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PubMed2005

Nicotine-induced embryonic malformations mediated by apoptosis from increasing intracellular calcium and oxidative stress.

BACKGROUND: Tobacco smoking by women during pregnancy increases the risk of congenital birth defects in the infants. Among the smoke products, nicotine is believed to be the major teratogenic factor that perturbs embryonic development. However, the role of nicotine in embryonic malformations has not been addressed, and the mechanisms by which nicotine affects embryonic development remain to be delineated. METHODS: To investigate the effects of nicotine on early embryogenesis, murine embryos at embryonic day (E) 8.5 were dissected out of the uteri, cultured in a roller bottle system, and treated with nicotine (0.6-6 microM) or vehicle. Embryonic morphogenesis and growth were examined in terms of structural morphology and crown/rump length, respectively. Programmed cell death (apoptosis) was assessed using LysoTracker Red staining of whole mount embryos and TUNEL assay of tissue sections. Changes in intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) production were assessed using fluorescent dyes (Flu-4, AM; H2DCFDA, respectively) under a confocal microscope. To further investigate the role of intracellular calcium and ROS in nicotine-induced embryopathy, embryos were treated with BAPTA-AM (2 microM) to inhibit [Ca2+]i elevation and ascorbic acid (vitamin C; 100 microg/ml) to scavenge ROS in presence of nicotine (6 microM). RESULTS: The embryos treated with nicotine in 3-6 microM were smaller than those treated with vehicle. Most of the embryos had open neural tube in the anterior (brain) regions. The embryos treated with 6 microM nicotine also exhibited severe defects in the posterior trunk, resembling caudal dysplasia. Excessive apoptosis was observed in the deformed structures. Significant increases in [Ca2+]i and ROS were seen in the tissues that had higher levels of apoptosis. Furthermore, inhibition of [Ca2+]i and scavenging of ROS significantly reduced embryonic malformation and apoptotic rates in the embryos. CONCLUSIONS: Nicotine affects embryonic development in a concentration-dependent manner. The nicotine-induced embryonic malformations are, in part, a result of excessive cell death. Nicotine increases [Ca2+]i and ROS level, which play a role in nicotine-induced embryonic apoptosis and malformations. These studies identify the molecular pathway of nicotine action in embryonic apoptosis and malformations, and provide a promising approach for ameliorating the teratogenic effects of nicotine.

Abnormalities, Drug-Induced↗
PubMed2005

Mitotic remodeling of the replicon and chromosome structure.

Animal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.

Acetylation↗
PubMed1990

Determinants of nerve cell patterns during development: a review.

The aim of the work reviewed is to define some of the mechanisms which are implicated in the control of neural cell pattern formation in the developing central nervous system. This question was examined by studies of brain embryonic development in normal and reeler mutant mice, which are characterized by profuse architectonic anomalies. The adult reeler phenotype is characterized by extreme abnormalities of cell positioning in the telencephalic and cerebellar cortices as well as by distinct architectonic anomalies in non-cortical structures such as the inferior olive, the facial nerve nucleus and other brainstem nuclei. Studies of the embryonic development of these structures reveal that neurons are generated at the normal time and migrate along normal pathways. Moreover, the processes of directional axonal growth, differentiation of class-specific features of neurons and glia, and synaptogenesis appear unaffected by the reeler mutation. In all instances, however, the early cell patterns formed by reeler neurons is consistently less regular than in normal embryos. These data indicate that brain architectonics does not exclusively result from the maturation of cells, neurites and connections, but is also contingent upon a specific stabilization of early neurons at the end of migration. One may infer that the presence of a normal allele at the reeler locus is necessary for this stabilization to occur normally, or that it is submitted to genetic control. Although the factor(s) responsible for the stable configuration of neural cell patterns are still unknown, several hypotheses can be considered. There is ample evidence against the role of diffusible factors, mesodermal components and afferent fiber systems. So far, most data point to the importance of cell-cell interactions which can be of three types: homophilic (neuronal-neuronal), heterophilic (neuronal-glial), or both. The cell-interaction mechanism could have been acquired during brain evolution of the mammalian lineage and the reeler gene could act by perturbing, directly or indirectly, these cell interactions. A better definition of the mechanisms responsible for the organization of nerve cell patterns is central to our understanding of brain development in normal as well as in pathological states. By following the example of recent successful research on invertebrate brain development, we believe that the genetic approach to this important question is a valuable one.

