Functional involvement of Xenopus LIM kinases in progression of oocyte maturation.

LIM kinases (LIMK), including LIMK1 and LIMK2, are unique LIM-family proteins containing a catalytic (kinase) domain. These kinases phosphorylate an actin-depolymerizing factor, cofilin, involved in the regulation of actin-filament dynamics. An unanswered question is the in vivo function of LIMK and how they contribute to development. When we cloned Xenopus homologues of mammalian LIMK, Xlimk1 and Xlimk2, we found that their mRNA and products were abundantly expressed in oocytes. In addition, we obtained evidence for the functional involvement of Xlimk1/2 during oocyte maturation. The microinjection of Xlimk1/2 mRNA into progesterone-treated oocytes significantly inhibited the appearance of a white maturation spot (WMS), an indicator of entry into meiosis. In oocytes lacking a WMS, the organization and/or migration of the microtubule-derived precursor of the meiotic spindle was predominantly affected. We also found that the ectopic expression of Xlimk1/2 clearly prevented dephosphorylation (activation) of Xenopus cofilin (XAC) during oocyte maturation. Furthermore, co-injection of Xlimk1/2 with the constitutively active type of XAC overcame the inhibitory effects by Xlimk1/2, suggesting that XLIMK-induced abnormality in oocyte maturation was mediated by XAC inactivation. Based on these findings, we propose that XLIMK is a putative regulator of cytoskeletal rearrangements during oocyte maturation, and the interaction between XLIMK activity and microtubule dynamics seems highly likely.

Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.

Enhancement by hepatocyte growth factor of bone and cartilage formation during embryonic mouse mandibular development in vitro.

To elucidate the possible roles of hepatocyte growth factor (HGF) in the early development of mouse mandible, HGF was applied to an organ-culture system with chemically defined media. Mandibular arches microdissected from mouse embryos at the 10th day of gestation were cultured for 10 days with or without HGF, HGF plus HGF-receptor (c-met) antisense oligodeoxyribonucleotide, or HGF plus c-met sense oligodeoxyribonucleotide in the media. The cultured mandibles were then analysed, histologically in serial paraffin sections. In the absence of HGF, the tooth organs of bud stage, Meckel's cartilage and the tongue were formed, whereas only a slight amount of bone tissue was formed in the cultured mandible. The expression of intrinsic HGF and c-met in the cultured mandibles was confirmed by reverse transcriptase-polymerase chain reaction. Furthermore, immunohistochemistry demonstrated that both HGF and c-met were localized in areas of the mesenchymal tissue forming bone and cartilage. With HGF in the medium, the volume of both bone and cartilage increased significantly and dose-dependently. HGF also increased the rate of proliferation of osteogenic cells and chondrocytes. Addition of c-met antisense oligodeoxyribonucleotide partially inhibited the HGF-induced enhancement of bone and cartilage formation, whereas addition of c-met sense oligodeoxyribonucleotide had no effect. These results revealed that exogenous HGF enhances bone and cartilage morphogenesis in the cultured mandibles, suggesting physiological roles for intrinsic HGF in the early development of mouse mandible.

Expression of a novel type of classic cadherin, PB-cadherin in developing brain and limb buds.

PB-cadherin is a novel classic type of cadherin predominantly expressed in brain of adult rats (Sugimoto et al. [1996] J. Biol. Chem. 271:11548-11556). To examine the spatial and temporal expression of PB-cadherin during development, we isolated full-length cDNA of mouse PB-cadherin and studied its expression pattern in mouse embryos. In Northern blots, PB-cadherin mRNA was detected at 9.5 days postcoitum (dpc) onwards. Whole-mount in situ hybridization showed that PB-cadherin signals mainly occurred in developing neural tissues, including brain and spinal cord, and limb buds in the 10.5 dpc embryo. In the brain, PB-cadherin mRNA were strongly expressed in the forebrain and midbrain-hindbrain boundary region (isthmus). In isthmus, PB-cadherin expression delineated the expression area of Wnt-1, a secreted signaling molecule essential for proper cerebellum development. In the developing limb, PB-cadherin mRNA was first localized in posterior part of buds at 10.5 dpc, and was thereafter distributed in a domain around the digit rudiments. This expression pattern is similar to that of BMP-2, a secreted signalling molecule involving limb patterning and morphogenesis. These findings suggested the possibility that PB-cadherin-mediated cell-cell adhesion has a functional role in pattern formation and morphogenesis of mouse embryonic brain and limb. Dev Dyn 1999;215:206-214.

