The EGF-like homeotic protein dlk affects cell growth and interacts with growth-modulating molecules in the yeast two-hybrid system

Levels of dlk, an EGF-like homeotic protein, are critical for several differentiation processes. Because growth and differentiation are, in general, exclusive of each other, and increasing evidence indicates that Dlk1 expression changes in tumorigenic processes, we studied whether dlk could also affect cell growth. We found that, in response to glucocorticoids, Balb/c 3T3 cells with diminished levels of dlk expression develop foci-like cells that have lost contact inhibition, display altered morphology, and grow faster than control cell lines. Balb/c 3T3 cells spontaneously growing more rapidly are also dlk-negative cells. Moreover, screening by the yeast two-hybrid system, using Dlk1 constructs as baits, resulted in the isolation of GAS1 and acrogranin cDNAs. Interestingly, these proteins are cysteine-rich molecules involved in the control of cell growth. Taken together, these observations suggest that dlk may participate in a network of interactions controlling how the cells respond to growth or differentiation signals.     

CD2-associated protein is widely expressed and differentially regulated during embryonic development

CD2-associated protein (CD2AP) is an adapter protein that is involved in various signaling and vesicular trafficking processes and also functions as a linker between plasma membrane proteins and the actin cytoskeleton. The protein is known to have important functions in T cells and glomerular podocytes, but it is also expressed by many other adult-type tissues and cells. Here we analyzed the expression of the protein during early embryonic development and organogenesis of the mouse. The results showed differential tissue-specific regulation of CD2AP in developing and maturing organs. In oocytes and pre-implantation embryos, CD2AP was located diffusely in the cytoplasm, whereas in late blastocysts it was concentrated to the intercellular contacts. During organogenesis, CD2AP was distinctly upregulated upon, e.g., the pretubular aggregation of metanephric mesenchyme cells and the appearance of the osteoblastic rim around cartilages during endochondral ossification. High CD2AP expression was also observed during epithelial-like conversion of some highly specialized secretory cell types such as the odontoblasts, the cells of the choroid plexus and the decidualized cells of the endometrial stroma. In other instances, such as the development of the proximal tubuli of the kidney and the flat alveolar epithelium of the lung, the protein was downregulated upon differentiation and maturation of the cells. Finally, certain cells, e.g., glomerular podocytes, those forming the collecting ducts of the kidney, and the urothelium of the kidney pelvis, expressed CD2AP throughout their differentiation and maturation. Multiple molecules and complex pathways regulate embryogenesis, and scaffolding proteins apparently have pivotal roles in targeting and finetuning, e.g., growth factor- or hormone-induced processes. The cell-type specific spatio-temporal regulation of CD2AP during development suggests that this adapter protein is a key regulatory partner in many signaling pathways and cellular processes governing differentiation and morphogenesis.     

Expression of nerve growth factor receptor during human peripheral nerve development

The expression of NGF receptors on human Schwann cells during development and myelination and in culture was analyzed using a murine monoclonal antibody to human NGF receptor. Nonmyelinated femoral nerves from 13- to 14-week fetuses stained strongly for NGF receptor, whereas tissues from later stages of development showed a decrease in the staining intensity. These changes correlated with the initiation of myelination (17-19 weeks), as observed by phase-contrast and electron microscopy, and the reactivity with monoclonal antibody 4C5, a marker of mature Schwann cells. In adult nerves, only the perineurium and few endoneurial cells were stained with anti-NGF receptor antibody. Cultured human fetal Schwann cells were positive for NGF receptor by immunofluorescence irregardless of donor age or length of time in culture. The decreased staining of NGF receptor with nerve maturation may reflect a dependence of antigen expression on Schwann cell differentiation and/or neuron-Schwann cell interaction.     

The mouse gap junction gene connexin29 is highly expressed in sciatic nerve and regulated during brain development

A novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28 981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelin-forming glial cells.     

Mepe is expressed during skeletal development and regeneration

Matrix extracellular phosphoglycoprotein (Mepe) is a bone metabolism regulator that is expressed by osteocytes in normal adult bone. Here, we used an immunohistochemical approach to study whether Mepe has a role in murine long bone development and regeneration. Our data showed that Mepe protein was produced by osteoblasts and osteocytes during skeletogenesis, as early as 2 days postnatal. During the healing of non-stabilized tibial fractures, which occurs through endochondral ossification, Mepe expression was first detected in fibroblast-like cells within the callus by 6 days postfracture. By 10 and 14 days postfracture (the hard callus phase of repair), Mepe was expressed within late hypertrophic chondrocytes and osteocytes in the regenerating tissues. Mepe became externalized in osteocyte lacunae during this period. By 28 days postfracture (the remodeling phase of repair), Mepe continued to be robustly expressed in osteocytes of the regenerating bone. We compared the Mepe expression profile with that of alkaline phosphatase, a marker of bone mineralization. We found that both Mepe and alkaline phosphatase increased during the hard callus phase of repair. In the remodeling phase of repair, Mepe expression levels remained high while alkaline phosphatase activity decreased. We also examined Mepe expression during cortical bone defect healing, which occurs through intramembranous ossification. Mepe immunostaining was found within fibroblast-like cells, osteoblasts, and osteocytes in the regenerating bone, through 5 to 21 days postsurgery. Thus, Mepe appears to play a role in both long bone regeneration and the latter stages of skeletogenesis.     

