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Insulin-like growth factor-2 regulation of conceptus composition: effects of the trophectoderm and inner cell mass genotypes in the mouse.

The purpose of this study was to measure the effect of insulin-like growth factor-2 deficiency on the growth of the mouse conceptus. Initial observations on normal development in the 129J/Sv strain established that wet and dry weights were reduced by 35% when the insulin-like growth factor-2 gene was inactive. The DNA contents were reduced by only 15%. We exchanged the inner cell mass and trophectoderm between mouse blastocysts that had or lacked an active insulin-like growth factor-2 gene. At embryonic Day 16.5, lack of this gene's activity in the derivatives of either tissue decreased the fluid volumes of the exocelomic and amniotic cavities. The wet weights of the "fetal placentas," the yolk sacs, and the fetuses were also decreased. However, the tissue wet weight decrease could not be accounted for by the change in DNA content, indicating that cell-associated biomass had changed. The conclusions are 1) that insulin-like growth factor-2 levels regulate the composition of the fetus and extra-embryonic tissues and 2) that trophectoderm and inner cell mass derivatives cooperate to control extra-cellular fluid volume in the conceptus.

Amnion↗

Amino acids and preimplantation development of the mouse in protein-free potassium simplex optimized medium.

Development of outbred CF1 mouse zygotes in vitro was studied in a chemically defined, protein-free medium both with and without amino acids. The addition of amino acids to protein-free potassium simplex optimized medium (KSOM) had little effect on the proportion of embryos that developed at least to the zona-enclosed blastocyst stage. In contrast, amino acids stimulated very significantly, in a dilution-dependent way, the proportion of blastocysts that at least partially or completely hatched. Amino acids also stimulated cell proliferation in both the trophectoderm and inner cell mass (ICM) cells, at rates that favored proliferation of cells in the ICM; had no effect on the incidence of cell death (oncosis or apoptosis); and improved development of the basement membranes, which form on the blastocoelic surface of the trophectoderm and between the primitive endoderm and the primitive ectoderm. Thus, KSOM, supplemented with amino acids but containing no protein supplements, supports development of a newly fertilized ovum to the late blastocyst stage, in which its normal, three-dimensional structure is preserved and in which the ICM has been partitioned into the primitive ectoderm and primitive endoderm.

Amino Acids↗

Expression of a Forssman antigenic specificity in the preimplantation mouse embryo.

A monoclonal antibody recognizing a Forssman antigenic specificity has been shown to react with cells of the preimplantation mouse embryo. The antigen is believed to be carried on glycolipid molecules on teratocarcinoma stem cells. This antigen is first detected on the trophectoderm of the early blastocyst. The topography of the expression on the trophectoderm is striking and novel. The antigen is no longer found on these cells after the blastocyst has hatched from the zona pellucida in utero. Inner cell masses are antigen-positive at all times. This is the first study of the distribution of a single antigenic determinant in early mouse embryogenesis.

Animals↗

FMRFamide immunoreactivity in the nervous system of the medusa Polyorchis penicillatus.

Three different antisera to the molluscan neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and two different antisera to the fragment RFamide were used to stain sections or whole mounts of the hydrozoan medusa Polyorchis penicillatus. All antisera stained the same neuronal structures. Strong immunoreactivity was found in neurons of the ectodermal nerve nets of the manubrium and tentacles, in neurons of the sensory epithelium, and in neurons at the periphery of the sphincter muscle. Strong immunoreactivity was also present in processes and perikarya of the whole outer nerve ring, in the ocellar nerves, and in nerve cells lying at the periphery of the ocellus. The inner nerve ring contained a moderate number of immunoreactive processes and perikarya, which were distinct from the swimming motor neurons. In contrast to the situation in the hydrozoan polyp Hydra attenuata, no immunoreactivity was found with several antisera to oxytocin/vasopressin and bombesin/gastrin-releasing peptide. The morphology and location of most FMRFamide-immunoreactive neurons in Polyorchis coincides with two identified neuronal systems, which have been recently discovered from neurophysiological studies.

Animals↗

The fate mapping of the eleventh and twelfth day mouse otocyst: an in vitro study of the sites of origin of the embryonic inner ear sensory structures.