A novel transcript encoding truncated LIM kinase 2 is specifically expressed in male germ cells undergoing meiosis.

LIM kinases, composed of LIMK1 and LIMK2, have unique structural features that contain two LIM motifs at the N-terminus and a catalytic domain at the C-terminus. We report evidence of a novel type of mouse LIMK2 (Limk2) transcript specifically expressed in testis. cDNA cloning showed this Limk2 variant, designated tLimk2, lacked LIM domains at the N-terminus, due to usage of a testis-specific, alternative initiation exon. In Northern blot analysis, tLimk2 was detected in intact adult testis, but not in germ-cell-deficient or immature testis, indicating the stage-specific expression of tLimk2 in spermatogenic cells. In situ hybridization clearly demonstrated that tLimk2 was restrictedly expressed in differentiated germ cells (pachytene spermatocytes to round spermatids) and not expressed in early stages of spermatogenic cells and somatic cells in testis. These results suggested the possibility that the tLimk2 product is involved in spermatogenesis, especially in meiotic and/or postmeiotic processes.

Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development.

Mesenchymal-epithelial tissue interactions are important for development of various organs, and in many cases, soluble signaling molecules may be involved in this interaction. Hepatocyte growth factor (HGF) is a mesenchyme-derived factor which has mitogenic, motogenic and morphogenic activities on various types of epithelial cells and is considered to be a possible mediator of epithelial-mesenchymal interaction during organogenesis and organ regeneration. In this study, we examined the role of HGF during lung development. In situ hybridization analysis showed HGF and the c-met/HGF receptor gene to be respectively expressed in mesenchyme and epithelium in the developing lung. In organ cultures, exogenously added HGF apparently stimulated branching morphogenesis of the fetal lung. In contrast, HGF translation arrest or neutralization assays resulted in clear inhibition of epithelial branching. These results suggest that HGF is a putative candidate for a mesenchyme-derived morphogen regulating lung organogenesis. We also found that HGF is involved in epithelial branching, in collaboration with fibroblast growth factor (FGF) family molecule(s). In mesenchyme-free culture, HGF alone did not induce epithelial morphogenesis, however, addition of both HGF and acidic FGF (aFGF) or keratinocyte growth factor (KGF), ligands for the KGF receptor, induced epithelial branching more extensively than that was observed in explants treated with aFGF or KGF alone. In addition, the simultaneous inhibition of HGF- and FGF-mediated signaling using neutralizing antibody and antisense oligo-DNA resulted in drastic impairment of epithelial growth and branching. Possible interactions between HGF and FGFs or other growth factors in lung development is given consideration.

Xenopus LIM motif-containing protein kinase, Xlimk1, is expressed in the developing head structure of the embryo.

The LIM double zinc finger motif locates in several developmentally functioning and cytoskeletal proteins, and is considered to act as a specific motif for protein-protein interactions. LIM kinase (LIMK) is a novel protein kinase containing two LIM motifs at the N-terminal, the function of which has yet to be clearly defined. In this study, we cloned a cDNA encoding Xenopus counterpart of human LIMK1 gene by RT-PCR mediated cloning, and designated in Xlimk1. Xlimk1 is highly homologous to mammalian LIMK1 in each structural domain, particularly in LIM and protein kinase domains. In Northern blot analysis, two distinct Xlimk1 transcripts of 9.0 Kb and 3.7 Kb were present in early cleavage stages of the embryo. Both mRNA species were subsequently decreased at the gastrula stages. The 9.0 Kb of Xlimk1 mRNA again appeared in late neurula stage, then the expression level gradually increased in later stages of the embryo. Whole-mount in situ hybridization analysis showed the localization of Xlimk1 transcripts in the animal half of the blastula embryo. In post-neurula stages, specific signals for Xlimk1 were predominant in the anterior (head) region of the embryo, including developing brain, hyoid and branchial arches, and anlagen of sensory organs. These results indicate that Xlimk1 may play an important role in neural development and formation of anterior (head) structures in the Xenopus embryo.

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

cDNA cloning, genomic organization, and chromosomal localization of the mouse LIM motif-containing kinase gene, Limk2.

LIM-kinases, including LIMK1 and LIMK2, are unique LIM-family proteins with two tandem LIM motifs at the N-terminal and a serine/threonine kinase domain at the C-terminal. In this study, we cloned two types of mouse Limk2 cDNA; one is an intact form (Limk2a) and the other has only one complete LIM domain (Limk2b). Northern blot analysis showed Limk2a mRNA was ubiquitously present in various adult tissues, while Limbk2b was predominantly expressed in brain. We also identified genomic organization of the Limk2 gene; it is similar to that of the related gene Limk1. The transcription unit contains 16 exons plus two alternative exons, thus, variation in the initiator exon usage gives rise to alternative transcripts of Limk2. Fluorescent in situ hybridization analysis showed the Limk2 gene were mapped to mouse chromosome 1D. These findings provide some important clues to the in vivo functions of LIM kinases.

