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PubMed2004

Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod.

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.

Amino Acid Sequence↗
PubMed2001

Microarray-based analysis of early development in Xenopus laevis.

In order to examine transcriptional regulation globally, during early vertebrate embryonic development, we have prepared Xenopus laevis cDNA microarrays. These prototype embryonic arrays contain 864 sequenced gastrula cDNA. In order to analyze and store array data, a microarray analysis pipeline was developed and integrated with sequence analysis and annotation tools. In three independent experimental settings, we demonstrate the power of these global approaches and provide optimized protocols for their application to molecular embryology. In the first set, by comparing maternal versus zygotic transcription, we document groups of genes that are temporally regulated. This analytical approach resulted in the discovery of novel temporally regulated genes. In the second, we examine changes in gene expression spatially during development by comparing dorsal and ventral mesoderm dissected from early gastrula embryos. We have discovered novel genes with spatial enrichment from these experiments. Finally, we use the prototype microarray to examine transcriptional responses from embryonic explants treated with activin. We selected genes (two of which are novel) regulated by activin for further characterization. All results obtained by the arrays were independently tested by RT-PCR or by in situ hybridization to provide a direct assessment of the accuracy and reproducibility of these approaches in the context of molecular embryology.

Animals↗
PubMed1999

Uterine and postnatal maternal effects in mice selected for differential rate of early development.

A series of mouse lines was produced by long-term restricted index selection for divergent rate of growth during early and late postnatal development. The selection program was based on the following treatments: E(+) and E(-) lines were selected to alter birth to 10-day weight gain while holding late gain for both lines constant and a control line was established via random selection. Using embryo transfer and crossfostering methodology, we partitioned postnatal growth for E(+), E(-), and C lines into progeny genetic, uterine maternal, and nurse maternal components. Selection for differential early growth resulted in correlated response in uterine and nurse maternal effects on body weights, with significant genetic-by-environment interactions. Significant uterine effects were also observed in tail length measurements. Direct uterine effects on body weight were relatively small and resulted in growth rate differences early in development. Nurse effects were large, resulting in modification of progeny growth trajectory especially during early postnatal development. Genetic-by-uterine interactions were large and demonstrate progeny-specific effects of the prenatal uterine environment.

Analysis of Variance↗
PubMed1994

Early development of Xenopus embryos is affected by simulated gravity.

Early amphibian (Xenopus laevis) development under clinostat-simulated weightlessness and centrifuge-simulated hypergravity was studied. The results revealed significant effects on (i) "morphological patterning" such as the cleavage furrow pattern in the vegetal hemisphere at the eight-cell stage and the shape of the dorsal lip in early gastrulae and (ii) "the timing of embryonic events" such as the third cleavage furrow completion and the dorsal lip appearance. Substantial variations in sensitivity to simulated force fields were observed, which should be considered in interpreting spaceflight data.

Animals↗
PubMed2000

Regeneration of descending spinal axons after transection of the thoracic spinal cord during early development in the North American opossum, Didelphis virginiana.

Opossums are born in an immature, fetal-like state, making it possible to lesion their spinal cord early in development without intrauterine surgery. When the thoracic spinal cord of the North American opossum, Didelphis virginiana, is transected on postnatal day 5, and injections of Fast Blue (FB) are made caudal to the lesion site 30-40 days or 6 months later, neurons are labeled in all of the spinal and supraspinal areas that are labeled after comparable injections in age-matched, unlesioned controls. Double-labeling studies document that regeneration of cut axons contributes to growth of axons through the lesion site and behavioral studies show that animals lesioned on postnatal day 5 use their hindlimbs in normal appearing locomotion as adults. The critical period for developmental plasticity of descending spinal axons extends to postnatal day 26, although axons which grow through the lesion site become fewer in number and more restricted as to origin with increasing age. Animals lesioned between postnatal day 12 and 26 use the hindlimbs better than animals lesioned as adults, but hindlimb function is markedly abnormal and uncoordinated with that of the forelimbs. We conclude that restoration of anatomical continuity occurs after transection of the spinal cord in developing opossums, that descending axons grow through the lesion site, that regeneration of cut axons contributes to such growth, and that animals lesioned early enough in development have relatively normal motor function as adults.

