Appearance and distribution of fetal brain macrophages in mice

Summary A study on the localization of fetal and neonatal brain macrophages of mice from embryonic day 10 (E10) to postnatal day 21 (P21) was carried out immunohistochemically using a monoclonal antibody against a macrophage differentiation antigen (Mac-1) and the labeled avidin-biotin technique. In the central nervous system, the macrophages recognized first were mainly located in the choroid plexuses of the fourth and lateral ventricles at E14. Their number increased at E17–P3 and gradually decreased thereafter. In the cerebral parenchyma, a few macrophages appeared at E14 in the matrix cell layer. They were also detected in the migrating zone at E15, E17 and in the cortical plate at E19. Mapping of positive cells at the stage of neuroblast formation (E15, E17, E19) disclosed the precise distribution of cerebral macrophages. The macrophages that appeared first in the choroid plexuses at E15 may be derived from the subarachnoid vessels, which extend into the stroma of the choroid plexuses when the matrix cell layer invaginates into the lateral ventricle to form the choroid plexuses. Almost all of the macrophages recognized in the cerebral parenchyma disappeared at P9 when the cytoarchitecture seemed to be completed. In the cerebellum, which develops later than the cerebrum, macrophages appeared after birth and were located mainly in the internal granular layer. The brain macrophages always appeared in the regions where cell proliferation and brain remodeling are most active at each stage. These findings suggest that fetal and neonatal brain macrophages may play an important role in scavenging degenerated cells and cell debris during histogenesis of the central nervous system.     

Transduction of the mammary epithelium with adenovirus vectors in vivo

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.     

Deletion of the mouse P450c17 gene causes early embryonic lethality

Dehydroepiandrosterone (DHEA), a 19-carbon precursor of sex steroids, is abundantly produced in the human but not the mouse adrenal. However, mice produce DHEA and DHEA-sulfate (DHEAS) in the fetal brain. DHEA stimulates axonal growth from specific populations of mouse neocortical neurons in vitro, while DHEAS stimulates dendritic growth from those cells. The synthesis of DHEA and sex steroids, but not mouse glucocorticoids and mineralocorticoids, requires P450c17, which catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities. We hypothesized that P450c17-knockout mice would have disordered sex steroid synthesis and disordered brain DHEA production and thus provide phenotypic clues about the functions of DHEA in mouse brain development. We deleted the mouse P450c17 gene in 127/SvJ mice and obtained several lines of mice from two lines of targeted embryonic stem cells. Heterozygotes were phenotypically and reproductively normal, but in all mouse lines, P450c17(-/-) zygotes died by embryonic day 7, prior to gastrulation. The cause of this early lethality is unknown, as there is no known function of fetal steroids at embryonic day 7. Immunocytochemistry identified P450c17 in embryonic endoderm in E7 wild-type and heterozygous embryos, but its function in these cells is unknown. Enzyme assays of wild-type embryos showed a rapid rise in 17-hydroxylase activity between E6 and E7 and the presence of C(17,20)-lyase activity at E7. Treatment of pregnant females with subcutaneous pellets releasing DHEA or 17-OH pregnenolone at a constant rate failed to rescue P450c17(-/-) fetuses. Treatment of normal pregnant females with pellets releasing pregnenolone or progesterone did not cause fetal demise. These data suggest that steroid products of P450c17 have heretofore-unknown essential functions in early embryonic mouse development.     

Gestational age-dependent effects of lipopolysaccharide on prostaglandin production by murine decidual caps

We sought to determine whether the gestational age of the pregnant mouse had any relationship with its lipopolysaccharide (LPS) responsiveness. Murine decidual caps from days 13, 15 and 17 of gestation (term is day 20) were dissected out, placed in inserts and equilibrated in media overnight. The following day, media were removed, replaced with fresh media (+/-LPS at 10 microg/mL). After LPS stimulation (24 h), prostaglandin (PG)E2 production by decidual caps from days 13 and 15 increased by 80-fold and 5-fold, respectively. PGF2alpha, 6-keto-PGF1a and TxB2 production also increased. Day 17 decidual caps were unaffected by LPS, pregnant mice inoculated i.p. with LPS (50 microg) at day 13 of gestation induced 100% delivery within 24 h. However, mice treated at days 15 and 17 had an equal occurrence of premature delivery or fetal resorption. This change in LPS responsiveness may indicate changes in the fetal-maternal immune system in late pregnancy.     

Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs

The reduced folate carrier (RFC1) is an important route by which the major blood folate, 5-methyltetrahydrofolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1(-)/- embryos examined at E18.5 by supplementation of pregnant RFC1(+/-) dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. These RFC1(-)/- mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.     