Alleles↗
PubMed2003

Preferential expression of the G90 gene in post-mitotic cells during mouse embryonic development.

G90 is a novel mouse gene that does not belong to any known gene family. It has previously been shown that this gene is expressed exclusively in post-mitotic cells of the adult mouse intestine and testis, therefore suggesting a role in the control of proliferation and/or differentiation. Here we report the detailed spatio-temporal expression pattern of G90 during mouse embryonic development. We found G90 expression in specific structures of the developing head, namely the brain, inner and middle ear, olfactory epithelium, vomeronasal organ, nasopharynx, oropharynx, papillae of the tongue and oral cavity, pituitary gland and epiglottis. Interestingly, there was a clear correlation between G90 expression and absence of proliferation in most of the cells showing expression of this gene during embryonic development; this finding supported our functional hypothesis.

Animals↗
PubMed1997

Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter.

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.

Animals↗
PubMed2000

Cadherins in the central nervous system.

The central nervous system (CNS) is divided into diverse embryological and functional compartments. The early embryonic CNS consists of a series of transverse subdivisions (neuromeres) and longitudinal domains. These embryonic subdivisions represent histogenetic fields in which neurons are born and aggregate in distinct cell groups (brain nuclei and layers). Different subsets of these aggregates become selectively connected by nerve fiber tracts and, finally, by synapses, thus forming the neural circuits of the functional systems in the CNS. Recent work has shown that 30 or more members of the cadherin family of morphoregulatory molecules are differentially expressed in the developing and mature brain at almost all stages of development. In a regionally specific fashion, most cadherins studied to date are expressed by the embryonic subdivisions of the early embryonic brain, by developing brain nuclei, cortical layers and regions, and by fiber tracts, neural circuits and synapses. Each cadherin shows a unique expression pattern that is distinct from that of other cadherins. Experimental evidence suggests that cadherins contribute to CNS regionalization, morphogenesis and fiber tract formation, possibly by conferring preferentially homotypic adhesiveness (or other types of interactions) between the diverse structural elements of the CNS. Cadherin-mediated adhesive specificity may thus provide a molecular code for early embryonic CNS regionalization as well as for the development and maintenance of functional structures in the CNS, from embryonic subdivisions to brain nuclei, cortical layers and neural circuits, down to the level of individual synapses.

Animals↗
PubMed1991

Freeze-fracture morphology of nuclear pockets.

Nuclear pockets (NP) are found in numerous human tumours and in certain non-neoplastic cells. This study concerns the structure of NP in cells from two malignant rhabdoid tumours, one embryonal rhabdomyosarcoma, two centroblastic/centrocytic lymphomas, one centrocytic lymphoma and one Ki-1 lymphoma, as well as in normal neutrophils. Structures were noted in freeze-fracture replicas that were interpreted as corresponding to the NP seen in ultrathin sections and were classified into four types. A lack of nuclear pores was common to all types. In addition, intramembranous particles were either absent or very scanty on both the E- and the P-faces of areas of the nuclear membrane involved in pocket formation. It can be concluded from the lack of nuclear pores that no interchange between the nucleus and cytoplasm takes place in these areas. The reason for the lack of intramembranous particles is not known. It is suggested that the nuclear lamina (intermediate filaments of the nuclear skeleton) is not in contact with the nuclear membrane here.

Adult↗
PubMed1998

Cytologic diagnosis and subtyping of rhabdomyosarcoma.

