Early Stages Of Organogenesis Research Articles

Patterning the zebrafish heart tube: Acquisition of anteroposterior polarity

The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10 −6–10 −5 M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10 −8–10 −7 M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage.

GENETICS OF SEX EXPRESSION IN FRAG ARIA SPECIES

Hermaphroditism is the normal mode of sex expression in diploid species of Fragaria (Rosaceae, 2n = 14, x = 7) with one known exception, gynodioecious F. vesca L. ssp. bracteata. The polyploid species of Fragaria are all trioecious. An extensive study involving appropriate hybridization, testcrossing, selling, and backcrossing revealed that the repression of sporangia and the inhibition of sporogenesis are controlled by a single gene (or a gene complex) with at least three alleles in the sporophytes of trioecious octoploid species (2n = 56, x = 7). A male suppressor (allele F) reduces microsporangia and represses microsporogenesis completely, but it allows normal development of the stigma, style, and ovary. A female suppressor (allele M), in the absence of modifier genes, inhibits megasporogenesis and drastically reduces the number of carpels and size of receptacles. The allele H, conferring perfect flowers at an early stage of flower organogenesis, acts as an inducer of microsporogenesis in females, but leaves both microand megasporangial development intact. At the sex locus, the F allele (femaleness) is dominant to H and M and the H allele (hermaphroditism) is dominant to M (maleness). Females are exclusively heterogametic (F/H or F/M), hermaphrodites may be homo‐ or heterogametic (H/H or H/M), and males are homogametic (M/M). The sex gene is expressed precisely in the genetic background of octoploid × diploid hybrids of Fragaria and their derivatives and in crosses with closely related hermaphroditic diploid Pontentilla glandulosa L. Gene dosage phenomena are absent. First generation progeny of colchi‐decaploids (F/F, –/–) are exclusively female, but all generations thereafter segregate in a normal diploidized manner (1:1). Application of phytohormones alters sex expression to a limited extent.

Vertebrate Craniofacial Development: The Relation between Ontogenetic Process and Morphological Outcome; pp. 190–207

Many structures that are present, often transiently, in the head of extant vertebrate embryos appear to be segmentally organized. These include the brain, particularly the hindbrain (e.g., rhombomeres), and adjacent axial structures such as paraxial mesoderm (e.g., somites, somitomeres) and neural crest cells. Also present in the head are additional sets of serially arranged structures that develop in more ventral and lateral locations. Examples of these are epibranchial placodes, aortic arches, and pharyngeal pouches. All these embryonic structures are frequently used both individually and collectively as characters to assist in defining homologies. New cell labeling and identification methods are providing detailed accounts of cell movements and tissue lineages that reveal a range of disparate behaviors not previously appreciated. The well-known migrations of neural crest cells bring all but the neurogenic members of this mesenchymal population form dorsal, axial locations into ventral and rostral locations where they largely surround the pharynx, stomdeum, and prosencephalon. Equally dramatic movements of neural plate cells, myoblasts, angioblasts, and placode-derived cells have recently been documented. These movements may occur in concert with those of other nearby tissues (e.g., branchiomeric myoblasts, neural crest cells, and surface ectoderm) or may be independent (e.g., placodal neuroblasts). Migrating cells may be clustered and follow definable pathways towards their destination (e.g., neural crest cells), or they may be solitary and wander invasively without a prespecified destination (e.g., angioblasts). These extensive morphogenetic movements bring cells into contact with a greater variety of other tissues and matrix environments than has heretofore been recognized. Moreover, because of these rearrangements, the cells present in a particular location, such as a branchial arch, may trace their ancestry to many axial levels, which complicates the analyses of segmental relations. Comparative morphological studies of craniofacial development have recently been augmented by descriptions of the sites and times of expression of many matrix components, growth factors and their receptors, and regulatory genes. Particularly important has been the discovery of a network of genes called the homeobox family. These genes are similar in their sequence and their organization along a chromosome to genes that establish the spatial identity of prospective body parts in drosophila. The combination of cellular and molecular descriptive studies of vertebrate craniofacial development provide exciting opportunities to catalogue patterns of gene expression and morphogenesis during the gastrula, neurula, and early organogenesis stages. Moreover, such data form the basis for proposing and then testing hypotheses about the mechanisms controlling cell movements, tissue formation, and the assembly of functionally integrated sets of structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Masculinizing effect of testes on developing rat ovaries in organ culture