Animals↗
PubMed1997

The effect of propofol on parthenogenetic activation, in vitro fertilization and early development of mouse oocytes.

OBJECTIVE: To assess the effect of propofol on fertilization and early embryo development in a mouse IVF model. DESIGN: Controlled study. SETTING: Mouse IVF. INTERVENTION(S): Mouse oocytes were exposed in vitro to propofol at a concentration of 0 (control), 50, 250, 500, 1,000, or 5,000 ng/mL for 30 minutes, washed, and inseminated. Thereafter, fertilization was assessed. Subsequent in vitro development to the blastocyst stage was monitored daily. The potential to activate parthenogenetically oocytes also was evaluated by looking for spontaneous extrusion of the second polar body or development to the two-cell stage. In a second step, a pure propofol solution was added to culture medium and used as a standard. MAIN OUTCOME MEASURE(S): Two-cell and blastocyst-stage embryo. RESULT(S): Where fertilization occurred, subsequent embryo cleavage and development up to the blastocyst stage was affected significantly by the presence of propofol solution in the medium, (i.e., 3% to 41%) in comparison with the control group (76%). Exposure of unfertilized oocytes for 30 minutes to propofol results in a parthenogenetic activation of 33% to 60%, which was significantly higher than the control (10%). When oocytes were kept in propofol for 24 hours, a mean of 30% of activation was observed as compared with 0.5% for the control. CONCLUSION(S): We can conclude from these experiments that even a brief exposure of cumulus-enclosed oocytes to a low concentration of propofol is deleterious to subsequent cleavage. Exposure of unfertilized oocytes to propofol results in a high degree of parthenogenetic activation.

Anesthetics, Intravenous↗
PubMedDate not supplied

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line.

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.

Animals↗
PubMed1993

Regulation of growth and differentiation in early development: of mice and models.

In this article we describe some of the fundamental processes occurring during early murine development, introduce cellular models used to investigate these processes and review some well-known factors that may be involved in their control. These include transforming growth factor beta, retinoic acid and leukaemia inhibitory factor. Refinements to the culture conditions of embryonic stem and embryonal carcinoma cells have enabled us to test the effects of these factors on growth and differentiation and in particular to establish that their interaction may determine the ultimate developmental state of the cell population. Preliminary studies using neutralizing antibodies in embryos are described that suggest that deregulation of normal expression can lead to a failure to implant. Insights into the events underlying normal embryonic development and implantation, yielded by the type of study described here, may contribute to an understanding of the mechanisms causing early embryonic loss and the role of toxicants in this process.

Animals↗
PubMed1991

The early development of the frog retinotectal projection.

The guidance of retinal ganglion cell axons has been investigated in embryos of the frog Xenopus. During the initial development of the brain a series of axon tracts are laid down forming a basic 'scaffold' or framework. Retinal axons grow through one of these tracts, the tract of the post-optic commissure (tPOC). This is the only tract that extends through the rostral part of the brain at these early stages of development. The origin and development of the tPOC has been studied using antibodies which label neurons at their earliest stages of differentiation. The first sign of the tPOC is a chain of neurons which differentiate simultaneously in the caudolateral part of the diencephalon. Axons from these neurons grow the short distance between adjacent cells interlinking the chain to form a descending tract. A series of other axon projections are then added to the tPOC, each of which is segregated into a particular subregion of the tract. Retinal axons are added to the tract approximately 18 h after its formation. They grow in the sub-pial part of the tract and always occupy the rostral-most edge. Retinal axons follow the tract to the region of the developing tectum where they leave, turn dorsally, and terminate. The reliance of retinal axons on this pre-existing pathway has been demonstrated by experimentally altering the course of the tPOC during its early development. The caudo-lateral wall of the diencephalon has been rotated through 90 degrees at a stage just before the tPOC neurons differentiate. Confirmation of the predicted alteration in the course of the tPOC has been made using immunocytochemistry. In such manipulated brains, retinal axons maintain their strong affinity for the rostral edge of the tPOC, following its altered course through the diencephalon.

