RNT-1, the C. elegans homologue of mammalian RUNX transcription factors, regulates body size and male tail development

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-beta (TGF-beta) signal mediators. However, the involvement of RUNX in TGF-beta signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-beta signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.     

Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development.

The Muc-1 mucin is found as a transmembrane protein in the apical surface of glandular epithelia. To provide insight into possible functions, we have assessed the timing of expression and the distribution of the Muc-1 protein during mouse embryogenesis using three different techniques: RT-PCR, northern blots and immunohistochemistry. Our results indicate that Muc-1 expression correlates with epithelial differentiation in stomach, pancreas, lung, trachea, kidney and salivary glands. Once started, Muc-1 synthesis continually increases with time, mainly due to epithelial area growth. Our data suggest that expression of the Muc-1 gene is under spatial and temporal control during organogenesis. Although Muc-1 is present in different organs, its expression is not induced systemically, but according to the particular onset of epithelial polarization and branching morphogenesis of each individual organ. It is of particular interest that Muc-1 protein can be detected lining the apical surfaces of the developing lumens when the epithelium of these organs is still undergoing folding and branching, and glandular activity has not yet started. We speculate that Muc-1 may participate in epithelial sheet differentiation/lumen formation during early development of the organs known to express it. This speculation is based on: (1) the detection of Muc-1 expression early during organogenesis, (2) the defined apical localization in different epithelia, (3) the decrease in cell-cell interactions when Muc-1 protein is highly expressed and (4) the possible interaction of its cytoplasmic tail with the actin cytoskeleton. However, it remains to be established using in vitro systems, whether the temporal and local expression of the Muc-1 gene coincident with the morphogenetic events described here is relevant for the process.     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

The expression of NOTCH2, HES1 and SOX9 during mouse retinal development

Notch signaling is an important regulator of both developmental and post-developmental processes. In the developing retina, Notch1 is required for the maintenance of retinal progenitor cells and for inhibiting photoreceptor cell fate, while Notch3 is required for inhibiting ganglion cell fate. Here we used immunolabeling coupled with a knock-in reporter approach to obtain a detailed spatiotemporal expression pattern of Notch2 during mouse retinal development. Although previous in situ hybridization studies did not reveal appreciable levels of Notch2 in the developing retina, we detected NOTCH2 protein and reporter expression in early embryonic retinal progenitors that also expressed the Notch downstream gene, HES1. In the postnatal retina, NOTCH2, as well as the Notch downstream genes, HES1 and SOX9, were detected in VSX2/Cyclin D1/SOX2-expressing cells in the postnatal retina, and in the mature retina NOTCH2 was most abundant in Müller glia. Our findings indicate a potential role for Notch2 in the developing and mature retina.     

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B,the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III–IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX–X. p19 mRNAs were present in PS from stages III–IV onward and in RS up to stages 1–2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15–16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2. Mol. Reprod. Dev. 51:22–35, 1998. © 1998 Wiley-Liss, Inc.     

The temporal and spatial expression pattern of myosin Va, Vb and VI in the mouse ovary

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

PKN-1, a homologue of mammalian PKN, is involved in the regulation of muscle contraction and force transmission in C. elegans

To examine the in vivo functions of protein kinase N (PKN), one of the effectors of Rho small guanosine triphosphatases (GTPases), we used the nematode Caenorhabditis elegans as a genetic model system. We identified a C. elegans homologue (pkn-1) of mammalian PKN and confirmed direct binding to C. elegans Rho small GTPases. Using a green fluorescent protein reporter, we showed that pkn-1 is mainly expressed in various muscles and is localized at dense bodies and M lines. Overexpression of the PKN-1 kinase domain and loss-of-function mutations by genomic deletion of pkn-1 resulted in a loopy Unc phenotype, which has been reported in many mutants of neuronal genes. The results of mosaic analysis and body wall muscle-specific expression of the PKN-1 kinase domain suggests that this loopy phenotype is due to the expression of PKN-1 in body wall muscle. The genomic deletion of pkn-1 also showed a defect in force transmission. These results suggest that PKN-1 functions as a regulator of muscle contraction-relaxation and as a component of the force transmission mechanism.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

C7, a novel nucleolar protein, is the mouse homologue of the Drosophila late puff product L82 and an isoform of human OXR1

The C7 gene was identified in a project aimed to characterize differential gene expression upon attachment of cells to extracellular matrix proteins in vitro. C7 is the homologue of Drosophila L82, a late puff gene (Stowers et al. (1999) Dev. Biol. 213, 116-130) and human OXR1, a gene, which protects cells against oxidation (Volkert et al. (2000) Proc. Natl. Acad. Sci. USA 97, 14530-14535). All are transcribed into multiple splice forms with a common 3' domain. Additional members of this novel gene family are found in a number of eukaryotic species. In the mouse, the C7 gene is highly and broadly expressed during development in at least 4 splice forms, 3 of which were sequenced. In the adult, the C7 gene is most highly expressed in brain and testis. Antibodies to recombinant C7 protein localized to nucleoli in a variety of cell types, suggesting that C7 may be involved in the formation or function of this important organelle.