Functional requirement of gp130-mediated signaling for growth and survival of mouse primordial germ cells in vitro and derivation of embryonic germ (EG) cells.

Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse primordial germ cells (PGC) in culture. The receptor complex for LIF comprises LIF-binding subunit and non-binding signal transducer, gp130. The gp130 was originally identified as a signal-transducing subunit of interleukin (IL)-6 and later also found to be a functional component of receptor complexes for other LIF-related cytokines (oncostatin M [OSM], ciliary neurotrophic factor [CNTF] and IL-11). In this study, we have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. OSM was able to fully substitute for LIF; both cytokines promoted the proliferation of migratory PGC (mPGC) and enhanced the viability of postmigratory (colonizing) PGC (cPGC) when cultured on SI/SI4-m220 cells. Interestingly, IL-11 stimulated mPGC growth comparable to LIF and OSM, but did not affect cPGC survival. IL-6 and CNTF did not affect PGC. In addition, a combination of IL-6 and soluble IL-6 binding subunit (sIL-6R), which is known to activate intracellular signaling via gp130, fully reproduced the LIF action of PGC. Both in the presence and absence of LIF, addition of neutralizing antibody against gp130 in culture remarkably blocked cPGC survival. These results suggest a pivotal role of gp130 in PGC development, especially that it is indispensable for cPGC survival as comparable to the c-KIT-mediated action. We have further demonstrated that a combination of LIF with forskolin or retinoic acid, a potent mitogen for PGC, supported the proliferation of PGC, leading to propagation of the embryonic stem cell-like cells, termed embryonic germ (EG) cells. Since EG cells were also obtained by using OSM or the IL-6/sIL-6R complex in place of LIF, a significant contribution of gp130-mediated signaling in EG cell formation was further suggested.

Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro.

Effects of retinoic acid (RA) on the growth of mouse primordial germ cells (PGC) were studied using an in vitro coculture system. Addition of RA to the culture medium markedly increased the number of PGC of the migratory phase and also significantly retarded the depletion of gonadal PGC. We observed stimulation of mitotic activity by RA treatment at all stages of PGC examined (8.5, 11.5, and 13.5 days postcoitum), even in the absence of feeder cells. From these results, we conclude that RA affects PGC directly to promote their survival and proliferation and that the RA-induced intracellular signal may have a crucial role in the development of PGC.

Insulin-like growth factor-I and transforming growth factor-alpha stimulate differentiation of type A spermatogonia in organ culture of adult mouse cryptorchid testes.

This study assessed the effect of growth factors on testicular germ cell differentiation in vitro. Testicular fragments of experimentally prepared cryptorchid testes of adult mice were cultured for 9 days in serum-free media containing various concentrations of IGF-I, TGF-alpha, FGF, and PDGF. Their histology was then examined under a light microscope. Each type of germ cell and mitotic cell in the seminiferous tubules was counted per 1000 Sertoli cells. IGF-I at a concentration of 10 ng/ml induced maximal differentiation of type A spermatogonia. TGF-alpha at concentrations ranging from 1 to 10 ng/ml also stimulated differentiation, whereas FGF and PDGF did not show any stimulation of spermatogonial differentiation in this experimental system.

Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis.

To study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N-glycanase, suggesting the presence of asparagine-linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present.

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level.

Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development.

During mammalian spermatogenesis, many specific molecules are expressed. We have recently identified a 93-kDa male meiotic germ cell-specific antigen (Meg 1) exclusively expressed in germ cells from the pachytene spermatocyte to the spermatid stage using the monoclonal antibody TRA 369 (Watanabe, D., Sawada, K., Koshimizu, U., Kagawa, T., and Nishimune, Y. (1992) Mol. Reprod. Dev. 33, 307-312). In this study, we cloned a cDNA representing this antigen from a mouse testis cDNA expression library, using the monoclonal antibody TRA 369. Northern blotting showed that this transcript was 2.3 kilobases in length and was expressed only in the testis and not in other somatic tissues or in the ovary. The expression of the mRNA was first detected at the pachytene spermatocyte stage of male germ cell development, and this expression was correlated with the expression of the protein. Sequence analysis of the cDNA revealed that the predicted protein consists of 611 amino acids, including a hydrophobic NH2 terminus characteristic of a signal peptide, two sets of internal repetitive sequences (four repeats of IPDPSAVKPEDWDD and GEWXPPMIPNPXYQ), and a hydrophilic COOH terminus. The deduced amino acid sequence has 58% homology with dog calnexin (the ER membrane phosphoprotein of pancreatic cells) and significant partial homology with calreticulin (high affinity Ca(2+)-binding protein of the ER membrane) at the repetitive sequence. Furthermore, we demonstrated the 45Ca2+ binding ability of this antigen by a 45Ca2+ overlay assay, and the name calmegin is proposed for this antigen. Calmegin is a novel Ca(2+)-binding protein that is specifically expressed in spermatogenesis. The highly regulated, specific, and abundant expression of calmegin suggests that it has important roles in spermatogenesis.

