Conserved expression of the PRELI domain containing 2 gene (<Emphasis Type="Italic">Prelid2</Emphasis>) during mid-later-gestation mouse embryogenesis

Prelid2, which belongs to the PRELI domain containing family, is identified as a conserved evolution gene. The expression and regulation during embryonic development of the prelid2 gene is unknown. In this study, we investigated the prelid2 gene expression and regulation using mouse embryos model, by in situ hybridization analysis, RT-PCR and bisulfite sequencing. In situ hybridization analysis showed that prelid2 gene expression were found in midbrain, spinal cord, optic eminence, otic vesicle and tail at E9.5 and E10.5 embryos, in forebrain, hindbrain, heart, lung, liver and kidney at E13.5 and E15.5 embryos. Real-time quantitative RT-PCR results verified the expression pattern in the four major mouse organs, brain, heart, lung, and liver during organs differentiation and formation. Bisulfite sequencing illustrated the consistent result of expression and its unmethylation status in the genomic promoter region at E12.5, E18.5, and new born. Thus, the prelid2 gene is a widely-spread, persistently expressed and unmethylated gene in mouse embryonic development. Our results suggest that the PRELI domain containing 2 gene is involved in mouse embryonic development.     

XRASGRP2 expression during early development of Xenopus embryos

Previously, we described the DNA microarray screening of vascular endothelial cells that were formed by treatment of aggregates prepared from Xenopus animal cap cells with activin and angiopoietin-2. One of the genes identified in this screening showed homology to human RASGRP2 which plays a role in the regulation of GTP-GDP exchange of the Ras and Rap proteins, and was named XRASGRP2. In the present study, we analyzed the expression pattern of xrasgrp2 during Xenopus embryogenesis. The xrasgrp2 mRNA was expressed after stage 24, as assessed by stage PCR analysis. Whole-mount in situ hybridization showed that xrasgrp2 mRNA was located in the vascular region of the embryo. Loss-of-function analysis revealed that the formation of blood and endothelial cells in the explants transplanted into Xenopus embryos was inhibited by antisense morpholino oligonucleotides that block xrasgrp2 translation. These results suggest that XRASGRP2 plays a role in angiogenesis in Xenopus embryos.     

Flounder (Paralichthys olivaceus) FoxD1 and its regulation on the expression of myogenic regulatory factor, MyoD

It has been reported that FoxD1 plays important roles in formation of several different tissues, such as retina and kidney in vertebrates. The function of FoxD1 in muscle development is, however, unclear although it is expressed in muscle cells in zebrafish. Muscles are the major tissue in fish, which serves as a rich protein source in our diet. To further understand the function of FoxD1 in fish muscle development, here we isolated and characterized the FoxD1 gene from flounder (Paralichthys olivaceus), a valuable sea food and an important fish species in aquaculture in Asia. We analyzed its expression pattern and function in regulating myogenic regulatory factor, MyoD, one of the earliest marker of myogenic commitment. In situ hybridization revealed that FoxD1 was expressed in the tailbud, adaxial cells, posterior intestine, forebrain, midbrain and half of the retina in flounder embryos. Functional studies demonstrated that when flounder FoxD1 was over-expressed in zebrafish by microinjection, MyoD expression was decreased, suggesting that FoxD1 may be involved in myogenesis by regulating the expression of MyoD.     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Temporal and tissue-specific expression of the tobacco ntf4 MAP kinase

The large number of mitogen-activated protein (MAP) kinase genes identified to date in plants suggests that their encoded proteins have a wide array of functions in development and physiological responses, as has been indicated by studies on the factors which lead to the activation of these kinases. Signalling pathways involving members of a multigene family employ a variety of mechanisms to ensure response specificity, one of which is via differential gene expression. We have performed detailed analyses of the expression of the tobacco ntf4 MAP kinase gene using a variety of approaches. The ntf4 gene promoter region was isolated and a chimeric ntf4 promoter-GUS fusion construct was introduced into plants. GUS expression was detected in pollen, in developing and mature embryos, and shortly after seed germination, but not in other floral tissues and tissues such as leaf, root, or stem. This expression pattern was confirmed by northern and western analyses. In situ hybridization and immunolocalization studies showed that the expression of the ntf4 gene and its encoded protein p45^Ntf4 occurred in embryos at least from the globular embryo stage until the mature seed, as well as in the seed endosperm. Taken together, the results show that the p45^Ntf4 MAP kinase has a very restricted expression pattern, being found only in pollen and seeds. These findings should be important when considering MAP kinase function in plants.     