An experiment was undertaken to determine which sensory structures of the mouse embryo inner ear developed from what portion of the mouse otocyst. Otocysts of gestation days 10, 11, 12, and 13 were divided by surgical dissection into six anatomical groups: dorsal, ventral, anterior, posterior, medial and lateral halves. They were organ cultured separately. After a period of ten days, the explanted tissues were harvested and processed histologically for microscopic analysis. The surgical control specimens fixed at the time of explantation were composed of undifferentiated ectodermal cells for tissues of gestation days 10, 11, and 12. Otocysts of gestation days 11, and 12 showed, during the course of their subsequent growth, that the three semicircular ducts and their associated cristae developed from the dorsal and lateral halves. Only the anterior and posterior canals and cristae originated from the medial portion. The posterior half gave rise to the posterior crista and the anterior half provided for the development of the anterior and lateral cristae. The cochlear duct and its sensory epithelium developed in all the anatomical groups except the dorsal half. The utricle developed in the dorsal section of the middle third of the otocyst, while the utricular macula developed in the anterior half of the same section of the otocyst. The saccule and its macula differentiated from the ventral section of the middle third of the anterior half.

Animals↗

Latent form of multiple dermoid cysts in the jaw bone.

Dermoid cysts of the jaw are exceedingly rare, and only six such cases have been documented. The present paper reports a case with multiple microcysts showing histological characteristics of dermoid cyst in the surgically resected mandibular bone. These cysts were located near the inferior border of the mandibular bone, and histologically composed of an inner thin layer of squamous epithelium with marked hyperorthokeratinization and an outer layer of fibrous tissue containing numerous mature sebaceous glands. In addition, a hair follicle-like epithelial sphere was seen in part. The present microcysts suggest that it arises de novo, and one possible cause of jaw cysts could be sequestration of the stomadeal ectoderm that may have been trapped deep in the surface and embedded in the developing mandibular bone during embryogenesis. It may not be possible that such cysts developed from the odontogenic epithelial remnant undergoing dermoid metaplasia.

Adult↗

Expression of the homeobox gene, Barx2, in wool follicle development.

We have cloned ovine Barx2, a member of the Bar class of homeobox genes, and present the first description of Barx2 expression in wool follicle development. Barx2 is uniformly expressed in the embryonic ectoderm but is transiently downregulated during the initiation of follicle morphogenesis. Subsequently, Barx2 is expressed throughout the epithelial component of the developing follicle except for a small group of cells at the leading edge of the follicle placode. These Barx2-negative cells are destined to form the follicle bulb and are the progenitors of the inner root sheath and hair shaft. In adult follicles, Barx2 is expressed throughout the outer root sheath but not in the inner root sheath or hair shaft, or in dermal cells associated with the follicle. The pattern of Barx2 expression in follicle morphogenesis is similar to that of the cell adhesion molecule E-cadherin, a similarity that echoes Barx2 coexpression with the L1 cell adhesion molecule in other tissues during mouse embryogenesis. Barx2 is also expressed in tongue and esophagus, two other keratinizing tissues, and we speculate that Barx2 may have a general function in controlling adhesive processes in keratinizing epithelia.

Animals↗

Prenatal expression of growth hormone receptor/binding protein and insulin-like growth factor-I (IGF-I) in the enamel organ. Role for growth hormone and IGF-I in cellular differentiation during early tooth formation?

Immunohistochemistry was used to study the ontogeny of GH receptor/binding protein (GHR/BP) and IGF-I from the 13-day-old embryo (E13) to the E19 rat fetus in the developing incisor and molar. Analysis of serial sections revealed diffuse staining of GHR/BP and IGF-I at the bud and early cap stages within both the mesenchyme of the dental papilla and the ectodermal-derived enamel organ. Just before transition to the cap stage, immunoreactivity of GHR/BP and IGF-I increased in the epithelial bud and extended to the condensed dental mesenchyme. At the cap stage, the dental epithelium showed an intense expression of GHR/BP and IGF-I, whereas the dental mesenchymal cells showed very weak staining. The inner enamel epithelium and the outer enamel epithelium were positive for both GHR/BP and IGF-I in the bell stage. Differentiating ameloblasts, odontoblasts and the secretory ameloblasts and odontoblasts continued to express GHR/BP and IGF-I in incisors. These findings support the premise that growth hormone and IGF-I may play a role in embryonic tooth development by regulating the epithelial-mesenchymal interactions that influence events in growth and cytodifferentiation.