Wn mutation of c-kit receptor affects its post-translational processing and extracellular expression.

The W locus of mice encodes the c-kit receptor tyrosine kinase. Recently, we characterized a novel mutant allele, Wn, and demonstrated that the c-kit protein synthesized in Wn/Wn cultured mast cells (CMC) was reduced in size and not expressed on their surface (Tsujimura et al., 1993). In this study, we further examined biochemical nature of the mutant form of c-kit protein, by using Wn/Wn CMC and 293T cells transfected with Wn-type c-kit cDNA (c-kitWn). The c-kit product synthesized in Wn/Wn CMC was truncated almost all cytoplasmic domain and was less glycosylated. In c-kitWn-transected cells, both glycosylation and extracellular expression of c-kit protein was also impaired, however, no truncation was detected. These results indicate that Wn-mutant form of c-kit product is insufficient in maturation, which is associated with impairments in the transport to the plasma membrane, and retention of the mutant protein in endoplasmic reticulum is suggested. This is the first demonstration of the c-kit mutation affecting posttranslational processing its product.

In utero and mammary transfer of hantavirus antibody from dams to infant rats.

The effects of maternal-infant transfer of specific antibody on infection attributable to hemorrhagic fever with renal syndrome-causing virus in the infant rat were studied. In cross-fostering experiments, maternal hantavirus-specific antibody was shown to be transferred equally effectively to infants either in utero or by breastfeeding. Both IgG- and IgA-specific antibodies were transferred. Infected infants that initially acquired high levels of maternal antibodies by either route survived lethal doses of the virus and did not show any signs of disease. Furthermore, 15 weeks later, these animals had no evidence of remaining antibodies or viral infection. These results indicate that maternal antibody, transferred to the infant rat in utero or by breastfeeding, is protective against hantavirus infection.

Effects of hormones, cyclic AMP analogues and growth factors on steel factor (SF) production in mouse Sertoli cell cultures.

Regulation of steel factor (SF) production in Sertoli cells from postnatal mouse testes was studied in a mast cell-Sertoli cell coculture system. Treatment of Sertoli cells with dibutyryl cAMP (50-1000 mumol l-1), forskolin (1-25 mmol l-1), and cholera toxin (10 micrograms ml-1) increased SF production, whereas FSH and theophylline had no significant effect. Furthermore, growth factors and testosterone, which would play some roles in spermatogenesis, were also tested, but none of these stimulated SF production. The constitutive production of SF may, rather, reflect the physiological condition of Sertoli cells in vivo.

A novel stage-specific differentiation antigen is expressed on mouse testicular germ cells during early meiotic prophase.

A murine cell surface antigen exhibiting stage-specific expression during spermatogenesis was detected with two monoclonal antibodies (mAbs), designated BC7 and CA12. In mouse testis, these mAbs recognized a small population of cells located near the periphery of seminiferous tubules at stages XII and I-VI, and these spermatogenic cells were identified as zygotene and early pachytene spermatocytes. Expression of the antigens was transient and was not detected in germ cells at more advanced stages of spermatogenesis such as late pachytene spermatocytes and round spermatids. Immunoprecipitation and immunoblotting studies showed that both mAbs CA12 and BC7 reacted with the same antigenic molecule, which had an estimated molecular mass of 95 kDa. CA12/BC7 antigen, detected in plasma membrane fraction, was a glycoprotein with sialic acid residues and had affinity with WGA lectin. Furthermore, intraperitoneal injection of mAb BC7 caused an apparent spermatogenic disturbance in prepubertal mice. These results suggested that CA12/BC7 antigen, a novel cell surface glycoprotein, is an essential molecule that plays an important role during early meiotic prophase of spermatogenesis.