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.     

Isolation of DNTNP, which encodes a potential nuclear protein that is expressed in the developing, dorsal neural tube.

We have performed a screen to identify genes expressed in a functionally significant anatomic region of the vertebrate dorsal neural tube, the dorsomedial roof of the third ventricle (DMRTV). The DMRTV includes the primordia of a series of circumventricular organs. The screen searched for genes preferentially expressed in the DMRTV of stage 18-25 chicken embryos, relative to their telencephala and ventral diencephalon. Through this screen, we have cloned a series of genes strongly expressed in the dorsal but not ventral neural tube. We describe here the first of these genes, DNTNP (dorsal neural tube nuclear protein). DNTNP is highly expressed in the dorsal regions of the diencephalon, the midbrain, the hindbrain, and the spinal neural tube in the chicken stage 18 embryo. Expression is also observed in the telencephalon, the branchial arches, the heart, and somites, but is absent from the presomitic mesoderm. The amino acid sequence of DNTNP reveals that it belongs to an uncharacterized protein family with at least two additional members. All the members of this family possess a basic region reminiscent of a nuclear localization signal (NLS). We demonstrate that the putative NLS of DNTNP can indeed direct nuclear localization of green fluorescent protein (GFP). The dorsal localization of DNTNP in the early embryonic central nervous system suggests roles for this molecule in specifying dorsal cell fates within the neural tube.     

The Drosophila gene Hairless encodes a novel basic protein that controls alternative cell fates in adult sensory organ development.

The mechanosensory bristles of adult Drosophila are composed of four cells that, in most cases, are progeny of a single sensory organ precursor (SOP) cell. Two sister cells in this lineage, the trichogen and tormogen, produce the external shaft and socket of the bristle, respectively. Loss-of-function mutations of Hairless (H) confer two distinct mutant phenotypes on adult bristles. The bristle loss phenotype results from the failure to specify and/or execute the SOP cell fate; the double socket phenotype results from the transformation of the trichogen (shaft) cell into a second tormogen (socket) cell. We have found that the H gene encodes a novel basic protein with a predicted molecular mass of 109 kD. Basal levels of expression of a transgene (P[Hs-H]) in which the H protein-coding region is under the control of the Hsp70 promoter are sufficient to provide full rescue of H mutant phenotypes. Heat shock treatment of P[Hs-H] transgenic animals as late larvae and early pupae produces a tormogen-to-trichogen (double shaft) cell fate transformation, as well as bristle multiplication and loss phenotypes very similar to those caused by loss-of-function mutations in the neurogenic gene Notch. Our results indicate that the SOP cell fate requires H to antagonize the activity of the neurogenic group of genes and that the expression of distinct cell fates by the trichogen/tormogen sister cell pair depends on an asymmetry in their levels of H+ activity or in their thresholds for response to H.     

Rat cytomegalovirus open reading frame R44 is an early-late gene that encodes a nuclear protein

Summary.  Rat cytomegalovirus (RCMV) open reading frame R44 is the homolog of human cytomegalovirus gene UL44, which encodes the DNA polymerase accessory protein. Here, we show that R44 is transcribed as a 3.6-kb mRNA within the early and late phases of infection in vitro. In order to find potential monoclonal antibodies (MoAbs) directed against the R44-encoded protein (pR44), a panel of anti-RCMV MoAbs was screened for binding to pR44 recombinant proteins. Thus, an anti-pR44 MoAb, termed RCMV8, was identified. By using this MoAb, pR44 could be detected as early as 8 h after RCMV infection in vitro. The pR44 protein was determined to have a molecular mass of approximately 55 kDa and was found to be localized to the nucleus of RCMV-infected cells. Received May 15, 2001 Accepted June 19, 2001     

The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb.

The Polycomb group (PcG) genes in Drosophila melanogaster are required for maintenance of correct spatial expression of homeotic genes, and their products are thought to form either a regulatory network or act as a multimeric complex. Recently, it has been suggested that because of homology between Polycomb (Pc) and Su(var)205, PcG genes encode chromatin proteins required for the maintenance of a determined state in chromatin. The polyhomeotic (ph) gene is a member of the PcG of genes. We present DNA sequence of a ph cDNA, which encodes a 169-kD protein with a single putative zinc finger, a serine/threonine-rich region, and has glutamine repeats, suggesting that ph is a DNA-binding protein. Polyclonal antisera directed against ph protein bind to approximately 80 sites on polytene chromosomes. Most of these sites appear to be the same as those recognized by antibodies to Pc protein. ph protein binds to insertion sites of constructs containing DNA from the bithoraxoid (bxd) region of the Bithorax complex, showing that ph binding to chromatin is DNA dependent. The same bxd constructs are recognized by Pc protein, strongly supporting the hypothesis that ph and Pc interact directly.     