Animals↗

Molecular cloning and developmental expression of two Chloride Intracellular Channel (CLIC) genes in Xenopus laevis.

CLIC proteins are components or regulators of novel intracellular anion channels in mammalian cells, and previous studies have suggested that human nuclear membrane-associated CLIC1 and mouse inner mitochondrial membrane CLIC4 are involved in cell division and apoptosis. We have isolated Xenopus homologues of CLIC1 and CLIC4 and shown them to be well conserved during chordate evolution, but poorly conserved in invertebrates. Consistent with fundamental cellular roles, Xenopus CLIC genes are expressed at every stage of embryonic development. Expression is localised to mesodermal and ectodermal tissues, with particularly marked expression of xCLIC4 in the developing nervous system. This is the first description of non-mammalian CLIC expression, and use of Xenopus laevis as a model organism may provide insights into the role of CLIC-associated ion channels in animal development.

Amino Acid Sequence↗

Involvement of the Edar signaling in the control of hair follicle involution (catagen).

Ectodysplasin (Eda) and its receptor (Edar) are required for normal development of several ectodermal derivatives including hair follicles (HFs). Here, we show that during the murine hair cycle the expression of Eda A1, Edar, Edaradd, and TRAF6 transcripts are minimal in the resting phase and maximal during HF transition from active growth to regression (catagen). Eda A1 mRNA and Edar proteins were expressed in the hair matrix and outer and inner root sheaths of anagen HFs. During catagen, Eda A1 mRNA and Edar protein were expressed in the outer and inner root sheaths and later in the secondary hair germ. Catagen development accompanied by increased apoptosis in the outer root sheath was significantly accelerated in downless mice or after treatment of wild-type mice by a fusion protein that inhibits Edar signaling, compared with the corresponding controls. Microarray, real-time polymerase chain reaction, and immunohistochemical analyses of skin of downless mice revealed a strong decrease of expression of X-linked inhibitor of apoptosis protein (XIAP), compared with the controls, suggesting XIAP as a target for Edar signaling. Thus, our data demonstrate that in addition to its well-established role in HF morphogenesis, Edar signaling is also involved in hair cycle control and regulates apoptosis in HF keratinocytes during catagen.

Animals↗

Placental permeability during early gestation in the hamster. Electron microscopic observations using horseradish peroxidase as a macromolecular tracer.

The permeabilities of the parietal yolk sac placenta and the preplacental region of the hamster conceptus during early postimplantation (day 8) were compared by means of electron microscopy and a macromolecular protein tracer, horseradish peroxidase (HRP). HRP was administered by injection into the maternal venous system; samples of the two placental tissues were obtained for examination at intervals between 4 minutes and 1 hour later. The three layers of the parietal yolk sac wall (from outer to inner: capsular trophoblast, Reichert's membrance, parietal endoderm) appeared to provide little impediment to the passage of HRP from perivitelline maternal blood spaces to the yolk sac cavity. HRP passed through the outer trophoblast layer, both by way of intracellular fenestrae (60-200 nm diameter) and narrower intercellular channels, and completely permeated the meshwork of Reichert's membrane within minutes after injection. The inner parietal endoderm cell layer was widely discontinuous and clearly presented no barrier to HRP movement. HRP reaching the yolk sac cavity was avidly endocytosed by the viceral yolk sac epithelium. In contrast to the parietal yolk sac, the preplacental region of the conceptus was impermeable to HRP. Zonular occluding junctions located between contiguous cells of the chorionic ectoderm layer of the preplacenta were the obvious barrier to the HRP molecules. These results suggest that in this rodent species, during the early postimplantation period of gestation, the pareital yolk sac placenta potentially plays a more important role in the maternal-embryonic transfer of macromolecular substances than does the preplacenta.

Animals↗

Nitric oxide regulates swimming in the jellyfish Aglantha digitale.

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative-resistant NADPH-diaphorase (NADPH-d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10(-4) M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2- and NO3- were present at high levels in the tentacles, but were not detectable in NADPH-d-negative areas. The NADPH-d-reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free-moving animals and semi-intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30-50 nM) and its precursor L-arginine (1 mM) stimulated swimming, and the effect was mimicked by 8-Br-cGMP (50-100 microM). The NO scavenger PTIO (10-100 microM) and a competitive inhibitor of NOS, L-nitroarginine methyl ester (L-NAME, 200 microM), significantly decreased the swimming frequency in free-moving animals, while its less-active stereoisomer D-nitroarginine methyl ester (D-NAME, 200 microM) had no such effect. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 5-20 microM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO-induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles.