Liver- and lobe-selective gene transfection following the instillation of plasmid DNA to the liver surface in mice

The present study has undertaken the liver- and lobe-selective gene transfections following the instillation of plasmid DNA (pDNA) to the liver surface in mice. The luciferase levels produced in the applied (left) liver lobe at 6 h after liver surface instillation of pDNA were significantly higher than those produced in the other tissues assayed, and ranged from 8.5-fold higher in other liver lobes to 320-fold higher in other tissues. After small intestine surface instillation of pDNA, the gene expression was a little detected in the tissues assayed. Following liver surface instillation of pDNA at a time from 2 to 48 h or at a volume from 15 to 120 microl, the gene expressions of the applied liver lobe were always significantly higher than those of other liver lobes and other tissues. We demonstrated the novel liver- and lobe-selective gene transfection utilizing the instillation to the liver surface.     

Hepatoblast-like cells populate the adult p53 knockout mouse liver: evidence for a hyperproliferative maturation-arrested stem cell compartment.

Although p53 regulates the cell cycle and apoptosis, gross embryonic development is normal in the p53 knockout (-/-) mouse. In this study, we comprehensively assessed liver development in p53 -/- mice (from embryonic day 15 to adult) for evidence of a cell cycle-induced perturbation in differentiation. Liver cell proliferation in the embryo and newborn is similar in p53 -/- and +/+ mice; in contrast, -/- adult hepatocytes divide at twice the rate of wild types. Developmental expression patterns of liver-specific markers that are up-regulated (e.g., phosphoenolpyruvate carboxykinase and aldolase B) and down-regulated (e.g., alpha-fetoprotein) are similar. Therefore, embryonic and perinatal liver development is normal in the absence of p53. However, the p53 -/- adult liver displays small blast-like cells, the majority being hepatic and some lymphoid. These cells appear in periportal regions and can infiltrate the parenchyma. The hepatic blast-like cells express both mature and immature liver markers, suggesting that differentiation of the liver stem cell compartment is blocked.     

DiGeorge syndrome critical region 8 (DGCR8) protein-mediated microRNA biogenesis is essential for vascular smooth muscle cell development in mice

DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.     

Neural control of the sequence of expression of myosin heavy chain isoforms in foetal mammalian muscles

The expression of myosin isoforms was studied during development of calf muscles in foetal and neonatal rats, using monoclonal antibodies against slow, embryonic and neonatal isoforms of myosin heavy chain (MHC). Primary myotubes had appeared in all prospective rat calf muscles by embryonic day 16 (E16). On both E16 and E17, primary myotubes in all muscles with the exception of soleus stained for slow, embryonic and neonatal MHC isoforms; soleus did not express neonatal MHC. In earlier stages of muscle formation staining for the neonatal isoform was absent or faint. Secondary myotubes were present in all muscles by E18, and these stained for both embryonic and neonatal MHCs, but not slow. In mixed muscles, primary myotubes destined to differentiate into fast muscle fibres began to lose expression of slow MHC, and primary myotubes destined to become slow muscle fibres began to lose expression of neonatal MHC. This pattern was further accentuated by E19, when many primary myotubes stained for only one of these two isoforms. Chronic paralysis or denervation from E15 or earlier did not disrupt the normal sequence of maturation of primary myotubes up until E18, but secondary myotubes did not form. By E19, however, most primary myotubes in aneural or paralyzed tibialis anterior muscles had lost expression of slow MHC and expressed only embryonic and neonatal MHCs. Similar changes occurred in other muscles, except for soleus which never expressed neonatal MHC, as in controls. Paralysis or denervation commencing later than E15 did not have these effects, even though it was initiated well before the period of change in expression of MHC isoforms. In this case, some secondary myotubes appeared in treated muscles. Paralysis initiated on E15, followed by recovery 2 days later so that animals were motile during the period of change in expression of MHC isoforms, was as effective as full paralysis. These experiments define a critical period (E15-17) during which foetuses must be active if slow muscle fibres are to differentiate during E19-20. We suggest that changes in expression of MHC isoforms in primary myotubes depend on different populations of myoblasts fusing with the myotubes, and that the normal sequence of appearance of these myoblasts has a stage-dependent reliance on active innervation of foetal muscles. A critical period of nerve-dependence for these myoblasts occurs several days before their action can be noted.     

Combined supplementation of folic acid and vitamin E diminishes diabetes-induced embryotoxicity in rats

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappaB (NF-kappaB) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappaB activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.     