Masculinizing effect of testes on developing rat ovaries was shown in vitro by culturing testes from 17.5-day-old fetuses in contact with female genital tracts from 14.5-day-old rat fetuses. The testes induced the differentiation of epithelial cells staining for cytokeratin in the ovarian blastema. These cells formed seminiferous cord-like structures delineated by a basement membrane, in a way that resembles early stages of testicular organogenesis. In addition to the morphological masculinization, functional masculinization was obtained since the ovaries produced the anti-Müllerian hormone as shown by bioassay and immunohistochemical procedures. Across a distance, testes from 17.5-day-old fetuses failed to induce masculinization. These results suggest that testes from 17.5-day-old fetuses produce a locally diffusible factor interfering with the development and the differentiation of the fetal ovaries. The possibility that the anti-Müllerian hormone secreted by the testes may be the factor involved is discussed comparing these results with those obtained with testes from different stages and with bibliographic data.

Cellular Pattern of Insulin-Like Growth Factor-I (IGF-I) and Type I IGF Receptor Gene Expression in Early Organogenesis: Comparison with IGF-II Gene Expression

To investigate the potential role(s) of the insulin-like growth factors (IGFs) in embryogenesis, we have used in situ hybridization histochemistry to localize mRNAs for IGF-I, IGF-II, and the type I IGF receptor during an early period in rat embryonic development (embryonic days 14 and 15). IGF-I and IGF-II mRNAs were found in distinctly different patterns of cellular distribution. IGF-I mRNA was particularly abundant in undifferentiated mesenchymal tissue in the vicinity of sprouting nerves and spinal ganglia, and in circumscribed regions of the developing face that corresponded to the target zones of the trigeminal nerve. IGF-I mRNA was also found in aggregations of mesenchyme surrounding, but not in developing muscle and cartilage. IGF-I mRNA was selectively concentrated in areas of active tissue remodeling, such as the cardiac outflow tract, and was undetectable in liver, pituitary, and nervous system at this early stage of organogenesis. IGF-II mRNA was abundant in developing muscle, cartilage, and vascular tissue, and in the embryonic liver and pituitary. IGF-II mRNA was also conspicuous in areas of vascular interface with the brain, such as the choroid plexus and the organum vasculosum of the lamina terminalis. Messenger RNA for the type I IGF receptor was widely distributed in embryonic tissues, but the highest level were seen in the ventral floorplate of the hindbrain, where specialized neuroepithelial cells act as guides for axonal targeting. In conclusion, the different cellular patterns of expression of genes for IGF-I and IGF-II indicate that these two IGFs are differently regulated and, thus, may have significantly different roles in the process of embryonic development. Furthermore, the early and widespread expression of the type-I IGF receptor gene, in contrast to the relatively limited and localized pattern of IGF-I gene expression, is consistent with the view that this receptor may mediate the effects of IGF-II as well as IGF-I during embryogenesis.

Effect of "in utero" exposure to low doses of low energy X-rays on the postnatal development of mouse.

Pregnant Swiss albino mice were exposed to a single dose of 9 mGy or 50 mGy of 70 kVp X-rays on day 3.5 (preimplantation), 6.5 (early organogenesis) or 11.5 (late organogenesis) of gestation. The offspring were observed for any radiation induced changes in litter size at birth and sex ratio at 4 wks of age, and postnatal mortality and growth retardation up to 6 wks of age. Irradiation at the early organogenesis stage produced an increase in the postnatal death, statistically significant in the 50 mGy group. A significant increase in the number of retarded offspring and decrease in body weight were observed in the groups exposed at the early and late organogenesis periods. The results indicate that a single exposure to low energy low dose X-rays can be harmful to the growing embryos and the type and extent of the injury will depend on the stage of development as well as the dose of radiation.

Gamma-irradiation of plant material in floriculture—Results and problems

γ-Irradiation of gladiolus corms before warm storage in a three-year-experiment resulted in an average yield increase of flower stems of 17.2% (0.75 Gy). An essential for the yield improvement in the early culture of gladiolus in a glass-house environment is considered the high shoot formation capacity of the gladiolus variety used, ”Frührot“, high quality of the plant material (corms), the irradiation at an early stage of organogenesis, marked suboptimum conditions for growth and high planting thickness.