Animals↗

Human embryonic stem cells: mother of all cell and tissue types.

Pluripotential embryonic stem (ES) cells have been derived very efficiently from spare human embryos produced by IVF and grown in culture to the nascent blastocyst stage. The inner cell mass (ICM) is isolated by immunosurgery and grown on selected embryonic fibroblast monolayer cultures. ICM cells lose their memory for axis during formation of ES cell colonies and are then unable to integrate tissue formation with a body plan. ES cells form teratomas in vivo with cells and tissues representative of the three major embryonic lineages (ectoderm, mesoderm, endoderm). The ES cells are continuously renewable and can be directed to differentiate into early progenitors of neural stem cells (Noggin cells) and from there into mature neurons and glia (astrocytes and oligodendrocytes). The neural stem cells formed from human ES cells repopulate the brains of newborn mice when injected into the lateral cerebral ventricles, forming astrocytes dominantly in the parenchyma. The human neural cells can be observed migrating from the subventricular areas along the rostral migratory stream. Human neurons can be found in the olfactory bulb. Human ES cells can also be directed into cardiomyocytes when co-cultured with visceral endoderm-like cells (END-2). These observations provide further scope to explore stem cell therapies, gene therapies and drug discovery. For compatible transplantation, ES may need to be derived with a range of HLA types or by nuclear transplantation or stem cell fusion.

Animals↗

Ontogeny of osmoregulation in embryos of intertidal crabs (Hemigrapsus sexdentatus and H. crenulatus, Grapsidae, Brachyura): putative involvement of the embryonic dorsal organ.

This study examined whether the existence of hyperosmotic internal fluids in embryos of euryhaline crabs (Hemigrapsus sexdentatus and H. crenulatus) in dilute seawater reflects osmotic isolation due to impermeability of the egg envelope, as proposed for other decapods, or active osmoregulation. When ovigerous crabs with eggs at gastrula stage were transferred from 100% seawater (osmolality 1000 mmol kg(-1)) to 50% seawater, embryogenesis and hatching of zoea were completed normally, but were delayed. Hatching failed if the transfer to 50% seawater occurred before gastrulation, and embryogenesis was abnormal in 25% seawater. In 100% seawater, embryos at all stages were internally hyperosmotic by 150-250 mmol kg(-1). On transfer to 50% seawater, osmolality initially decreased but remained 200-350 mmol kg(-1) hyperosmotic to the medium for several weeks until hatching. High efflux rates of tritium-labelled water (t((1/2)) 16-75 min) and (22)Na (t(1/2) 109-374 min) from H. crenulatus embryos were inconsistent with the osmotic isolation hypothesis. It is concluded that post-gastrula embryos were actively hyper-osmoregulating. The diffusional water permeability of the embryos decreased during development while the sodium efflux rate increased 10-fold. Very rapidly exchanging pools of water and sodium (t(1/2) a few seconds to minutes) probably corresponded to peri-embryonic fluid and implied that the egg envelope was a negligible barrier to diffusion of water and salts. Higher Na(+)/K(+)-ATPase activities in late embryos of H. crenulatus incubated in 50% seawater than in embryos incubated in full strength seawater were consistent with an acclimation response. An area of the embryonic surface located over the yolk in the region of the embryonic dorsal organ stained with AgNO(3). Staining appeared at gastrulation, persisted throughout development and was lost at hatching. Deposits of AgCl between the outer and inner membranes, identified by X-ray microanalysis, suggest that the dorsal organ was a site of chloride extrusion. A model for osmoregulation in post-gastrula embryos is proposed: osmotic uptake of water is balanced by excretion of water and salts via the dorsal organ and salt loss is balanced by active uptake over the general embryonic ectoderm.

Animals↗

Both blastomeres of the mouse 2-cell embryo contribute to the embryonic portion of the blastocyst.

To track the lineage of both blastomeres of 2-cell embryos during mouse preimplantation development, each cell was injected with dextran solutions conjugated with different fluorochromes. The fate of the progeny of the first two blastomeres was followed with confocal microscopy during cleavage and during the formation of the blastocyst. We observed that in most of cleaving embryos the cells derived from the two first blastomeres intermingled in both the trophectoderm and the inner cell mass (ICM) and did not form two discrete groups. We conclude that embryonic parts of blastocysts contain descendants of both blastomeres of 2-cell embryo.