Animals↗
PubMed1991

Early development of the human thalamus: Golgi and Nissl study.

Qualitative and quantitative parameters of the early neuronal development of the thalamus were examined in human embryos and fetuses ranging in age from 5-16 gestational weeks (g.w.) using Nissl stained and Golgi impregnated tissue. In the earliest period analyzed, from 5-6 weeks of gestation, dorsal and ventral parts of the human thalamus were poorly differentiated structures forming the sides of the third ventricle. At 6 g.w., the prospective thalamic anlage consisted of the ventricular (VZ), the intermediate (IZ) and the marginal zones (MZ). The neurons in the VZ were bipolar and radially arranged to the ventricular surface. From 6-8 gestational weeks detached bipolar or branched bipolar cells with horizontal or oblique orientation were encountered in the IZ and occasionally in the MZ. In the period between 10-14 weeks of gestation, segregation of neurons into different thalamic nuclei began. The lateral geniculate nucleus, centrum medianum and mediodorsal nucleus were the first to be distinguished. In comparison with the previous period, the neurons throughout the rostro-caudal axis of the thalamus were more differentiated, with a higher number of primary dendrites that were often bifurcated. This resulted in various shapes of immature neurons: simple uni- and bipolar (type 1), branched uni- and bipolar (type 2) and young multipolar (type 3). In addition, the first spines were frequently observed on dendrites at 12 weeks of gestation. At 14-16 weeks of gestation, segregation of the neurons into thalamic nuclei continued. The neurons exhibited further dendritic differentiation that followed the caudo rostral gradient. For the first time, multipolar neurons, especially in the caudal part, could be subdivided into two classes: multipolar with a 'bushy' appearance, the result of many primary dendrites with several bifurcations and 'simpler' multipolar neurons with fewer and less branched primary dendrites. Neuronal differentiation was further expressed by an increase in the total dendritic length and somatic surface, up to six and three times, respectively, from the earliest period analyzed to 16 weeks of gestation. Dendritic length increased initially by the formation of new primary branches and their elongation and secondly by formation of higher branch orders. At 16 g.w., a group of immature cells were observed dorsally, on the frontal sections through the rostral thalamus. Its ganglionic eminence origin is discussed.

Analysis of Variance↗
PubMed2002

Essential role of the transcription factor Ets-2 in Xenopus early development.

The fibroblast growth factor (FGF)/MAPK pathway plays an important role in early Xenopus developmental processes, including mesoderm patterning. The activation of the MAPK pathway leads to induction of Xenopus Brachyury (Xbra), which regulates the transcription of downstream mesoderm-specific genes in mesoderm patterning. However, the link between the FGF/MAPK pathway and the induction of Xbra has not been fully understood. Here we present evidence suggesting that Ets-2 is involved in the induction of Xbra and thus in the development of posterior mesoderm during early embryonic development. Overexpression of Ets-2 caused posteriorized embryos and led to the induction of mesoderm in ectodermal explants. Expression of a dominant-negative form of Ets-2 or injection of antisense morpholino oligonucleotides against Ets-2 inhibited the formation of the trunk and tail structures. Overexpression of Ets-2 resulted in the induction of Xbra, and expression of the dominant-negative Ets-2 inhibited FGF- or constitutively active MEK-induced Xbra expression. Moreover, overexpression of Ets-2 up-regulated the transcription from Xbra promoter reporter gene constructs. Ets-2 bound to the Xbra promoter region in vitro. These results taken together indicate that Xenopus Ets-2 plays an essential role in mesoderm patterning, lying between the FGF/MAPK pathway and the Xbra transcription.

Animals↗
PubMed2003

Prolonged neurogenesis during early development of gonadotropin-releasing hormone neurones in sheep (Ovis Aries): in vivo and in vitro studies.