Embryonic expression of a Drosophila src gene: alternate forms of the protein are expressed in segmental stripes and in the nervous system

Expression of the Drosophila src-related gene, Dsrc28C has been investigated at the protein level using monoclonal antibodies. This analysis has revealed that the Dsrc28C gene encodes two protein forms: a 66-kD doublet predicted from the sequence of a cDNA clone and an additional 55-kD form. The 66-kD protein doublet is observed first at the cellular blastoderm stage and is not detectable in embryos after 12 hr of development. Expression of the 55-kD protein lags behind that of the 66-kD doublet and then persists throughout embryogenesis. Indirect immunofluorescence microscopy reveals that Dsrc28C protein is localized to the cell periphery during cellular blastoderm and gastrulation. The cell periphery-associated staining is then resolved into ectodermal stripes along the fully extended germ band. After the stripes fade, cytoplasmic staining of the majority of cells within the central and peripheral nervous system is observed. The 66-kD protein was shown to represent the cell periphery-associated form of the protein through antibody staining of larval salivary glands expressing a heat shock promoter-driven, full-length Dsrc28C cDNA. Staining of embryos with a monoclonal antibody specific for the 66-kD protein indicates that the 55-kD protein is the nervous system form. Thus, the 66- and 55-kD proteins are products of the Dsrc28C gene, which exhibit different temporal and spatial patterns of expression in the embryo.     

The Mycobacterium tuberculosis complex-restricted gene cfp32 encodes an expressed protein that is detectable in tuberculosis patients and is positively correlated with pulmonary interleukin-10

Human tuberculosis (TB) is caused by the bacillus Mycobacterium tuberculosis, a subspecies of the M. tuberculosis complex (MTC) of mycobacteria. Postgenomic dissection of the M. tuberculosis proteome is ongoing and critical to furthering our understanding of factors mediating M. tuberculosis pathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. The cfp32 gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, the cfp32 gene sequence is conserved within the MTC, as no polymorphisms were found in the tested cfp32 PCR products upon sequence analysis. Western blotting of M. tuberculosis subcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to the M. tuberculosis pathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies.     

C10 is a novel chemokine expressed in experimental inflammatory demyelinating disorders that promotes recruitment of macrophages to the central nervous system

Chemokines may be important in the control of leukocytosis in inflammatory disorders of the central nervous system. We studied cerebral chemokine expression during the evolution of diverse neuroinflammatory disorders in transgenic mice with astrocyte glial fibrillary acidic protein-targeted expression of the cytokines IL-3, IL-6, or IFN-alpha and in mice with experimental autoimmune encephalomyelitis. Distinct chemokine gene expression patterns were observed in the different central nervous system inflammatory models that may determine the phenotype and perhaps the functions of the leukocytes that traffic into the brain. Notably, high expression of C10 and C10-related genes was found in the cerebellum and spinal cord of GFAP-IL3 mice with inflammatory demyelinating disease and in mice with experimental autoimmune encephalomyelitis. In both these neuroinflammatory models, C10 RNA and protein expressing cells were predominantly macrophage/microglia and foamy macrophages present within demyelinating lesions as well as in perivascular infiltrates and meninges. Intracerebroventricular injection of recombinant C10 protein promoted the recruitment of large numbers of Mac-1(+) cells and, to a much lesser extent, CD4(+) lymphocytes into the meninges, choroid plexus, ventricles, and parenchyma of the brain. Thus, C10 is a prominent chemokine expressed in the central nervous system in experimental inflammatory demyelinating disease that, we show, also acts as a potent chemotactic factor for the migration of these leukocytes to the brain.     

SPARC is expressed by macroglia and microglia in the developing and mature nervous system.

SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that is highly expressed during development, tissue remodeling, and repair. SPARC produced by olfactory ensheathing cells (OECs) can promote axon sprouting in vitro and in vivo. Here, we show that in the developing nervous system of the mouse, SPARC is expressed by radial glia, blood vessels, and other pial-derived structures during embryogenesis and postnatal development. The rostral migratory stream contains SPARC that becomes progressively restricted to the SVZ in adulthood. In the adult CNS, SPARC is enriched in specialized radial glial derivatives (Müller and Bergmann glia), microglia, and brainstem astrocytes. The peripheral glia, Schwann cells, and OECs express SPARC throughout development and in maturity, although it appears to be down-regulated with maturation. These data suggest that SPARC may be expressed by glia in a spatiotemporal manner consistent with a role in cell migration, neurogenesis, synaptic plasticity, and angiogenesis.     

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.