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.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Elfin is expressed during early heart development

Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.     

The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development. At embryonic day (E) 5.5, Sfrp5 is expressed in the visceral endoderm at the distal tip region of the embryo and at E6.5 in the AVE opposite the primitive streak. In Lim1 embryos, which lack anterior neural tissue and sometimes form a secondary body axis, Sfrp5-expressing cells fail to move towards the anterior and remain at the distal tip of E6.5 embryos. When compared with Dkk1, which encodes another secreted Wnt antagonist molecule present in the visceral endoderm, Sfrp5 and Dkk1 expression overlap but Sfrp5 is expressed more broadly in the AVE. Between E7.5 and 8, Sfrp5 is expressed in the foregut endoderm underlying the cardiac mesoderm. At E8.5, Sfrp5 is expressed in the ventral foregut endoderm that gives rise to the liver. Additional domains of Sfrp5 expression occur in the dorsal neural tube and in the forebrain anterior to the optic placode. These findings identify a gene encoding a secreted Wnt antagonist that is expressed in the extraembryonic visceral endoderm and anterior definitive endoderm during axis formation and organogenesis in the mouse.     

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.     

Laminin alpha5 is essential for the formation of the zebrafish fins

The vertebrate fin fold, the presumptive evolutionary antecedent of the paired fins, consists of two layers of epidermal cells extending dorsally and ventrally over the trunk and tail of the embryo, facilitating swimming during the embryonic and larval stages. Development of the fin fold requires dramatic changes in cell shape and adhesion during early development, but the proteins involved in this process are completely unknown. In a screen of mutants defective in fin fold morphogenesis, we identified a mutant with a severe fin fold defect, which also displays malformed pectoral fins. We find that the cause of the defect is a non-sense mutation in the zebrafish lama5 gene that truncates laminin α5 before the C-terminal laminin LG domains, thereby preventing laminin α5 from interacting with its cell surface receptors. Laminin is mislocalized in this mutant, as are the membrane-associated proteins, actin and β-catenin, that normally form foci within the fin fold. Ultrastructural analysis revealed severe morphological abnormalities and defects in cell-cell adhesion within the epidermis of the developing fin fold at 36 hpf, resulting in an epidermal sheet that can not extend away from the body. Examining the pectoral fins, we find that the lama5 mutant is the first zebrafish mutant identified in which the pectoral fins fail to make the transition from an apical epidermal ridge to an apical fold, a transformation that is essential for pectoral fin morphogenesis. We propose that laminin α5, which is concentrated at the distal ends of the fins, organizes the distal cells of the fin fold and pectoral fins in order to promote the morphogenesis of the epidermis. The lama5 mutant provides novel insight into the role of laminins in the zebrafish epidermis, and the molecular mechanisms driving fin formation in vertebrates.     

Alpha-fetoprotein is dynamically expressed in rat pancreas during development

To identify proteins involved in pancreatic development, we used a differential proteomics approach by comparing pancreatic extracts from four biologically significant stages of development: embryonic day (E) 15.5, E18.5, postnatal (P) days 0 and adult. By two-dimensional gel electrophoresis (2D-E) and MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) following database searching and protein annotation, 15 proteins were identified as being differently expressed in the pancreas between the four phases. The expression pattern and the localization of alpha-fetoprotein (AFP), one of significant changed proteins observed, were further determined. Four isoforms of AFP (72 kDa, 60 kDa, 48 kDa and 37 kDa) were found by Western blotting in the pancreas tested, most of them showed a stronger signal in E18.5 followed by a steady decrease and only a 60-kDa isoform was detected in the adult pancreas. Immunolocalization for AFP revealed that a positive reactivity was detectable at E15.5 pancreas, became stronger in the cytoplasm of mesenchyme cells at E18.5, and declined after birth to a nearly undetectable level in adults. The dynamic expression of AFP in rat pancreas from different stages indicates that AFP might be involved in some aspects of pancreatic development.     

Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.     

Systematic screening for genes specifically expressed in the anterior neuroectoderm during early Xenopus development