Animals↗

Developmental analysis of the Hba(th-J) mouse mutation: effects on mouse peri-implantation development and identification of two candidate genes.

The Hba(th-J) mouse mutation is a deletion on chromosome 11 that spans the alpha-globin complex and causes alpha-thalassemia in heterozygous animals and in utero death of embryos homozygous for the deletion. We hypothesised that one or more genes closely linked to the Hba locus are also deleted in these mutant mice and that deletion of these additional genes is responsible for the embryo lethality. We have analysed the developmental profile of mutant embryos using a PCR assay to distinguish homozygous embryos from wild-type and heterozygous embryos. No homozygous embryos are detectable on Day 6.5 of gestation and morphological analysis of embryos on Day 5.5 shows that both the embryonic and extraembryonic ectoderm of the egg cylinder are reduced in size and contain degenerate cells. Preimplantation homozygous embryos are morphologically normal with the same proportion developing to the blastocyst stage as control embryos. However, the cell number of homozygous embryos on Day 4.5 is significantly reduced due predominantly to a decrease in the cell number of the trophectoderm and not the inner cell mass. When homozygous blastocysts are plated in vitro, outgrowth of giant trophectoderm cells appears similar to that of wild-type embryos but outgrowth of the inner cell mass is affected. Cells from the inner cell mass of homozygous embryos appear to undergo necrosis and dissociate from the trophectoderm outgrowth after 3 to 4 days in culture. These studies demonstrate that the development of both the inner cell mass and the trophectoderm of embryos homozygous for the Hbath-J deletion is affected by the mutation. We have used quantitative Southern blotting to show that 3-methyladenine glycosylase (mpg) and dist1, two genes closely linked to the Hba locus on chromosome 11, are also deleted in this mutation. Reverse transcriptase-polymerase chain reaction analyses demonstrate that mpg and dist1 are normally expressed by preimplantation and early postimplantation embryos, whereas alpha-globin transcripts from the Hba locus are not detected until Day 7.5 of gestation. These studies demonstrate that deletion of the mpg or dist1 genes is likely to be responsible for the homozygote embryo lethality and the potential roles of these gene products in early embryogenesis are discussed.

Animals↗

Expression of Forssman antigen in the post-implantation mouse embryo.

The expression of Forssman antigen on the surface of cells of post-implantation mouse embryos between 5 and 7 1/2 days old and of cells of the gonads from 10 1/2 days has been followed using the monoclonal antiserum M1/22.25. In the early post-implantation embryo a lineage-related distribution is found. The inner cell mass of the blastocyst was previously shown to be Forssman antigen positive and its derivative tissues the epiblast of the 5-day embryo and the primary embryonic endoderm are also positive. The endoderm cells remain positive both over the embryonic and extraembryonic portions of the embryo but the epiblast becomes Forssman antigen negative as it differentiates into embryonic ectoderm. The extraembryonic ectoderm which is derived from the Forssman negative trophectoderm remains negative throughout. The primordial germ cells are Forssman antigen positive from their first appearance in the germinal ridge until day 14 when they become negative but after that time it is other cells not related by direct lineage which become Forssman antigen positive. These are tentatively identified as Sertoli cells precursors as it is the Sertoli cells which are the antigen-positive population in the adult testis.

Animals↗

Lens specification is the ground state of all sensory placodes, from which FGF promotes olfactory identity.

The sense organs of the vertebrate head comprise structures as varied as the eye, inner ear, and olfactory epithelium. In the early embryo, these assorted structures share a common developmental origin within the preplacodal region and acquire specific characteristics only later. Here we demonstrate a fundamental similarity in placodal precursors: in the chick all are specified as lens prior to acquiring features of specific sensory or neurogenic placodes. Lens specification becomes progressively restricted in the head ectoderm, initially by FGF and subsequently by signals derived from migrating neural crest cells. We show that FGF8 from the anterior neural ridge is both necessary and sufficient to promote olfactory fate in adjacent ectoderm. Our results reveal that placode precursors share a common ground state as lens and progressive restriction allows the full range of placodal derivatives to form.

Animals↗