Sox15 is up regulated in the embryonic mouse testis

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.     

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.     

Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

New morphological aspects of blood islands formation in the embryonic mouse hearts

Vasculogenesis in embryonic hearts proceeds by formation of aggregates consisting of erythroblasts and endothelial cells. These aggregates are called blood-islands or blood-island-like structures. We aimed to characterize blood islands in mouse embryonic hearts at stages spanning from 11 dpc through 13 dpc, i.e. prior to the establishment of the coronary circulation. Our observations suggested that there are two types of blood islands. One formed by migrating nucleated erythroblasts, which associated with migrating endothelial cell and the second by in situ emergence of two kinds of cells belonging to separate populations: one resembling an erythroblast progenitor and the second resembling an endothelial-cell progenitor. The subepicardial blood islands contain nucleated erythroblasts, undifferentiated mesenchymal cells, platelets, and early lymphocytes. The subepicardial blood islands resemble vesicles with protruding prongs directed toward the myocardium. Ahead of the prongs, angiogenic sprouting and degradation of fibronectin is observed. Vesicles gradually change their shape from spherical to tubular at 13 dpc and grow and extend along the interventricular sulcuses forming vascular tubes. We presume that the vascular tubes located within the interventricular sulcuses are precursors of coronary veins. Our data seems to indicate that embryonic heart vasculogenesis is accompanied by hematopoiesis     

Expression profiles of extracellular superoxide dismutase during mouse organogenesis

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.     

Developmental methylation of the regulatory region of HoxB5 gene in mouse correlates with its tissue-specific expression

We have studied the dynamics of de novo CpG methylation in the regulatory region of one of the homeobox gene HoxB5 during mouse development by sodium bisulfite sequencing. Methylation pattern was examined at embryonic day 18.5 and adult in kidney and spleen while in the liver the same exercise has been done in 11.5 dpc, 18.5 dpc, 5 dpp and in adult. In the liver at 11.5 dpc, all the 47 contiguous sites (including a CpG island from 2035 to 2330 bp) at 5' regulatory region of HoxB5 were unmethylated. Random methylation commences from 18.5 dpc and continues in 5 dpp and in the adult. In the kidney at 18.5 dpc, 26 CpGs were examined (excluding the CpG island region) and all of them were unmethylated but the fetal spleen had at least a few sites considerably methylated. In the adult there was a low level methylation in the kidney, on the other hand, in the spleen, all the CpGs were methylated except a few sites and certain sites were totally methylated. Thus in the adult, the level of methylation was much higher than in the fetal stage. On the other hand semi-quantitative RT-PCR revealed that the extent of expression of HoxB5 was higher in embryonic stages than in the adult. Thus HoxB5 is a good paradigm to support that the developmental methylation of HoxB5 and its expression pattern show an inverse correlation.     

Developmental expression of plasma glutathione peroxidase during mouse organogenesis

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5–18.5. In whole embryos of E7.5–8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5–18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.     

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from We/We embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood.     

Effect of avermectin (AVM) on the expression of γ‐aminobutyric acid A receptor (GABAAR) in Carassius gibelio (Bloch, 1782)

This study describes the effect of avermectin (AVM) on the expression of γ‐aminobutyric acid A receptor (GABA_AR) in Carassius gibelio. To assess the specific expression of GABA_AR in the brain, gonads, liver, kidneys, heart, muscles, and skin of C. gibelio, the expression of GABA_AR α1 subunit (GABA_ARα1) was measured by Western blotting. To study the effects of AVM on the expression of GABA_AR, the median lethal concentration (LC₅₀) at 24, 48, and 96 h of AVM was determined and the expression of GABA_AR in the brain, liver, and kidneys of the corresponding C. gibelio evaluated by Western blotting and immunohistochemistry. The results show that GABA_AR was expressed in the brain, gonads, liver, kidneys, heart, intestines, muscles, and skin, while primarily distributed in the central nervous system and moderately distributed in peripheral tissues. The expression of GABA_AR in the brain, liver, and kidney tissues of C. gibelio was increased with the treatment of AVM at 24 h LC₅₀, but attenuated by the treatment of AVM at 48 h LC₅₀ and 96 h LC₅₀. This suggests a threshold effect of AVM.     

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.