Rat Embryogenesis Research Articles

Immunoelectron microscopic analysis of a novel carbohydrate differentiation antigen (CDA-3C2) in the developing rat olfactory and otic systems.

A carbohydrate differentiation antigen (CDA-3C2) exhibits a highly specific and restricted pattern of expression during rat embryogenesis. In the periphery of the embryo, this antigen is associated transiently with the lateral ectoderm but is retained only in the olfactory and otic epithelium throughout morphogenesis. At the light microscopic level, CDA-3C2 immunoreactivity appears mostly along cell periphery and in the extracellular matrix. The aim of the present study was to determine the specific cellular and subcellular distribution of CDA-3C2 in vivo in order to identify potential sites of cellular and tissue function of the antigen during embryogenesis. There was a strikingly similar subcellular distribution of CDA-3C2 in the developing otic and olfactory systems, found mostly along cell membranes, microvillar projections and acellular secretions of the epithelium. Mature sensory components of the epithelia were not immunoreactive, whereas supportive cells and their secreted structures were densely stained. The highly coincident nature of CDA-3C2 in both sensory epithelia suggests that this carbohydrate epitope, and possibly its carrier macromolecule, participate in a morphogenetic function common to these two sensory epithelia.

A singularity of PDGF alpha-receptor expression in the dorsoventral axis of the neural tube may define the origin of the oligodendrocyte lineage.

During rat embryogenesis, PDGF alpha receptor (PDGF-alpha R) mRNA is expressed in the ventral half of the spinal cord in two longitudinal columns, one each side of the central canal. Initially, these columns are only two cells wide but the cells subsequently appear to proliferate and disseminate throughout the spinal cord. Our previous studies of PDGF-alpha R expression in the developing CNS suggested that PDGF-alpha R may be a useful marker of the oligodendrocyte lineage in situ. The data presented here complement those studies and lead us to propose that the earliest oligodendrocyte precursors in the spinal cord originate in a very restricted region of the ventricular zone during a brief window of time around embryonic day 14 (E14). In the embryonic brain, migrating PDGF-alpha R+ cells appear to originate in a localized germinal zone in the ventral diencephalon (beneath the foramen of Monro). Our data demonstrate that gene expression and cell fate can be regulated with exquisite spatial resolution along the dorsoventral axis of the mammalian neural tube.

Spatiotemporal patterns of expression of NGF and the low-affinity NGF receptor in rat embryos suggest functional roles in tissue morphogenesis and myogenesis

We show here that NGF and its low-affinity receptor (p75NGFR) are expressed during rat embryogenesis at sites that are known to have important roles in tissue morphogenesis and myogenesis. The developing skin of the maxilla, the mandible, and the limb showed very similar patterns of NGF and p75NGFR expression. However, NGF and p75NGFR expression in the developing limb initiated at the limb bud stage and was concentrated at proximal and distal developmental sites that have been reported to be involved in limb morphogenesis. Expression at the proximal/distal ends of the limb persisted throughout limb development, with some of the highest levels of expression occurring at the limb axillary sites, which were not highly innervated. We have also found p75NGFR expression at sites of mesenchymal/epithelial interactions in several developing organs that do not appear to have an adjacent source of NGF and may therefore be sites that bind and respond to the other members of the NGF family (brain-derived neurotrophic factor and neurotrophin-3). These organs include the lung, testes, and kidney, where expression of p75NGFR occurred during the morphogenesis of specific epithelial structures and was coexpressed with the cell adhesion molecule NCAM. In addition, we found that NGF and p75NGFR were expressed during myogenesis. p75NGFR was observed in myoblast cells expressing MyoD1, a myoblast differentiation marker, and NGF transcripts in cells just adjacent to the developing myoblasts. When the myoblasts differentiate into myotubes, p75NGFR and MyoD1 cease to be expressed and the adjacent cells concomitantly cease to be make NGF. However, NGF and p75NGFR were not present in the early muscle precursor cells of the myotome of the somites but were observed in the dermatome and sclerotome, respectively. These results suggest that NGF and p75NGFR have functional roles in developmental processes that affect morphogenesis and cell differentiation.

Expression of the IGFBP-2 gene in post-implantation rat embryos.