Testis developmental phenotypes in neurotropin receptor trkA and trkC null mutations: role in formation of seminiferous cords and germ cell survival

The objective of the present study was to determine if the neurotropin receptors trkC and trkA are involved in embryonic testis development. These receptors bind neurotropin 3 and nerve growth factor, respectively. The hypothesis tested was that the absence of trkC or trkA receptors will have detrimental effects on testis development and morphology. The trkA and trkC homozygote knockout (KO) mice generally die either at or shortly after birth. Therefore, heterozygote mice were mated to obtain homozygote gene KO mice at Embryonic Day (E) 13, E14, E17, and E19 of gestation, with E0 being the plug date. Gonads from approximately 80 embryos were collected and fixed, and each embryo was genotyped. To determine gonadal characteristics for each genotype, the number of germ cells, number of seminiferous cords, seminiferous cord area, and interstitial area were calculated at each developmental age. Germ cell numbers varied in trkA gene KO mice from those of wild-type mice at each age evaluated. In trkC gene KO mice, differences were detected in germ cell numbers when compared to wild-type mice at E17 and E19. At E19, germ cell numbers were reduced in both trkA and trkC gene KO mice when compared to wild-type animals. Apoptosis was evaluated in testes of wild-type, trkC gene KO, and trkA gene KO mice to determine if the alteration in germ cell numbers at each developmental age was influenced by different patterns of germ cell survival or apoptosis. No differences were found in germ cell apoptosis during embryonic testis development. Interestingly, trkA gene KO mice that survived to Postnatal Day 19 had a 10-fold increase in germ cell apoptosis when compared to germ cells in wild-type mice. Evaluation of other morphological testis parameters demonstrated that trkC KO testes had reduced interstitial area at E13, reduced number of seminiferous cords at E14, and reduced seminiferous cord area at E19. The trkA gene KO testes had a reduction in the number of seminiferous cords at E14. Histology of both trkA and trkC gene KO testes demonstrated that these gonads appear to be developmentally delayed when compared to their wild-type testis counterparts at E13 during testis development. The current study demonstrates that both trkA and trkC neurotropin receptors influence germ cell numbers during testis development and events such as seminiferous cord formation.     

Diffusible rod-promoting signals in the developing rat retina.

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5-6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.     

Cellular and subcellular localization of aquaporins 1, 3, 8, and 9 in amniotic membranes during pregnancy in mice

The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (E12), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between E10 and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at E14 in the lateral membrane and apical junction; at E16 in the lateral membrane alone; at E17 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes.     

Mouse placenta is a major hematopoietic organ

Placenta and yolk sac from 8- to 17-day-old (E8-E17) mouse embryos/fetuses were investigated for the presence of in vitro clonogenic progenitors. At E8-E9, the embryonic body from the umbilicus caudalwards was also analysed. Fetal liver was analysed beginning on E10. At E8, between five and nine somite pairs (sp), placenta, yolk sac and embryonic body yielded no progenitors. The first progenitors appeared at E8.5 at the stage of 15 sp in the yolk sac, 18 sp in the embryonic body, 20 sp in the placenta and only at E12 in the fetal liver (absent at E10, at E11 not determined). Progenitors with a high proliferation potential that could be replated for two months, as well as the whole range of myeloid progenitors, were found at all stages in all organs. However, the earliest of these progenitors (these yielding large, multilineage colonies) were 2-4 times more frequent in the placenta than in the yolk sac or fetal liver. In the fetal liver, late progenitors were more frequent and the cellularity increased steeply with developmental age. Thus, the fetal liver, which is a recognized site for amplification and commitment, has a very different hematopoietic developmental profile from placenta or yolk sac. Placentas were obtained from GFP transgenic embryos in which only the embryonic contribution expressed the transgene. 80% of the colonies derived from these placental cells were GFP+, and so originated from the fetal component of the placenta. These data point to the placenta as a major hematopoietic organ that is active during most of pregnancy.     

Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.     

Effects of 17-hydroxyprogesterone caproate (17-OHPC) administration to pregnant squirrel monkeys (Saimiri boliviensis boliviensis)

Poor reproductive performance of the squirrel monkey (Saimiri boliviensis boliviensis) in captivity and a relative progesterone (P) deficiency in pregnancy have been reported. Since premature births may contribute to pregnancy wastage, we attempted to measure the effectiveness of 17-hydroxyprogesterone caproate (17-OHPC) treatment of pregnant squirrel monkeys to prevent early deliveries. Based on clearance studies of nonpregnant animals, 25 mg of 17-OHPC was administered at 6-day intervals to a test group of 31 pregnant monkeys while the control group of 29 received saline. Venous blood was obtained at 6- to 12-day intervals for measurement of 17-hydroxyprogesterone (17-OHP), P, 17-B estradiol (E), and androstenedione (A), and dihydroepiandrosterone (DHEA) levels by radioimmunoassays. The treated group had a significant increase in serum 17-OHP (P < 0.001), P (P < 0.01), and DHEA (P < 0.05) levels compared to controls. The numbers of live births, stillbirths, or neonatal deaths did not differ significantly between groups. Although 17-OHPC administration appeared to increase P and 17-OHP levels, this did not alter the duration of pregnancy nor delay the onset of labor. A significant fall in 17-OHP, P, and E levels was observed 6–12 days before delivery.