various stages. This investigation revealed expression of PlxnD1 in cells from the central nervous system (CNS) and in vascular endothelium. Early expression in the CNS is located in the ganglia, cortical plate of the cortex, and striatum. At later embryologic stages, neural expression was also seen in the external granular layer of the cerebellum and several nerve nuclei. The expression in the vascular system resides solely in the endothelial cells of developing blood vessels. Based on our results, we suggest that this expression of a member of the plexin family in vascular endothelium could point toward a role in embryonic vasculogenesis.     

ARX, a novel Prd-class-homeobox gene highly expressed in the telencephalon, is mutated in X-linked mental retardation

Investigation of a critical region for an X-linked mental retardation (XLMR) locus led us to identify a novel Aristaless related homeobox gene (ARX ). Inherited and de novo ARX mutations, including missense mutations and in frame duplications/insertions leading to expansions of polyalanine tracts in ARX, were found in nine familial and one sporadic case of MR. In contrast to other genes involved in XLMR, ARX expression is specific to the telencephalon and ventral thalamus. Notably there is an absence of expression in the cerebellum throughout development and also in adult. The absence of detectable brain malformations in patients suggests that ARX may have an essential role, in mature neurons, required for the development of cognitive abilities.     

Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation

BACKGROUND: Whereas multiple basic helix-loop-helix (bHLH) genes are expressed in the developing nervous system, they account for the differentiation of only subsets of neurones, suggesting that there may be as-yet unidentified bHLH genes. RESULTS: We have isolated a novel bHLH gene, designated Math6, a distant mammalian homologue of the Drosophila proneural gene atonal. Structural analysis of the Math6 gene demonstrated that the coding region is divided into three exons, whereas that of other atonal homologues is present in a single exon, indicating that the genomic structure of Math6 is unique among the atonal homologues. Math6 is initially expressed by neural precursor cells in the ventricular zone, but later by subsets of differentiating and mature neurones such as hippocampal neurones and cerebellar Purkinje cells. Mis-expression of Math6 with retrovirus in the developing retina induced neurogenesis, while inhibiting gliogenesis, without affecting cell proliferation and death. CONCLUSIONS: These results show that cells which would normally differentiate into glia adopted the neuronal fate by mis-expression of Math6, indicating that Math6 promotes neuronal vs. glial fate determination in the nervous system.     

C16orf5, a novel proline-rich gene at 16p13.3, is highly expressed in the brain

A novel gene has been characterized, designated C16orf5, with an unusually high content of proline residues (40% over 104 residues) at the N-terminus of the protein. The C-terminus of the protein is also cysteine rich with 14 cysteine residues present. Analysis using Northern and dot blots showed that the highest expression of this gene is in the brain. The gene was located on chromosome 16 at band p13.3 by FISH to metaphase chromosomes. Southern blot analysis with a human–rodent somatic cell hybrid panel showed a location between the somatic hybrid breakpoints 23HA and CY196. This gene comprises at least four exons and an open reading frame of 786 bp encoding a predicted protein of 261 amino acids. Analysis of this protein using PSORTII predicted a nuclear localization. Received: April 12, 1999 / Accepted: June 4, 1999     

CTX,a novel molecule specifically expressed on the surface of cortical thymocytes in Xenopus

CTX, a novel developmentally regulated type-I transmembrane protein is expressed specifically by a large fraction of cortical thymocytes in the amphibian Xenopus. This apparently monomeric 55-kDa glycoprotein is composed of two immunoglobulin domains, one variable (V) and one constant (C2 type), followed by a transmembrane and a 64-amino acid cytoplasmic domain. The first immunoglobulin domain is a V-J segment that is generated without gene rearrangement. In the genome, the V and C2 domains are both encoded by two half-domain exons. Two CTX loci are found per haploid genome, and they exhibit sequence differences with a high replacing/silent ratio in the CDR1-like region of the V domain, suggesting that these differences were selected. The cytoplasmic domain contains a motif that is highly conserved evolutionarily in several types of proteins, including adenylyl cyclases. Based on its unique tissue distribution, the variability of its V region and the motif of its cytoplasmic domain, CTX is a candidate for a new type of specific signaling molecule involved in thymocyte selection.     

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis.

Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.     

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis

The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system (CNS) of tadpoles and adult frogs. This cDNA (xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal (Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine (His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cell-specific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15-16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.     

Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.     

In silico study of a novel gene evolved from an ancestral SVIP gene and highly expressed in the adult mouse testes

We found that a cDNA clone isolated from a mouse testis cDNA library, 1700015G11 (Mmu_15G11), corresponded to the most highly expressed testis-specific mRNA in the adult mouse. Although the Mmu_15G11 cDNA is predicted to encode a small protein consisting of 67 amino acid residues, it has not yet been functionally annotated and has been designated as an unclassifiable clone. Since the Mmu_15G11 protein possibly has a pivotal role in spermatogenesis, we initiated an in silico study of this clone, and revealed that an ancestral gene of 15G11 genes evolved from an ancestral gene for mammalian small valosin-containing protein-interacting protein (SVIP) genes by gene duplication. Although SVIP protein reportedly participates in endoplasmic reticulum-related protein degradation, 15G11 protein is predicted to be a nuclear protein and possibly participates in the interaction between proteins and nuclear DNA.     

Isolation and characterization of a novel gene, xMADML, involved in Xenopus laevis eye development.

We have identified Xenopus MADM-like (xMADML), a Xenopus laevis gene related to the murine MADM and the human NRBP genes. xMADML is expressed throughout early development and is expressed most strongly in the developing lens and more weakly in the retina and other anterior tissues. We demonstrate that disruption of xMADML translation by means of morpholino injection results in impaired retina and lens development. Reciprocal transplantation of the presumptive lens ectoderm between morpholino-injected embryos and those injected solely with a dextran lineage tracer demonstrates that xMADML is necessary in both the lens and the retina for correct development of these eye tissues. Analysis of gene expression after knockdown of xMADML revealed significant alterations in the expression of some genes, including Pax6, xSix3, Sox2, and Sox3, suggesting that xMADML plays a role in regulating gene expression during development of the eye. This investigation is the first in vivo study examining the developmental role of this novel gene and reveals an important role of xMADML in eye tissue development and differentiation.     

Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes.

A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA). The apparent affinity (EC50) for QA (0.1 microM) was higher than that for KA (50 microM). The maximal response to QA was about 1/10 of that to KA. QA inhibited the KA induced current. The N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3 dione (DNQX) competitively blocked the effects of both agonists. Currents induced by KA, QA and D in oocytes expressing GluR1 showed identical voltage sensitivities. GluR1 and KA receptor-channels expressed from rat striatum poly(A)+ RNA showed the same ionic selectivity, being permeable mostly to Na+ and K+. The current-voltage relationships of GluR1 showed a strong inward rectification, whereas those of KA receptor-channels expressed from poly(A)+ RNA from various rat brain regions were more linear.