Transforming growth factor-beta5 expression during early development of Xenopus laevis

Members of the transforming growth factor-beta (TGF-beta) superfamily play various roles during development in both vertebrates and invertebrates. Two isoforms, TGF-beta2 and -beta5, have been isolated from Xenopus laevis. We describe here the localization of TGF-beta5 mRNA in early embryos of X. laevis, assessed by whole-mount in situ hybridization. The first detectable expression of TGF-beta5 was seen in the stage 14 embryo at the posterior tip of notochord, which continued to later stages, accompanied by the expression in bilateral regions of posterior wall in the tail region next to the notochord. At later stages, transient expression was seen in the cement gland (around stage 21) and in the somites (stages 24-27). In addition, expression was present in the branchial arches (stage 29-36) and olfactory placodes (stage 36).     

cDNA cloning and mRNA expression of Transformer 2 (Tra 2) in chicken embryo

We report the cloning of a chicken Transformer 2 (Tra 2) cDNA that encodes a protein of 289 amino acids which are 97.9% identical to those of mammalian splicing factor, Tra 2. Tra 2 mRNA was expressed in chicken embryonic tissues and was observed as a band of 1.5 kb by Northern blot analysis. Whole mount in situ hybridization showed an mRNA expression of Tra 2 in telencephalon, mandible, hyoid arch, wing and leg buds as early as day 3.5 of incubation. These results suggest that the Tra 2 gene may play a role in organogenesis in the chicken embryo.     

RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis

The RNA interference technique is a powerful tool to understand gene function. Intriguingly, RNA interference cannot only be used for cells in vitro, but also in living organisms. Here, we have adapted the method for use in the chick embryo. However, this technique is limited by the uncertainty in predicting the RNAi transfection efficiency and site in the embryo. Hence, we elaborated a modified vector system, pEGFP-shRNA, which can coexpress enhanced green fluorescent protein (EGFP) and short hairpin RNA (shRNA) simultaneously to facilitate analysis of gene silencing in chicken embryos. We tested the silencing of two highly conserved genes (cAxin2, cParaxis), which play crucial roles in chicken embryonic developmental processes. For each target gene, four to five small DNA inserts, each of them encoding one shRNA, were selected and cloned individually to the vector downstream of the Pol III promoter (either human H1 or U6 promoter), which shared with highly conserved motifs in human and chicken. The pEGFP-shRNA constructs were electroporated into the neural tube or somites. After subsequent re-incubation of 24 h, the EGFP expression, with green fluorescent signal, indicated the transfected regions in the neural tube or somites. The EGFP expressing embryos were further submitted into the process of in situ hybridization for examination of the silencing effects. The results show that the EGFP signal in transfected areas correlated with the silencing of the target genes (cAxin2, cParaxis). The cAxin2 expression was inhibited by shRNAs of either targeting the RGS domain or the DAX domain coding region. The cParaxis mRNA level in transgenic somites and the related migratory myogenic population was also reduced. The results suggest that our novel dual expression EGFP-shRNA system opens a new possibility to study gene function in a convenient and efficient way.     

Expression of transforming growth factor-beta s 1-4 in chicken embryo chondrocytes and myocytes

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.     

Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation.     

Molecular differences between the rostral and caudal halves of the sclerotome in the chick embryo

It is known that both neural crest cell migration and motor axon outgrowth in most vertebrate embryos are segmented because of restrictions imposed upon their distribution by the neighbouring sclerotomes, each of which is divided into a rostral and a caudal half. The caudal half does not allow crest migration or axon outgrowth, while the rostral half does. In this paper, we investigate the expression of proteins and glycoproteins in the two halves of the sclerotome of the chick embryo at stages between 20 and 32 pairs of somites by two-dimensional SDS-polyacrylamide gel electrophoresis. We find that the patterns of expression are complex, and that polypeptides and glycoproteins vary both spatially and temporally: of those that are expressed differentially by the sclerotome, some differ quantitatively and others qualitatively. Some macromolecules change their spatial distribution with developmental age, and some appear or disappear as the embryos become older.     

一种适于植物幼胚mRNA整体原位杂交的方法

A mRNA whole-mount in situ hybridization method is reported here for quick, direct analysis of the spatial and temporal mRNA expression patterns in plant young embryos. A cDNA clone THE3 (tobacco heart embryo 3) was isolated by differential screening from tobacco (Nicotiana tabacum L.) heart embryo cDNA library as compared with the globular embryo cDNA library. The distribution of THE3 mRNA in tobacco heart embryos and globular embryos was investigated by a whole-mount in situ hybridization technique, showing that THE 3 is preferentially expressed in heart embryos.     

Expression of Lrrn1 marks the prospective site of the zona limitans thalami in the early embryonic chicken diencephalon

An unknown chicken gene selected from a published substractive hybridization screen (GenBank Accession No. ; [Christiansen, J.H., Coles, E.G., Robinson, V., Pasini, A., Wilkinson, D.G., 2001. Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development. Mech. Dev. 102, 119-133.]) was deemed of interest because of its dynamic pattern of expression across the forebrain and midbrain regions. A 528bp fragment cloned from early chick embryo cDNA and used for in situ hybridization corresponded to part of the 3' untranslated region of the chicken gene Leucine-rich repeat neuronal protein 1 (Lrrn1). The expression of this gene, mapped in the embryonic chick brain between stages HH10 and HH26, apparently preconfigures the zona limitans thalami site before overt formation of this boundary structure. Apart of colateral expression in the forebrain, midbrain and hindbrain basal plate, the most significant expression of Lrrn1 was found early on across the entire alar plate of midbrain and forebrain (HH10). This unitary domain soon divides at HH14 into a rostral part, across alar secondary prosencephalon and prospective alar prosomere 3 (prethalamus; caudal limit at the prospective zona limitans), and a caudal part in alar prosomere 1 (pretectum) and midbrain. The rostral forebrain domain later downregulates gradually most extratelencephalic signal of Lrrn1, but the rostral shell of zona limitans retains expression longer. Expression in the caudal alar domain also changes by downregulation within its pretectal subdomain. Caudally, the midbrain domain ends at the isthmo-mesencephalic junction throughout the studied period. Embryonic Lrrn1 signal also appears in the somites and in the otic vesicle.