The insect immune protein hemolin is expressed during oogenesis and embryogenesis

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.     

Novel nucleolar protein, midnolin, is expressed in the mesencephalon during mouse development

Using the gene trap method and the selection of embryonic stem cells in vitro, we have identified several novel genes involved in mouse development. The detailed analysis of one of these, named midnolin (midbrain nucleolar protein), is reported here. Expression of the midnolin gene is developmentally regulated: it is strongly expressed at the mesencephalon (midbrain) of the embryo in day 12.5 (E12.5) mice. The midnolin encodes a protein of 508 amino acids (aa), which contains a Ubiquitin-like domain. The intracellular distribution of the midnolin was studied by using midnolin-green fluorescent protein (GFP) fusion proteins. Midnolin was found to be localized in the nucleus and nucleolus, but not in the cytoplasm. The nucleolar localization signal was determined to be a 28aa peptide (440-QQKRLRRKARRDARGPYHWTPSRKAGRS-467) located at the C-terminal region of the midnolin. Our results suggest that midnolin is involved in regulation of genes related to neurogenesis in the nucleolus.     

Pax-5 is expressed at the midbrain-hindbrain boundary during mouse development.

The murine paired-box-containing gene 5, Pax-5, is highly homologous to two other Pax genes, Pax-2 and Pax-8. The expression pattern of Pax-5 during mouse embryogenesis was examined by in situ RNA hybridization and compared to those of Pax-2 and Pax-8. Beginning at day 9.5 postcoitum (p.c.), Pax-5 was expressed in the developing brain, predominantly at the midbrain-hindbrain boundary, and in the neural tube. While the neural tube expression pattern overlapped completely with Pax-2 and Pax-8, the expression pattern in the brain was only partially overlapping. Unlike Pax-2 and Pax-8, Pax-5 was not expressed in the developing excretory system, thyroid, eye or ear. Our data suggest that Pax-5 has a role in the development of the central nervous system.     

Adamts9 is widely expressed during mouse embryo development

ADAMTS metalloproteases constitute a family of 19 secreted protein or proteoglycan processing enzymes. ADAMTS9 and its closest mammalian relative, ADAMTS20, are related to gon-1, a metalloprotease required for gonadal morphogenesis in Caenorhabditis elegans. Although expressed at generally low levels in embryonic subectodermal mesenchyme, ADAMTS20 is required for melanoblast colonization of skin. Mutations in Adamts20 cause Belted, one of several white spotting alleles in the mouse. In contrast to Adamts20, we previously showed by Northern blotting that Adamts9 was expressed highly throughout mouse development. Using RNA in situ hybridization, we determined the spatial and temporal regulation of Adamts9 during mouse embryogenesis. At 7.5 dpc Adamts9 is expressed in the allantois, trophoblast, parietal endoderm and decidual tissue. At 9.5 dpc it is expressed in head mesoderm and in the developing heart. From 11.5 to 12.5 dpc, Adamts9 is strongly expressed in posterior mesoderm, in the craniofacial region, ventral body wall and diaphragm. After 14.5 dpc, Adamts9 was highly expressed in the mesenchyme of developing lung, kidney, and mesentery. It is expressed during skeletogenesis, being present from 13.5 dpc in perichondrium, in the proliferation zone of growth plates after 15.5 dpc and it is highly expressed in newly formed bone. It is expressed in vascular endothelium and during formation of the pituitary and cochlea, but expression in the central nervous system is limited to the floor plate of the diencephalon, to the ventricular zone of the cerebral cortex and to the choroid plexus.     

Drosophila protein phosphatase 5 is encoded by a single gene that is most highly expressed during embryonic development

A putative Drosophila melanogaster homologue of mammalian PP5, termed Dm PP5, was identified from cDNA. Dm PP5 comprises a phosphatase catalytic domain preceded by an amino terminal domain containing three tetratricopeptide repeat motifs and shares 60% overall amino acid identity with human PP5. Genomic restriction analysis identified a single Dm PP5 gene that was mapped to the third chromosome at locus 85E10-12 and a strain carrying a deletion that encompasses this gene was identified. Dm PP5 mRNA and protein are more highly expressed in the embryo than at later developmental stages, but their expression levels do not always change synchronously. Dm PP5 protein localises to both the nucleus and the cytoplasm of cells at the periphery of newly cellularized embryos.     

Two novel transmembrane protein tyrosine kinases expressed during Caenorhabditis elegans hypodermal development.

We describe our characterization of kin-15 and kin-16, a tandem pair of homologous Caenorhabditis elegans genes encoding transmembrane protein tyrosine kinases (PTKs) with an unusual structure: the predicted extracellular domain of each putative gene product is only about 50 amino acids, and there are no potential autophosphorylation sites in the C-terminal domain. Using lacZ fusions, we found that kin-15 and kin-16 both appear to be expressed during postembryonic development in the large hypodermal syncytium (hyp7) around the time that specific hypodermal cells fuse with hyp7. kin-15 and kin-16 were positioned on the genetic and physical maps, but extrachromosomal arrays containing wild-type kin-15 and/or kin-16 genes were unable to complement candidate lethal mutations. The results suggest that kin-15 and kin-16 may be specifically involved in cell-cell interactions regulating cell fusions that generate the hypodermis during postembryonic development.