DANCER: a program for digital anatomical reconstruction of gene expression data

A digital anatomy construction (DANCER) program was developed for gene expression data. DANCER can be used to reconstruct anatomical images from in situ hybridization images, microarray or other gene expression data. The program fills regions of a drawn figure with the corresponding values from a gene expression data set. The output of the program presents the expression levels of a particular gene in a particular region relative to other regions. The program was tested with values from experimental in situ hybridization autoradiographs and from a microarray experiment. Reconstruction of in situ hybridization data from adult rat brain made by DANCER corresponded well with the original autoradiograph. Reconstruction of microarray data from adult mouse brains provided images that reflect actual expression levels. This program should help to provide visualization and interpretation of data derived from gene expression experiments. DANCER may be freely downloaded.     

The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos

Myogenesis is facilitated by four myogenic regulatory factors and is significantly inhibited by myostatin. The objective of the current study was to examine embryonic gene regulation of myostatin/myogenic regulatory factors, and subsequent manipulations of protein synthesis, in broiler embryos under induced hyperammonemia. Broiler eggs were injected with ammonium acetate solution four times over 48 h beginning on either embryonic day (ED) 15 or 17. Serum ammonia concentration was significantly higher (P<0.05) in ammonium acetate injected embryos for both ED17 and ED19 collected samples when compared with sham-injected controls. Expression of mRNA, extracted from pectoralis major of experimental and control embryos, was measured using real-time quantitative PCR for myostatin, myogenic regulatory factors myogenic factor 5, myogenic determination factor 1, myogenin, myogenic regulatory factor 4 and paired box 7. A significantly lower (P<0.01) myostatin expression was accompanied by a higher serum ammonia concentration in both ED17 and ED19 collected samples. Myogenic factor 5 expression was higher (P<0.05) in ED17 collected samples administered ammonium acetate. In both ED17 and ED19 collected samples, myogenic regulatory factor 4 was lower (P⩽0.05) in ammonium acetate injected embryos. No significant difference was seen in myogenic determination factor 1, myogenin or paired box 7 expression between treatment groups for either age of sample collection. In addition, there was no significant difference in BrdU staining of histological samples taken from treated and control embryos. Myostatin protein levels were evaluated by Western blot analysis, and also showed lower myostatin expression (P<0.05). Overall, it appears possible to inhibit myostatin expression through hyperammonemia, which is expected to have a positive effect on embryonic myogenesis and postnatal muscle growth.     

Developmentally regulated expression of a cell surface protein,neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons. © 1996 John Wiley & Sons, Inc.     

Mustn1 is essential for craniofacial chondrogenesis during Xenopus development

Mustn1 is a vertebrate-specific protein that, in vitro, was showed to be essential for prechondrocyte function and thus it has the potential to regulate chondrogenesis during embryonic development. We use Xenopus laevis as a model to examine Mustn1 involvement in chondrogenesis. Previous work suggests that Mustn1 is necessary but not sufficient for chondrogenic proliferation and differentiation, as well as myogenic differentiation in vitro. Mustn1 was quantified and localized in developing Xenopus embryos using RT-PCR and whole mount in situ hybridization. Xenopus embryos were injected with either control morpholinos (Co-MO) or one designed against Mustn1 (Mustn1-MO) at the four cell stage. Embryos were scored for morphological defects and Sox9 was visualized via in situ hybridization. Finally, Mustn1-MO-injected embryos were co-injected with Mustn1-MO resistant mRNA to confirm the specificity of the observed phenotype. Mustn1 is expressed from the mid-neurula stage to the swimming tadpole stages, predominantly in anterior structures including the pharyngeal arches and associated craniofacial tissues, and the developing somites. Targeted knockdown of Mustn1 in craniofacial and dorsal axial tissues resulted in phenotypes characterized by small or absent eye(s), a shortened body axis, and tail kinks. Further, Mustn1 knockdown reduced cranial Sox9 mRNA expression and resulted in the loss of differentiated cartilaginous head structures (e.g. ceratohyal and pharyngeal arches). Reintroduction of MO-resistant Mustn1 mRNA rescued these effects. We conclude that Mustn1 is necessary for normal craniofacial cartilage development in vivo, although the exact molecular mechanism remains unknown.     

The Chromosomal Organization of the Human Endogenous Retrovirus-like Sequence HERV-H: Clustering of the HERV-H Sequences in a 300-kb Region Close to the GRPR Locus on the X Chromosome

Within the haploid genome there are approximately 1,000 copiesof the human endogenous retroviruslike sequence, HERV-H. Althoughthese sequences are scattered throughout the entire genome,in situ hybridization experiments revealed that there are discreteclusters positioned on chromosomes 1p and 7q. In this study,we have located three HERV-H sequences which were unexpectedlyclustered within a 300-kilobase region close to the GRPR locuson the X chromosome. In previous studies, no clusteringof thissequence has been reported at this locus. Our finding demonstratesthat, like other repetitive sequences, clustering of HERV-Hoccurs in the human genome, although these sequences may notalways be detected by in situ hybridization methods.