Position-specific expression of the annulin protein during grasshopper embryogenesis.

Annulin, named for its annular expression in developing limb buds, is a approximately 100 kDa membrane-associated protein that is expressed in a complex and changing pattern during grasshopper embryogenesis. Its expression is dynamic along the developing midline and in the mesoderm, transient in neuroepithelial sheath cells around mitotic neuroblasts, and position-specific in circumferential stripes in each limb bud segment. Annulin expression begins along the midline of the embryo at the onset of gastrulation. Mesoderm cells express the protein as they migrate away from the midline as do new cells that come to lie at the midline. During neurogenesis, annulin expression disappears from many midline cells until only a specific subset of midline glial cells expresses high levels of the protein. Starting at the beginning of neurogenesis, sheath cells express annulin in correlation with the mitotic activity of the neuroblasts they surround.     

Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo.

Monoclonal antibody 6F8 was used to characterize and clone fasciclin IV, a new axonal glycoprotein in the grasshopper, and to study its function during growth cone guidance. Fasciclin IV is dynamically expressed on a subset of axon pathways in the developing CNS and on circumferential bands of epithelial cells in developing limb buds. One of these bands corresponds to the location where the growth cones of the Ti1 pioneer neurons make a characteristic turn while extending toward the CNS. Embryos cultured in the 6F8 antibody or Fab exhibit aberrant formation of this axon pathway. cDNA sequence analysis suggests that fasciclin IV has a signal sequence; long extracellular, transmembrane, and short cytoplasmic domains; and shows no homology with any protein in the available data bases. Thus, fasciclin IV appears to be a novel integral membrane protein that functions in growth cone guidance.     

Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex

We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P. Sugrue. 1992. J. Cell Biol. 118:1477-1488). We suggest that the function of pinin is to pin intermediate filaments to the desmosome. Therefore, pinin may play a significant role in reinforcing the intermediate filament- desmosome complex. cDNA clones coding for pinin were identified, using degenerative oligonucleotide probes that were based on the internal amino acid sequence of pinin for the screening of a cDNA library. Immunoblotting of expressed recombinant proteins with the monoclonal 08L antibody localized the 08L epitope to the carboxyl end of the protein. Polyclonal antibodies directed against fusion proteins immunoidentified the 140-kD protein in tissue extracts. Immunofluorescence analysis, using the antifusion protein antibody, demonstrated pinin at lateral epithelial boundaries, which is consistent with desmosomal localization. The conceptual translation product of the cDNA clones contained three unique domains: (a) a serine- rich domain; (b) a glutamine-proline, glutamine-leucine repeat domain; and (c) an acidic domain rich in glutamic acid. Although the 3' end of the open reading frame of the clone for pinin showed near identity to a partial cDNA isolated for a pig neutrophil phosphoprotein (Bellavite, P., F. Bazzoni, et al. 1990. Biochem. Biophys. Res. Commun. 170:915- 922), the remaining sequence demonstrated little homology to known protein sequences. Northern blots of mRNA from chicken corneal epithelium, MDCK cells, and various human tissues indicated that pinin messages exhibit tissue-specific variation in size, ranging from 3.2 to 4.1 kb. Genomic Southern blots revealed the existence of one gene for pinin, suggesting alternative splicing of the mRNA. Expression of the full-length cDNA clones in human 293 cells and monkey COS-7 cells demonstrated that a 140-kD immunoreactive species on Western blots corresponded to pinin. Pinin cDNA transfected into the transformed 293 cells resulted in enhanced cell-cell adhesion. Immunofluorescence staining revealed that the expressed pinin protein was assembled to the lateral boundaries of the cells in contact, which is consistent with the staining pattern of pinin in epithelial cells.     

Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system

The murine genome contains multiple genes with protein domains homologous to the Drosophila paired box, present in certain segmentation genes. At least one of these murine paired box (Pax) genes is associated with a developmental mutation. This report, in conjunction with the accompanying paper, describes a second member of this gene family, Pax2, that is also expressed during embryogenesis. Two overlapping cDNA clones were isolated and sequenced. At least two forms of the Pax2 protein can be deduced from the cDNA sequence. In addition to the highly conserved paired domain, an octapeptide sequence is located downstream. Expression of Pax2 is primarily restricted to the developing embryo in the excretory and central nervous systems. The transient nature of Pax2 expression during kidney organogenesis correlates with polarization and induction of epithelial structures and may indicate an important morphogenetic role for this gene.     

A new Arabidopsis nucleic-acid-binding protein gene is highly expressed in dividing cells during development

An Arabidopsis thaliana cDNA encoding a new RNA-binding protein (RBP37) was cloned from a silique cDNA library. The predicted amino acid sequence corresponds to a RBP containing two RNA recognition motifs (RRM) and a basic domain. An affinity for nucleic acids was confirmed in binding assays using in vitro synthesised AtRBP37 protein. In situ hybridisation experiments on sections of flowers and siliques showed expression only in growing organs: gynoecium, petals, filaments and during early-embryogenesis expression is located in the embryo proper and the suspensor up to late heart stage. Expression is not detected in the embryo during maturation.This results suggests an expression pattern correlated with dividing cells.     

Gene sequence,developmental expression,and protein phosphorylation of RAB-17 in maize

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17 164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279–2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.     

The EGF-inducible protein EIP-1 of migrating normal and malignant rat liver epithelial cells is identical to plasminogen activator inhibitor 1 and is a component of the ECM migration tracks.

Induction of rat liver epithelial cell migration by epidermal growth factor (EGF) changes the expression pattern of secreted proteins. The expression of the early induced glycoprotein EGF-inducible protein No. 1 (EIP-1) correlates with the migratory behavior of both normal and Ha-ras-transformed, tumorigenic cells and is deposited into the ECM migration tracks. The sequence of two clones from a cDNA library of EGF-induced cells and the amino terminal sequence of the purified protein revealed that EIP-1 is identical to rat plasminogen activator inhibitor 1 (PAI-1). Based on the migration-linked expression pattern of EIP-1/PAI-1 it is proposed that the inhibitor is required for the migration of these cells, but not sufficient to stimulate it.     

Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors.

The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.     

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.     

Dynamic expression of a cell surface protein during rearrangement of epithelial cells in the Manduca wing monolayer.

The expression of cell surface protein 2F5 changes dynamically in space and time during morphogenesis of the Manduca wing pattern. Two cell types (generalized epithelial cells and scale precursors) rearrange within each of the two epithelial monolayers of the wing to form periodic rows of scale cells. These two monolayers also interact with each other during a brief period of adult development. Each cell type shows a different pattern of protein 2F5 expression during cell rearrangement and during interaction of the two wing monolayers. Before and after these morphogenetic movements of epithelial cells, the protein is expressed on only a small population of wing cells. In abdominal epithelia where scale cells are also present but are not arranged in periodic rows, the expression pattern of the surface protein is temporally and spatially very different. An earlier study (Nardi and Magee-Adams, Dev. Biol., 116, 278-290, 1986) had shown that basal processes only extend from epithelial cells during their period of rearrangement within a monolayer and during the transient apposition of the wing's upper and lower monolayers. The differential distribution of protein 2F5 on lateral surfaces and basal processes of scale precursor cells and generalized epithelial cells may account in part for their orderly segregation into alternating rows as well as for the transient interaction of the two wing monolayers.     

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.     

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.     

Molecular cloning and expression of a novel adhesion molecule, SC1

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.