Integrin expression patterns during early limb muscle development in the mouse

Cell-extracellular matrix interactions play crucial roles in limb muscle development but practically nothing is known on what integrins are involved before the differentiation of muscle precursor cells (MPCs) in the limb muscle masses. In this study we determine the expression patterns of integrins during early forelimb muscle development in the mouse. alpha6beta1 integrin is downregulated in the lateral dermomyotome when delamination of MPCs occurs. In late E9.5 embryos, alpha1beta1 and alpha5beta1 are expressed in a pattern very similar to pax3, which marks MPCs migrating to the limb bud. After myf5 upregulation in the limb bud, alpha1beta1 and alpha5beta1 expression is maintained and the alpha4beta1 integrin starts being expressed.     

Analysis of Nedd4 expression during skeletal development in the mouse limb

Nedd4, a ubiquitin-protein ligase, was originally identified as being down-regulated during development of the mouse brain (Nedd denotes neural precursor cell expressed developmentally down-regulated) (Kumar, S., Tomooka, Y., Noda, M., 1992. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem. Biophys. Res. Commun. 185, 1155-1161). Subtractive hybridization was used in an attempt to identify genes that are preferentially expressed early in skeletogenesis. Using this technique Nedd4 was identified multiple times. Northern blot analysis confirmed that Nedd4 is down-regulated in the forelimb and hind limb. In situ hybridization was carried out to identify regions of the limb bud expressing Nedd4. Nedd4 is expressed weakly in condensing mesenchyme, and abundantly in proliferating and prehypertrophic chondrocytes, but is undetectable in hypertrophic chondrocytes. Primary cultures, which closely mimic in vivo chondrogenesis, were also used to demonstrate the stage-specific expression of Nedd4 during early skeletal development.     

Characteristic tetraspanin expression patterns mark various tissues during early Xenopus development

The tetraspanins (Tspans) constitute a family of cell surface proteins with four transmembrane domains. Tspans have been found on the plasma membrane and on exosomes of various organelles. Reports on the function of Tspans during the early development of Xenopus have mainly focused on the expression of uroplakins in gametes. Although the roles of extracellular vesicles (EVs) including exosomes have been actively analyzed in cancer research, the contribution of EVs to early development is not well understood. This is because the diffusivity of EVs is not compatible with a very strict developmental process. In this study, we analyzed members of the Tspan family in early development of Xenopus. Expression was prominent in specific organs such as the notochord, eye, cranial neural crest cells (CNCs), trunk neural crest cells, placodes, and somites. We overexpressed several combinations of Tspans in CNCs in vitro and in vivo. Changing the partner changed the distribution of fluorescent-labeled Tspans. Therefore, it is suggested that expression of multiple Tspans in a particular tissue might produce heterogeneity of intercellular communication, which has not yet been recognized.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Telokin expression is restricted to smooth muscle tissues during mouse development

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.     

Integrin expression during epithelial migration and restratification in the tenascin-C-deficient mouse cornea.

In the unwounded cornea, tenascin-C localizes to a short stretch of the basement membrane zone at the corneoscleral junction or limbus. To determine whether the function of the limbus is affected by the absence of tenascin-C, mice possessing a deletion of tenascin-C and strain-matched wild-type mice are used in corneal debridement wounding experiments. The expression of integrins (alpha3, alpha9, and beta4) in the tenascin-C knockout corneas is evaluated by producing polyclonal cytoplasmic domain antipeptide sera and performing immunofluorescence microscopy. In addition, we evaluate the localization of several other proteins involved in wound healing, including fibronectin, laminin beta1, nidogen/entactin, and VCAM-1, in both the tenascin knockout and wild-type mice. There are no differences in healing rate, scarring, or neovascularization after corneal debridement wounds. alpha9 integrin is expressed at the limbal border of unwounded tenascin-C knockout animals and is upregulated during migration only after the larger wounds. At 8 weeks after larger wounds, the localization of alpha9 again becomes restricted to the limbal border. Results show that tenascin-C is not required for development or maintenance of the corneal limbus or for normal re-epithelialization of corneal epithelial cells after debridement wounding.     

Non-random X-chromosome expression early in mouse development

We have used a sensitive electrophoretic technique for estimating the activity, or ratio, of two allozymes of the X-chromosome-linked enzyme phosphoglycerate kinase (PGK-1), in order to investigate the randomness of X-chromosome expression in the derivatives of the three primary cell lineages of the early mouse conceptus. The maternally derived Pgk-1 allele is preferentially expressed in the derivatives of the primitive endoderm and trophectoderm lineages at 6 1/2 days post coitum in Pgk-1^a/Pgk-1^b heterozygous conceptuses, and in the one informative 5 1/2-day heterozygous conceptus analysed. This evidence for preferential expression of the maternally derived X chromosome (X^m), so soon after the time of X-chromosome inactivation, favors the possibility that the preferential expression of X^m is a consequence of primary non-random X-chromosome inactivation, rather than a secondary selection phenomenon. The majority of embryos analysed at 4 1/2 and 5 1/2 days pc produced only a single PGK-1 band, corresponding to the allozyme produced by the Pgk-1 allele on X^m, although 50% of these embryos should have been heterozygous females. Possible explanations are discussed.     