Gonadotropin-releasing hormone (GnRH) neurons involved in controlling the reproductive function in vertebrates are derived from the olfactory placode. However, in the sheep and the rat species, GnRH-immunoreactive (GnRH-IR) neurons could not be detected in the olfactory region during the earliest phase of GnRH system development. Using in situ hybridization (ISH) and immunohistochemistry (IHC) in sheep embryos ranging from 26 to 53 days' gestational age (G26-G53), the present work confirmed that GnRH expression could not be detected during the earliest steps of migration. The first ISH+ cells were detected in the nasal septum and at the entrance of the telencephalon at G33 stage. [(3)H]-thymidine pulses applied to in vitro olfactory explant cultures showed that GnRH neuron precursor cells have an extended multiplication period corresponding to G26-G36 before entering the neuronal differentiation process. Therefore, the lack of GnRH neuron detection during the early phase of development in the sheep compared to the mouse and other vertebrates represents a major difference in the early development of GnRH neurons. In the mouse, GnRH neuron precursors have a limited multiplication period in the vomeronasal pit and only postmitotic neurons start migration, whereas in the sheep embryo, the multiplication period is extended to about 10 days as demonstrated in olfactory explant cultures.

Animals↗
PubMed1991

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development.

Animals↗
PubMed2006

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus.

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.

Analysis of Variance↗
PubMed1999

Maturation of neuromuscular transmission during early development in zebrafish.

We have examined the rapid development of synaptic transmission at the neuromuscular junction (NMJ) in zebrafish embryos and larvae by patch-clamp recording of spontaneous miniature endplate currents (mEPCs) and single acetylcholine receptor (AChR) channels. Embryonic (24-36 h) mEPCs recorded in vivo were small in amplitude (<50 pA). The rate of mEPCs increased in larvae (3.5-fold increase measured by 6 days), and these mEPCs were mostly of larger amplitude (10-fold on average) with (</=5-fold) faster kinetics. Intracellular labeling with Lucifer yellow indicated extensive coupling between muscle cells in both embryos and larvae (</=10 days). Blocking acetylcholinesterase (AChE) with eserine had no effect on mEPC kinetics in embryos at 1 day and only partially slowed (by approximately 1/2) the decay rate in larvae at 6 days. In acutely dissociated muscle cells, we observed the same two types of AChR with conductances of 45 and 60 pS and with similar, brief (<0.5 ms) mean open times in both embryos and larvae. We conclude that AChR properties are set early during development at these early stages; functional maturation of the NMJ is only partly shaped by expression of AChE and may also depend on postsynaptic AChR clustering and presynaptic maturation.

Animals↗
PubMed1997

GABA immunoreactivity in hypothalamic neurons and growth cones in early development in vitro before synapse formation.

GABA (gamma-aminobutyrate) is the most prevalent inhibitory transmitter in the mature hypothalamus. In contrast, in the developing hypothalamus, GABA may exert depolarizing actions leading to neuronal excitation. To determine whether GABA is present in hypothalamic neurons early in development, and whether there is a preferential expression in axonal growth cones, immunogold and peroxidase studies were used with light and whole mount transmission electron microscopy. At embryonic day 15, a stage of development at the beginning of hypothalamic neurogenesis, histological sections showed GABA immunoreactivity in fibers and weakly stained perikarya. Hypothalamic neurons (13%) cultured at embryonic day 15 were immunoreactive after 1 day in vitro. The percentage of neurons stained, and the intensity of staining increased during the next few days to 39% at 4 days in vitro. Neuritic growth cones, including lamellipodia and long filopodia, showed strong immunoreactivity before synaptogenesis. By using neuronal whole mounts studied with transmission electron microscopy and GABA silver-enhanced immunogold staining, a quantitative comparison of growth cones after a day and a half in culture revealed that the growth cone of the longest process, the putative axon, had a greater level of immunogold labeling than that of the shorter processes, the putative dendrites. This finding is one of the earliest biochemical differences between putative axons and dendrites. Astrocytes in the same cultures showed no immunolabeling. These results indicate that GABA is present very early in the development of hypothalamic neurons and is in a position to be released.

Animals↗