A cDNA library derived from the anterior neuroectoderm (ANE) of Xenopus late-gastrula embryos was systematically screened to isolate novel developmental regulatory genes involved in early brain development. We isolated 1,706 5 expressed sequence tags (ESTs), which were subdivided into 1,383 clusters and categorized into 19 classes based on predicted functions according to their similarities to other known genes. Of these, 757 clusters that were considered possible novel regulatory genes or unknown genes were subjected to expression pattern analysis using whole-mount in situ hybridization. Genes from 69 clusters (9%) were expressed in the ANE region. Based on their expression patterns and predicted amino acid sequences, 25 genes were selected for further analysis as novel Xenopus genes expressed broadly or region-specifically in the ANE. Eighteen genes were expressed in postulated patterning centers in the neuroectoderm, including the anterior (four genes) and lateral (nine genes) neural ridges, the midbrain-hindbrain boundary region (one gene) and the midline region of the neural plate (two genes), whereas 13 genes were expressed in the eye anlagen. Therefore, early regionalization of the neuroectoderm appears to occur mainly in those neural patterning centers and the eye anlagen. We determined the entire coding regions of p54nrb, Semaphorin 6D and a novel gene designated scribble-related protein 1 (SCRP1). Interestingly, Semaphorin 6D is expressed in the mesoderm with a dorsoventral gradient, as well as in the ectoderm at the gastrula stage, implying a new role for this protein in development other than in axon guidance.     

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.     

Morphogenesis of the anterior segment in the zebrafish eye

Background  

The ocular anterior segment is critical for focusing incoming light onto the neural retina and for regulating intraocular pressure. It is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. During development, cells from diverse embryonic lineages interact to form the anterior segment. Abnormal migration, proliferation, differentiation, or survival of these cells contribute to diseases of the anterior segment such as corneal dystrophy, lens cataract, and glaucoma. Zebrafish represent a powerful model organism for investigating the genetics and cell biology of development and disease. To lay the foundation for genetic studies of anterior segment development, we have described the morphogenesis of this structure in zebrafish.     

alpha4 integrin is expressed in a subset of cranial neural crest cells and in epicardial progenitor cells during early mouse development

By RNA in situ hybridization and immunohistochemical analyses of early stage mouse embryos, we find that alpha 4 integrin gene is expressed in migratory cranial neural crest cells originating from the presumptive forebrain, midbrain, and rhombomeres 1 and 2 of the presumptive hindbrain. alpha 4 is also expressed in epicardial progenitor cells in the septum transversum that migrate to the heart.     

Selenoprotein N is dynamically expressed during mouse development and detected early in muscle precursors

Background  

In humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles.     

Lectin binding in the anterior segment of the bovine eye

Summary Eleven different fluorescent lectin-conjugates were used to reveal the location of carbohydrate residues in frozen sections of the anterior segment of bovine eyes. The lectins were specific for the following five major carbohydrate groups: (1) glucose/mannose group (Concanavalin A (Con A)); (2)N-acetylglucosamine group (wheat germ agglutinin (WGA)); (3) galactose/N-acetylgalactosamine group (Dolichos biflorus agglutinin (DBA),Helix pomatia agglutinin (HPA),Helix aspersa agglutinin (HAA),Psophocarpus tetragonolobus agglutinin (PTA),Griffonia simplicifolia agglutinin-I-B₄ (GSA-I-B₄),Artocarpus integrifolia agglutinin (JAC), peanut agglutinin (PNA) andRicinus communis agglutinin (RCA-I)); (4)l-fucose group (Ukex europaeus agglutinin (UEA-I)); (5) sialic acid group (wheat germ agglutinin (WGA)). All the studied lectins except UEA-I reacted widely with different structures and the results suggest that there are distinct patterns of expression of carbohydrate residues in the anterior segment of the bovine eye. UEA-I bound only to epithelial structures. Some of the lectins reacted very intensely with apical cell surfaces of conjunctival and corneal epithelia suggesting a different glycosylation at the glycocalyx of the epithelia. Also, the binding patterns of conjunctival and corneal epithelia differed with some of the lectins: PNA and RCA-I did not bind at all, and GSA-I-B₄ bound only very weakly to the epithelium of the cornea, whereas they bound to the epithelium of the conjunctiva. In addition, HPA, HAA, PNA and WGA did not bind to the corneal basement membrane, but bound to the conjunctiva and vascular basement membranes. This suggests that corneal basement membrane is somehow different from other basement membranes. Lectins with the same carbohydrate specificity (DBA, HPA, HAA and PTA) reacted with the sections almost identically, but some differences were noticed: DBA did not bind to the basement membrane of the conjunctiva and the sclera and did bind to the basement membrane of the cornea, whereas other lectins with same carbohydrate specificities reacted vice versa. Also, the binding of PTA to the trabecular meshwork was negligible, whereas other lectins with the same carbohydrate specificities reacted with the trabecular meshwork. GSA-I-B₄ reacted avidly with the endothelium of blood vessels and did not bind to the stroma, so that it made blood vessels very prominent and it might be used as an endothelial marker. This lectin also reacted avidly with the corneal endothelium. Therefore, GSA-I-B₄ appears to be a specific marker in bovine tissues for both blood vessel and corneal endothelium cells.     

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.     

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.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.