We reviewed the cytological findings of 38 cases of rhabdomyosarcoma (RMS) with histological confirmation performed during a period of 15 years and proposed a morphological subtyping based on the most prominent cytologic features. Seventeen of these cases were alveolar, 14 cases embryonal, and seven botryoid subtypes. From these cases, a total of 43 samples, of which 37 were fine needle aspiration (FNA) biopsies and six were touch imprints, were evaluated. Detailed cellular features were identified which enabled differentiation into histological subtypes. In the alveolar RMSs, most tumour cells were small and lymphocyte-like, having finely granular chromatin. The finding of cells with more abundant cytoplasm, eccentrically located nuclei and bi/multinucleated tumour cells in a background of mucosubstance helped in the differential diagnosis. Two cell types, including large, tadpole or ribbon-shaped tumour cells and small, round cells with scant cytoplasm, were seen in embryonal RMSs. In botryoid RMSs, a cell type with tightly grouped nuclei within elongated cytoplasm similar to a myotubular structure was observed in addition to the two cell types of embryonal RMSs. We conclude that with experience it will be possible to subtype these tumours by cytologic examination alone.

Adolescent↗
PubMed1999

Developmental expression of protein phosphatase 2A in the kidney.

Although a number of growth and transcription factors are known to regulate renal growth and development, the signal transduction molecules necessary to mediate these developmental signals are relatively unknown. Therefore, the activity and mRNA and protein expression of the signal transduction molecule protein phosphatase 2A (PP2A) were examined during rat kidney development. Northern analysis of total kidney RNA or Western analysis of kidney protein homogenates from embryonic day 15 to 90-d-old adults demonstrated developmental regulation of the catalytic, major 55-kD B regulatory subunit and A structural subunit with the highest levels of expression in late embryonic and newborn kidneys. Similarly, okadaic acid-inhibitable phosphatase enzyme activity was highest in the embryonic and newborn kidney. To map cell-specific expression of PP2A in the developing kidney, in situ hybridization with a catalytic subunit digoxigenin-labeled cRNA was performed on embryonic day 20 and newborn kidneys. PP2A was found predominately in the nephrogenic cortex and particularly in the developing glomeruli and non-brush border tubules in the embryonic day 20 and newborn kidneys. Similarly, immunocytochemistry with a specific PP2A catalytic subunit polyclonal anti-peptide antibody demonstrated catalytic subunit protein particularly concentrated in the podocytes of glomeruli in the newborn kidney. In the adult kidney, PP2A protein was no longer detectable except in the nuclei of distal tubular cells. Therefore, the developmental regulation of PP2A activity and protein during kidney development and its mapping to the nephrogenic cortex, developing glomeruli, and tubules suggests a role for PP2A in the regulation of nephron growth and differentiation.

Aging↗
PubMed1985

Quinolinate. A selective neurotoxin in embryonic and posthatching chicken retinas.

Quinolinate (QUIN), an endogenous dicarboxylic amino acid, structurally related to the putative retinal neurotransmitter aspartate, acts as a specific neurotoxin in the chick neural retina. Qualitative analysis of QUIN's neurotoxic effects reveals that sensitivity to the amino acid is first detected in the 9-day-old embryonic chick retina. Nuclei and cytoplasm of some cells in the inner region of the inner nuclear layer and in the ganglion cell layer appear hypochromatic or electron lucent when examined by light or electron microscopy, respectively. Between day 10 and 12, the sensitivity of the embryonic retina to QUIN increases and remains around the day 12 level throughout the remaining embryonic and initial posthatching period. Cells in the inner half of the inner nuclear layer continue to be the most severely affected throughout retinal development, ganglion cells less so. Photoreceptor and most cells in the outer region of the inner nuclear layer remain undamaged. QUIN effects are partially reversible: retinas exposed to QUIN briefly in vitro and then transferred to fresh QUIN-free medium are not as severely affected as those allowed no recovery time. In day 1 posthatching chick retinas, similar patterns of QUIN-toxicity were observed in vitro (0.5-5 mM QUIN; 5-30 min) and in vivo (200-600 micrograms QUIN/eye; 0.5-24 hr following intravitreal injection).

Animals↗
PubMed1990

Marked regional heterogeneity of 125I-Bolton Hunter substance P binding and substance P-induced activation of phospholipase C in astrocyte cultures from the embryonic or newborn rat.