The insulin-like growth factors (IGFs) stimulate mitogenesis in a variety of cell types both in vitro and in vivo. These effects are mediated by both IGF receptors and a family of IGF binding proteins (IGFBPs), which are found complexed with the IGFs in serum and tissue fluids. Here we compare the sites of expression during early rat embryogenesis of the genes encoding the RGD-containing IGF binding protein IGFBP-2 and IGF-II. At all ages from early post-implantation through mid-gestation, the expression of IGFBP-2 was highly complementary to IGF-II. IGFBP-2 mRNA was detected throughout the epiblast of the egg cylinder as early as e7, when IGF-II expression was restricted to trophectoderm and other extraembryonic cells. As gastrulation proceeded, IGFBP-2 expression ceased as IGF-II expression began in the newly formed embryonic and extra-embryonic mesoderm, but was retained in other epiblast derivatives including the surface ectoderm and neuroectoderm, throughout its rostral-caudal extent. By e10-e11, IGFBP-2 expression in neuroectoderm was restricted to the rostral brain of the primary neural tube and was found in the new population of neuroepithelium formed in the tail bud during secondary neurulation. IGFBP-2 expression remained high in the ventricular layer of the rostral brain into mid-gestation ages but decreased or disappeared as cells entered the mantle layer and began to express the neurofilament-related gene alpha-internexin. IGFBP-2 mRNA was abundant in surface ectoderm, particularly that of the branchial arches, and all ectodermal placodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).

Expression of Inhibin/Activin Subunit Messenger Ribonucleic Acids during Rat Embryogenesis*

Inhibin (alpha/beta) and activin (beta/beta) were first recognized as gonadal hormones that regulate the production and release of FSH from the anterior pituitary gland. Studies now show that these proteins, which are members of the transforming growth factor-beta (TGF beta) superfamily, and their corresponding messenger RNAs have a broad anatomical distribution and may regulate the growth and differentiation of a variety of cell types. To determine whether inhibin and activin may also play a role in embryonic development, in situ hybridization techniques were utilized to examine the localization of the mRNAs encoding the inhibin/activin subunits (alpha, beta A, beta B) in rat embryos from 12 days post coitum (p.c.) until birth. The beta A-subunit message was localized in the heart at 12 days p.c. and in the dermal layer of the skin starting at 13 days p.c. At 14 days p.c. this mRNA was first observed in the whisker follicles, in the developing skeleton of the snout, limbs, and in the intervertebral disks. At 16 days p.c. the beta A-message was found in the striatum of the brain, and at 18 days p.c. it was also detected in the cerebral cortex. The beta A-mRNA signal appeared in hair bulbs at 17 days p.c., in teeth at 18 days p.c., and in tendons and gonads just before birth. Expression of beta A-mRNA was no longer detected in the skin or intervertebral disks after 17 days p.c. The beta B-subunit message was found in the area of rapidly dividing cells surrounding the forebrain ventricle, starting at 14 days p.c., in the gonads from the time of gonadal sexual differentiation, at 14 days p.c., and in the salivary gland as early as 17 days p.c. The beta B-message continued to be expressed in these areas throughout embryogenesis. The inhibin-alpha subunit message was also detectable in the gonads from 14 days p.c. until birth. These data suggest that 1) inhibin and activin may be produced in the gonads and possibly play a hormonal role in the embryonic rat during the last trimester of pregnancy, and 2) activin may regulate aspects of the embryonic development of the heart, skin, hair and whiskers, cartilage, bone, tendons, teeth, salivary gland, brain, and gonads, possibly in coordination with other members of the TGF beta superfamily whose mRNAs are expressed in some of these same tissues during development.

The Teratogenic Effects of E‐64 on Rat Embryogenesis

To examine the effects of E-64 (a thiol protease inhibitor) on the development of rat embryos, pregnant Wistar rats were injected with 10-30 mg/kg of E-64 intra-peritoneally on days 9 and 10 of gestation. On day 21, the rats were killed and the fetuses were examined for malformations. When 30 mg/kg of E-64 was administered, the incidence of resorptions and malformations was 25.7% and 60.4%, respectively. The most common malformations were hydrocephaly, exencephaly, anophthalmia, microphthalmia, hydronephrosis and renal hypoplasia. These results suggest that thiol protease inhibitor is teratogenic during early rat embryogenesis.

Effects of thyroid antagonists on rat embryos cultured in vitro

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.

Changes of serum thyroid hormone levels induce malformations on early embryogenesis in rats

The incidence of malformation is increased in infants of hyperthyroid or hypothyroid woman. Although many papers reported that the fetus is insulted from maternal thyroid hormone, the placenta (maternal-fetal barrier) is not yet fully developed before 11.5 days of gestation in rat embryos, suggesting the effect of thyroid hormone on early rat embryogenesis. This study was, therefore, undertaken to investigate whether excess or lack of thyroid hormones would affect early embryogenesis in rat embryo culture. Malformations including open neuropore and microencephaly were observed in 10 of 30 embryos incubated in hyperthyroid serum, and in 12 of 42 cultured in T3-enriched normal serum. Similar malformations were observed on 14 of 42 embryos cultured in hypothyroid serum and in 10 of 30 cultured in hypothyroid serum supplemented with T3. The frequencies of these malformations were significantly higher than in the control embryos (0 in 72 embryos) cultured with normal rat serum. These results suggest that the maternal thyroid status might play an important role for the complication of fetal malformations during early gestational period.

