Differential Expression of c-myc and N-myc during Oral Organogenesis of the Mouse Embryo

The expressions of the c- and N- myc proto-oncogenes during oral development of midgestational mouse embryos were examined by in situ hybridization in order to analyze their roles. In the mandibular rudiment, c- myc RNA was strongly expressed in the mesenchymal condensation around the ossification center in which high-level expression of 2 ar (osteopontin) was detected. In tooth germs, c- myc was strongly expressed in the epithelia at the bud stage, and its expression gradually became restricted to the inner enamel epithelia from the cap to bell stages. In contrast, N- myc expression was detected in the undifferentiated mesenchymal cells of the dental papilla. Incorporation of BrdU was examined immunohistochemically to study the relationship between the expressions of c- and N- myc and cell proliferation. Unexpectedly, the distribution of BrdU labelled regions was not coincident with the expressions of c- and N- myc . These results suggest that the level of myc expression is not necessarily related to cell proliferation.     

Structure and site of expression of a murine type II hair keratin

We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Transient expression of a novel serine protease in the ectoderm of the ascidian Herdmania momus during development

 We have studied gene expression during ascidian embryonic development using the technique of differential display and isolated partial cDNA sequences of 12 genes. Developmental regulation of these genes has been confirmed by northern hybridization analysis. Further cDNA cloning and sequence analysis of an mRNA that is present during gastrulation, neurulation and tailbud formation reveals that it encodes a novel serine protease containing a single kringle motif and catalytic domain. The spatial expression of this gene, designated Hmserp1, is restricted to precursor cells of the epidermis. The structure and expression of Hmserp1 is discussed in relation to possible functions during development. Received: 8 October 1996 / Accepted: 17 December 1996     

Type I and type II cytokeratin cDNAs from the zebrafish (Danio rerio) and expression patterns during early development

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1,426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2,398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73%-91% and 87%-93% similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184-205 and 191-212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.     

Expression of HGF/SF,HGFI/MSP,and c-met suggests new functions during early chick development

We report the cloning of full-length cDNAs for a plasminogen-related growth factor, hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor, c-met, and a close member of the same family, hepatocyte growth factor-like/macrophage stimulating protein (HGFI/MSP), from the chick. We have used these cDNAs to provide the first report of the expression of this family of growth factors and the c-met receptor at early stages of vertebrate development. RNAase protection and wholemount in situ hyb ridization were used on chick embryos between formation of the primitive streak and early organogenesis. We find patterns of expression for HGF/SF and its receptor c-met consistent with their known roles in ep ithelial-mesenchymal transformation and angiogenesis. In addition, these genes and HGFI/MSP are expressed in discrete locations within developing somites, suggesting a role in paraxial mesodermal development. Very strong and early expression of HGF/SF in the elevating limb buds suggests its involvement in limb outgrowth. HGFI/MSP is expressed in the notochord and then in the prospective floor plate region and could play a role in development of the neural tube. Interestingly, c-met is often more closely as sociated with HGFI/MSP than with its known ligand, HGF/SF, raising the possibility that c-met expression may be induced by HGFI/MSP. © 1995 Wiley-Liss, Inc.     

Differential expression of mRNAs for sialyltransferase isoenzymes induced in the hippocampus of mouse following kindled seizures

Sialic acids play important roles in various biological functions. In the brain, evidence suggests that sialylation of glycoproteins and glycolipids affects neural plasticity. While the 18 sialyltransferase isoenzymes (STs) identified to date synthesize individual sialyl-oligosaccharide structures, they each exhibit activity toward more than one substrate and can overlap in their specificity. Therefore, the distribution of STs is a secondary factor in the study of specific sialylation. Here, seven STs; ST3Gal I-IV, ST8Sia IV, ST6Gal I and ST6GalNAc II, the expressions of which were identified in the adult hippocampus by RT-PCR, showed diverse localization patterns in the hippocampus on in situ hybridization, suggesting that the individual cells expressed relevant STS: Furthermore, to assay activity-related changes in ST expression, we used amygdaloid-kindling among models of neural plasticity. Differential expression of the STs participating in the kindling, notably, up-regulation of ST3Gal IV and ST6GalNAc II mRNAs, and down-regulation of ST3Gal I and ST8Sia IV mRNAs, were observed in the hippocampus following kindled seizures. These results indicate that ST expressions are regulated by physiological activity and may play a role in neural plasticity.     

Region-specific and epileptogenic-dependent expression of six subtypes of alpha2,3-sialyltransferase in the adult mouse brain

Sialylated glycoconjugates play important roles in various biological functions. The structures are also observed in brains and it has been proposed that sialylation may affect neural plasticity. To clarify the effects of sialylation in the brain, particular neurons that exhibit sialylation should first be determined. Using in situ hybridization, we performed systematic surveys of the localization of mRNAs encoding the six alpha2,3-sialyltransferases (ST3Gal I-VI) in the adult mouse brain with or without physiological stimulation. First, striking region-specific patterns of expression were observed: While ST3Gal II, III, and V mRNAs were in neuronal cells throughout the brain, ST3Gal I, IV, and VI mRNAs were in restricted brain regions. Next, to assess whether the expression of the six mRNAs can be regulated, we examined the effect of kindling epileptogenesis on the six mRNA levels. Of the six subtypes, upregulation in the ST3Gal IV level in the thalamus was most pronounced; the number of ST3Gal IV-expressing neurons in the anterior thalamic nuclei increased from 2% to 21% in a time-dependent manner during epileptogenesis. Western blot analysis evaluated the increase of the end-products in the thalamus. These findings provide a molecular basis to clarify when and where sialylated glycoconjugates function accompanied by neural plasticity.     

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.     

Differential expression of two spermidine synthase genes during early fruit development and in vegetative tissues of pea

Two cDNAs from young pea fruits coding for functional spermidine synthases (EC 2.5.1.16) were isolated. The corresponding genes were named psSPDSYN1 and psSPDSYN2. Both cDNAs complemented spe3 gene when introduced into the Y480 strain of Saccharomyces cerevisiae, which is a null mutant for the spermidine synthase gene. psSPDSYN1 and psSPDSYN2 are regulated differentially. psSPDSYN1 is up-regulated early after fruit set whereas psSPDSYN2 is expressed later. Spermidine synthase activity was detected in pea ovaries, and correlates with the pattern of expression of psSPDSYN1. In the pea plant, psSPDSYN1 is highly expressed in actively growing tissues, whereas the highest level of psSPDSYN2 mRNA was detected in fully elongated stem.     

Temporal and spatial gene expression of cytochrome B5 during flower and fruit development in olives

We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4–6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 °C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.