The specific binding of 125I-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 microM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Chromatin structure and developmental expression of the human alpha-globin cluster.

The human alpha-like globins undergo a switch from the embryonic zeta-chain to the alpha-chain early in human development, at approximately the same time as the beta-like globins switch from the embryonic epsilon-to the fetal gamma-chains. We investigated the chromatin structure of the human alpha-globin gene cluster in fetal and adult erythroid cells. Our results indicate that DNase I-hypersensitive sites exist at the 5' ends of the alpha 1- and alpha 2-globin genes as well as at several other sites in the cluster in all erythroid cells examined. In addition, early and late fetal liver erythroid cells and adult bone marrow cells contain hypersensitive sites at the 5' end of the zeta gene, and in a purified population of 130-day-old fetal erythroid cells, the entire zeta-to alpha-globin region is sensitive to DNase I digestion. The presence of features of active chromatin in the zeta-globin region in fetal liver and adult bone marrow cells led us to investigate the transcription of zeta in these cells. By nuclear runoff transcription studies, we showed that initiated polymerases are present on the zeta-globin gene in these normal erythroid cells. Immunofluorescence with anti-zeta-globin antibodies also showed that late fetal liver cells contain zeta-globin. These findings demonstrate that expression of the embryonic zeta-globin continues at a low level in normal cells beyond the embryonic to fetal globin switch.

Adult↗
PubMed1996

The neostriatal mosaic: basis for the changing distribution of neurokinin-1 receptor immunoreactivity during development.

The pattern of neurokinin-1 receptor-like immunoreactivity (NK-1Rir) was mapped in perinatal and adult mouse striatum by using a new polyclonal antiserum. NK-1Rir was detected in the differentiating regions of the ganglionic eminences on embryonic day 12.5 (E12.5). NK-1Rir structures were enriched in the striatal patch compartment between E16.5 and approximately postnatal day 3 (P3); distributed more uniformly, within portions of both the patch and matrix compartments on P7; and enriched in the matrix compartment in the adult. Analysis of the phenotype of NK-1Rir cells on P2, P7, and in the adult suggested that cholinergic cells accounted for the majority of NK-1Rir cells early postnatally, with increasing contributions from somatostatinergic cells later postnatally. In the adult, approximately half of NK-1Rir cells were cholinergic and half were somatostatinergic. The transient enrichment of NK-1R-bearing cells and processes in the patch compartment which contains cells that express substance P (SP), a putative ligand for the NK-1R, may be a consequence of compartment formation or may be functionally important for compartment development.

Amino Acid Sequence↗
PubMed1978

Significance of maternal uterine proteins in the establishment of pregnancy.

Mammalian embryonic development depends on extrinsic support by the maternal organism. During the preimplantation period the growing blastocyst needs favourable environmental conditions, which are provided by endometrial transformations and secretion. The macromolecular composition of uterine secretion at different times before implantation is characterized by a spectrum of protein patterns which change daily. In an attempt to shed light on the significance of the uterine secretion proteins, particularly uteroglobin in the rabbit, in the establishment of early pregnancy, analyses are reported which demonstrate the origin and endocrine control of the synthesis and release of these proteins. In a comparative study of blastocyst development in vivo and in vitro the significance and consequences of asynchrony between the embryonic and maternal reproductive phases are investigated. Particular attention is drawn to uterine influences on the rabbit blastocyst coverings, and how embryonic development can be retarded by abnormal structural transformation of the zona pellucida and the mucin coat. Finally, the covenient experimental systems of pseudopregnancy and of delayed uterine secretion in the rabbit permit an approach the question of whether the uterine macromolecular components constitute a maternal response to the presence of the as yet unimplanted blastocyst.

Animals↗
PubMed1991

Effects of early partial decapitation on the ontogenic development of chicken lymphoid organs. I. Thymus.