Possible Recruitment of Osteoblastic Precursor Cells from Hypertrophic Chondrocytes during Initial Osteogenesis in Cartilaginous Limbs of Young Rats

The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by auto radiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic procursor cells will have to be explored further.

An in situ-hybridization study of the localization of retinol-binding protein and transthyretin messenger RNAs during fetal development in the rat

Abstract The patterns, and the cellular sites, of expression of the genes for retinol-binding protein (RBP) and transthyretin (TTR) were studied during early- to mid-stages of rat embryogenesis. In situ hybridization of single-stranded RNA probes to rat embryo sections revealed specific sites at which RBP messenger RNA (mRNA) and TTR mRNA were localized in rat conceptuses from 7 to 13 days of gestation. RBP and TTR mRNAs were both observed in the visceral endoderm as early as at 7 days of gestation. The transcripts were not expressed in the parietal endoderm. At 9 days of gestation, TTR mRNA was strongly detected in the visceral extraembryonic endoderm and in the foregut endoderm, whereas RBP mRNA was detected only in the visceral extraembryonic endoderm. From the 10th day to the 13th day of gestation, both transcripts were increasingly expressed in the visceral yolk sac endoderm and in the developing fetal liver, and gradually decreased in the foregut. At 11 days of gestation, TTR mRNA was first detected in the tela choroidea of the fourth ventricle in the brain; and at 13 days, the TTR mRNA was strongly evident in the developing choroid plexus of the fourth and lateral ventricles. These in situ hybridization studies with embryos at different developmental stages show that RBP and TTR mRNAs are transcribed quite early during embryogenesis. The protein products of these transcripts may play important roles in vitamin A and thyroid hormone metabolism, and in the functions that these regulatory compounds play, in the developing rat embryo.

Diabetes affects sorbitol and myo-inositol levels of neuroectodermal tissue during embryogenesis in rat

Diabetes affects sorbitol and myo-inositol levels of neuroectodermal tissue during embryogenesis in rat

Pattern of the insulin-like growth factor II gene expression during rat embryogenesis.

The rat insulin-like growth factor II (IGF-II) gene, encoding a fetal somatomedin, expresses a family of transcripts in embryonic/fetal tissues, and also in the adult choroid plexus and the leptomeninges. We have localized IGF-II gene transcripts in sections of rat embryos of embryonic days 10-16 by performing in situ hybridization. These transcripts are present in the head mesenchyme, formed from both the mesoderm and the cephalic portion of the neural crest, and also in the majority of other tissues of mesodermal origin, predominantly those derived from the somites and the lateral mesoderm. Intense labelling was detected in muscle cells, and their precursors, throughout the examined stages, whereas in chondrocytes the initial high level of hybridization declined substantially prior to ossification. IGF-II gene transcripts are also present in derivatives of other germ layers, but in restricted sites. Thus, from the derivatives of the endoderm, only the liver and the bronchial epithelium yielded hybridization signals. Ectoderm-derived tissues, including the central and peripheral nervous system, were negative for hybridization, with the exception of the choroid plexus, the newly forming pituitary rudiment and, to a lesser extent, the auditory placode. The pattern of IGF-II gene expression during embryogenesis overlaps significantly with the reported distribution of immunohistochemically detected TGF-beta 1. A paracrine/autocrine role for IGF-II in the developmental process is discussed.

Rat embryogenesis following exposure to alcohol and nicotine.

Pregnant Sprague-Dawley rats were treated from day 9 through 12 of gestation with either ethanol (12.5% v/v in physiological saline, 0.015 ml/g, i.p.) and nicotine (16.7 mg/kg, s.c.), alone or in combination. Corresponding groups of animals received physiological saline in a similar manner and these served as controls. Embryos were recovered on gestational day 12 and were quantitatively assessed on the basis of 16 recognizable developmental endpoints (morphological scores). The head and the crown-rump lengths of the embryos were also measured. Embryos exposed to nicotine and to ethanol-nicotine were significantly affected in their development whereas those exposed to ethanol alone were spared.

Future investigations onboard Soviet biosatellites of the Cosmos series

Many rat experiments onboard Cosmos biosatellites have furnished information concerning the effects of weightlessness, artificial gravity, and ionizing radiation combined with weightlessness on structural and biochemical parameters of the animal body. The necessity to expand the scope of physiological investigations has led to the project of flight primate studies. It is planned to carry out the first primate experiments onboard the Cosmos biosatellite in 1982. At present investigations of weightlessness effects on the cardiovascular and vestibular systems, higher nervous activity, skeletal muscles and biorhythms of two rhesus monkeys are being developed and tested. It is also planned to conduct a study of weightlessness effects on embryogenesis of rats and bioenergetics of living systems onboard the same biosatellite. Further experiments onboard Cosmos biosatellites are planned.