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic development

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) is an enzyme which is known to help build up the GlcAbeta1-3GalNAc(4,6-bisSO4) unit of chondroitin sulfate E (CS-E). This enzymatic activity has been reported in squid cartilage and in human serum, but has never been reported as an enzyme required during early mouse development. On the other hand, CS-E has been shown to bind with strong affinity to Midkine (MK). The latter is a heparin-binding growth factor which has been found to play important regulatory roles in differentiation and morphogenesis during mouse embryonic development. We have analyzed the expression pattern of the GalNAc4S-6ST gene during early mouse embryonic development by whole mount in situ hybridization. The results show that GalNAc4S-6ST is differentially expressed in the anterior visceral ectoderm at stage E5.5 and later becomes restricted to the embryonic endoderm, especially in the prospective midgut region. During the turning process, expression of GalNAc4S-6ST gene is detected in the forebrain, branchial arches, across the gut tube (hindgut, midgut and foregut diverticulum), in the vitelline veins and artery and in the splanchnopleure layer. These results open the possibility of a role for GalNAc4S-6ST during early mouse development.     

FACS-array gene expression analysis during early development of mouse telencephalic interneurons

Cortical interneuron dysfunction has been implicated in multiple human disorders including forms of epilepsy, mental retardation, and autism. Although significant advances have been made, understanding the biologic basis of these disorders will require a level of anatomic, molecular, and genetic detail of interneuron development that currently does not exist. To further delineate the pathways modulating interneuron development we performed fluorescent activated cell sorting (FACs) on genetically engineered mouse embryos that selectively express green fluorescent protein (GFP) in developing interneurons followed by whole genome microarray expression profiling on the isolated cells. Bioinformatics analysis revealed expression of both predicted and unexpected genes in developing cortical interneurons. Two unanticipated pathways discovered to be up regulated prior to interneurons differentiating in the cortex were ion channels/neurotransmitters and synaptic/vesicular related genes. A significant association of neurological disease related genes to the population of developing interneurons was found. These results have defined new and potentially important data on gene expression changes during the development of cortical interneurons. In addition, these data can be mined to uncover numerous novel genes involved in the generation of interneurons and may suggest genes/pathways potentially involved in a number of human neurological disorders.     

Cytokeratin expression during mouse embryonic and early postnatal mammary gland development

Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6⁺ and K8⁺/K14⁺ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.     

p38在小鼠着床前胚胎中的表达（简报）

探讨p38 MAPK在小鼠着床前胚胎期的表达图式,并对其作用作初步分析。用免疫印迹法分析胚胎全裂解物中的p38蛋白。为考察p38在着床前发育中的作用,在胚胎培养液中添加p38专一性抑制剂SB203580。此外对同位素标记的胚胎作双向电泳分析,示踪ZGA(zygotic gene activation,合子型基因激活)标志物TRC的表达情况。在卵母细胞中能检测到低水平的p38蛋白,而在合子中的检测度更低,表明p38是贮存于卵母细胞内的母型转录物,自减数分裂期随其它母型转录物一起逐步降解。到2细胞中期p38蛋白的表达量开始恢复,在4细胞时达到顶峰,在8细胞时又跌落。D38蛋白在2到4细胞期的表达量上升提示该蛋白在小鼠着床前胚胎发育中可能发挥一定作用。经与p38抑制剂SB203580共培养后的2细胞中期胚胎中仍能清晰检测到TRC,因而以TRC为标志的ZGA对SB203580不敏感。SB203580同样不能阻止胚胎发育到桑椹胚期。     

Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse.

Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.     

The dynamic expression of tenascin-C and tenascin-X during early heart development in the mouse

One of a family of extracellular matrix proteins, tenascin-C (TNC) is expressed in a spatiotemporally restricted pattern associated with tissue remodeling during embryonic development, wound healing, cancer invasion and tissue regeneration. Another form, tenascin-X (TNX), is found in most tissues but most predominantly in heart and muscle, often complementarily to TNC. The present analysis demonstrated their expression during early heart development, using mouse lines containing the lacZ gene targeted to the TNC locus, by RT-PCR, immunohistochemistry, and in situ hybridization. TNC was transiently expressed at important steps during heart development: (1) precardiac mesodermal cells differentiating to cardiomyocytes and endocardial cells at E 7.5 - 8.5; (2) cardiomyocytes in the outflow tract at E 8.5 - 12; (3) endocardial cells forming cushion tissue at E 9.5 - 13; and (4) mesenchymal cells in the proepicardial organ (PEO), the precursors of coronary vessels, at E 9.5. When PEO cells were transferred onto the heart surface, the expression of TNC was downregulated, while TNX was upregulated at E 11. Initially, epicardial cells around the AV groove and atrium started to express TNX. TNX-positive cells then gradually spread all over the entire surface of the heart and invaded and formed primitive vascular channels in the myocardium. Despite restricted expression at important sites and steps during cardiogenesis, the hearts of TNC deficient mice developed normally. No difference in the expression pattern of TNX were observed in TNC knockout and wild mice. These results suggest; (1) TNC could play important roles in the differentiation of cardiomyocytes and the early morphogenesis of the heart; (2) TNX could be involved in coronary vasculogenesis; (3) TNX does not compensate for the loss of TNC.