For the present study we used the classic model of early partial decapitation (DCx) of chicken embryos (Fugo, J. Exp. Zool., 85: 271-298, 1940; Betz, Gen. Comp. Endocrinol., 9: 172-186, 1967) in an attempt to analyze the neuroendocrine immune relationships during ontogeny. The elimination of the prosencephalon in chickens at 33-38 hr of incubation induced profound structural, histochemical, and morphometric changes in the embryonic development of the thymus gland. These included a greater development of the cortex than of the medulla, an increased mitotic index, high numbers of pyknotic cells, and enlarged connective tissue trabeculae containing numerous large lymphoid cells; hypertrophied reticular-epithelial cells; delayed appearance of medullary epithelial cysts; and intrathymic granulopoiesis. Furthermore, preliminary radioimmunoassays revealed a sharp increase in the values of circulating thymic hormones, mainly thymosin beta 4 in 17-day-old embryos. The results are discussed with regard to the possible role of prolactin, thyroxine, testosterone, and thymic hormones in the ontogenic development of the chicken thymus.

Adenosine Triphosphatases↗
PubMed2005

Stomodeal and neurohypophysial placodes in Ciona intestinalis: insights into the origin of the pituitary gland.

The ascidian larva has a central nervous system which shares basic characteristics with craniates, such as tripartite organisation and many developmental genes. One difference, at metamorphosis, is that this chordate-like nervous system regresses and the adult's neural complex, composed of the cerebral ganglion and associated neural gland, forms. It is known that neural complex differentiation involves two ectodermal structures, the neurohypophysial duct, derived from the embryonic neural tube, and the stomodeum, i.e. the rudiment of the oral siphon; nevertheless, their precise role remains to be clarified. We have shown that in Ciona intestinalis, the neural complex primordium is the neurohypophysial duct, which in the early larva is a short tube, blind anteriorly, with its lumen in continuity with that of the central nervous system, i.e. the sensory vesicle. The tube grows forwards and fuses with the posterior wall of the stomodeum, a dorsal ectodermal invagination of the larva. The duct then loses posterior communication with the sensory vesicle and begins to grow on the roof of the vesicle itself. The neurohypophysial duct differentiates into the neural gland rudiment; its dorsal wall begins to proliferate neuroblasts, which migrate and converge to build up the cerebral ganglion. The most anterior part of the neural gland organizes into the ciliated duct and funnel, whereas the most posterior part elongates and gives rise to the dorsal strand. The hypothesis that the neurohypophysial duct/stomodeum complex possesses cell populations homologous to the craniate olfactory and adenohypophysial placodes and hypothalamus is discussed.

Animals↗
PubMed2006

Image contrast using the secondary and tertiary eigenvectors in diffusion tensor imaging.

Diffusion tensor imaging (DTI) is a new imaging modality that can provide unique information on brain white matter anatomy. Measurements of water diffusion constant along multiple axes are fitted to a tensor model, from which the diffusion anisotropy and dominant fiber orientation can be estimated. Even though the tensor model is an oversimplification of the underlying neuroanatomy, information within the tensor has not been fully utilized in routine research and clinical studies. In this study we proposed and examined the properties and anatomical contents of several DTI-derived image contrasts that utilize all three eigenvectors. The new contrasts are studied and validated using known anatomical structures in ex vivo mouse brain and embryonic mouse cortex. Application to human white matter is illustrated. Our results suggest that when these contrasts are combined with a priori anatomical knowledge, they reveal neuroanatomical information that is useful for tissue segmentation and diagnosis of white matter lesions.

Animals↗
PubMed1997

Lhx4, a LIM homeobox gene.

LIM homeobox genes are well conserved in evolution and play important roles as transcriptional regulators of embryonic development. Here we report on the structure of LIM domains of the mouse Lhx4 (Gsh4) gene. The cDNA was generated by modified reverse transcription-PCR from midgestation embryo templates, using a degenerate consensus primer. The deduced amino acid sequence of the first LIM domain reveals 77% identity and that of the second domain reveals 86% identity with the corresponding sequences of the closely related Lhx3 gene. In addition, there is 38-56% similarity to other members of the Lhx gene family. The LIM consensus sequence is well conserved in Lhx4.

Amino Acid Sequence↗