Genesis of the Golgi complex

In the early period of rat and chicken liver embryogenesis, the Golgi apparatus is formed through the budding of the vesicles from the outer part of the nuclear hepatocyte membrane. In the later period of embryogenesis and after birth, the Golgi apparatus is supplemented with the vesicles that bud from smooth enlarged parts of granular endoplasmic reticulum cisterns. The dimensions of the Golgi apparatus depend on the intensity of hepatocyte functioning.

α-difluoromethylornithine as a postcoitally effective antifertility agent in female rats

Intraperitoneal injection of alpha-difluoromethylornithine (DFMO) at a dose of 200 mg/kg, twice a day, during days 4-7 of pregnancy caused inhibition of embryogenesis in rats. These animals did not deliver any pups at term. However, there was no effect of DFMO on pregnant rats following treatment during day 1-3 of pregnancy. It was observed that DFMO completely abolished ornithine decarboxylase levels in the pregnant uterus. The action of DFMO appears to be due to the inhibition of putrescine and polyamines during a critical period of early embryo development. We suggest that this drug may act as a postcoitally effective antifertility agent in females.

Transplacental action of sex hormones in embryogenesis and early postnatal development of rats

More than 200 pregnant rats were used in the experiments: 53 received testosterone propionate, 84 received estrogens, and the rest were controls (Table i). The hormones were injected on the last 3 days of pregnancy (the 19th,* 20th, and 21st days) subcutaneously as a suspension in aqueous alcohol or as an aqueous solution in sessional doses of: testosterone propionate I, 5, I0, and 50 mg, dihydrostilbestrol (DHS) 0.2 mg, diethylstilbestrol (DES) 0.2 and 1 mg, and sigetin* 0.2, 2, and 20 mg.

Autonomic receptors of the early rat embryo heart growth and development.

The adult heart rate responds to stimulation of the sympathetic and parasympathetic nervous system. These responses are mediated by the release of norepinephrine and acetylcholine (ACh) which act on beta-adre-nergic and on muscarinic-cholinergic receptors, respectively. During rat embryogene-sis, the heart commences to beat during Day 10 and circulation is established before these nerves reach and innervate the heart on Day 15 (1-3). The effect of ACh on the beat rate of excised rat embryo hearts has been reported by two groups of workers and their conclusions differed; Hall (4) observed a decrease in rate in the Day 11 ½ and older embryos but Pager et al. (5) could not demonstrate this effect until Day 16½. Our studies using the whole explanted embryo suggest an explanation for this discrepancy. Cholinergic receptors can be experimentally distinguished into two types by (a) their response to atropine, which selectively blocks muscarinic receptors, and (b) to curare, which selectively blocks nicotinic receptors. The experiments reported in this paper demonstrate the presence of cholinergic receptors in the preinnervated intact embryonic heart, consistent with Hall's result in the excised heart, classify them as being of the muscarinic type and show that they are independent of the adrenergic receptors in their action and time of functional appearance. We have found (6) in the explanted whole embryo that the adrenergic response of the heart appears during Day 10 concom-mitant with the onset of heart function, a result similar to that of Hall's with excised hearts. Our findings indicate that cholinergic response first appears approximately ½ day later in development than the adrenergic response, a finding also first observed by Hall. Methods. Embryos were explanted at either Day 10 1/2 (10–15 somites) or Day 11 (21-26 somites) of gestation. The explantation procedure followed was as described in New (7), Shepard et al. (8), and Robkin et al. (9).

Experimental study of the effect of a series of phosphoroorganic pesticides (Dipterex and Imidan) on embryogenesis.

Experiments conducted on pregnant Wistar rats show that chlorophos (Dipterex) has embroyotoxic and teratogenic effects after oral introduction in a 80 mg/kg dose during a critical period of embryogenesis. Embryotoxic and teratogenic effects are absent during the introduction of 8 mg/kg of the pesticide. The oral introduction of phthalophos (Imidan) in a 30 mg/kg dose once on day 9 of pregnancy and the introduction of a 1.5 mg/kg dose daily throughout the course of pregnancy caused increased postimplantation mortality of embryos. A dose of 30 mg/kg of phthalophos on day 9 or day 13 of pregnancy causes developmental abnormalities, including hyponathia and hydrocephaly. A 0.06 mg/kg phthalophos dose does not affect the course of embryogenesis in white rats. Thus the organophosphate pesticides Dipterex and Imidan exhibit embryotoxic and teratogenic effects at doses which significantly exceed the acutal amounts of the pesticide that can enter the human organism.