Behavioral changes and structural defects in rats irradiated in utero

Pregnant rats were irradiated with whole-body doses (0.25–1.25 Gy) of Cs-137 γ-rays on gestational day 15, or with 1.0 Gy on gestational days 11, 13,15, or 17. Postnatal growth (body weight) and several preweaning behaviors (righting reflex, negative geotaxis, reflex suspension, modified open field activity, spatial maze exploration, continuous corridor activity, and gait) of the offspring were monitored prior to sacrifice on post-parturition day 28. Brain (sensorimotor cortex) and pituitary tissues were processed for histological evaluation and morphometric analysis. For most behavioral endpoints, there were dose-dependent changes produced by irradiation on gestational day 15, with one endpoint (continuous corridor activity) demonstrating changes after 0.25 Gy that were significantly different from control values. The data indicate that the most sensitive organ showing radiation-induced alterations changes from the pituitary at gestational day 11 to the primitive cortex of the brain at days 13 to 17 with a peak of sensitivity at day 15. These results demonstrate that a spectrum of related functional and morphological deficits can be produced by even low-dose in utero irradiation, with the specific endpoint showing the greatest change being determined by the specific day of gestation on which irradiation occurs. Extrapolating from these experimental data with rats to the human situation, it is recommended that care be taken, when possible, to avoid exposure of the fetus, even after the early stages of organogenesis.

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds.

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Contrasting effects of estradiol-17β and 17α-ethinyl estradiol-17β on cultured whole embryos

Estradiol-17β (E 2) and 17α-ethinyl estradiol-17β (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E 2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E 2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E 2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E 2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransfonnation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.

Postimplantation development of mitomycin C-treated mouse blastocysts.

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.

Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis

Abstract We have established the time and position of expression for multiple enamel proteins during the development of the mouse molar tooth organ. Using high-resolution two-dimensional gel electrophoresis coupled with immunoblotting and immunocytochemistry, a 46-kDa enamel protein (pI, 5.5) was detected during late cap stage (18-days gestation, E18d) within differentiation-zone-II inner enamel epithelia associated with an intact basal lamina. At E19d a second enamel polypeptide of 72 kDa (pI, 5.8) was identified at the time and position of initial biomineralization in differentiation zone V. At 20 days, differentiation-zone-VI ameloblasts without basal lamina (late bell stage) expressed 46- and 72-kDa enamel proteins and, in addition, expressed a relatively more basic 26-kDa enamel protein (pI, 6.5–6.7); detected after initial formation of calcium hydroxyapatite crystals. Antibodies raised against chemically synthesized enamel peptides cross-reacted with both the 72-kDa and 26-kDa polypeptides, but did not cross-react with the 46-kDa enamel polypeptide. The sequential expression of multiple enamel proteins suggests several functions: (a) the anionic enamel proteins may provide an instructive template for calcium hydroxyapatite crystal formation; (b) the more neutral proteins possibly serve to regulate size, shape and rates of enamel crystal formation. We suggest that initial expression of enamel gene products during mouse tooth development possibly recapitulates ancestral features of amelogenesis documented in prereptilian vertebrates. These results imply that multiple instructive signals may be responsible for mammalian enamel protein induction and that the sequential expression of a family of enamel proteins reflects the evolutionary acquisition of a more complex genetic program for amelogenesis.

Post-implantation embryo culture: validation with selected compounds for teratogenicity testing.

1. Some chemical compounds selected by experts for the validation of in vitro teratogenicity testing were investigated in whole rat embryos cultured during the early stages of organogenesis. All sixteen known in vivo teratogens tested also induced specific malformations in embryos grown in culture. 2. Of the nine compounds which were negative in in vivo rat teratogenicity studies, none provoked dysmorphogenic effects in cultured embryos. Abnormal development of the embryos was only observed with these compounds at concentrations also high enough to affect significantly overall growth and/or differentiation. 3. The results showed a high predictability of this system for the compounds tested and suggest that the post-implantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Developmental toxicity of four model alkylating agents on japanese medaka fish (Oryzias latipes) embryos

As public and scientific interest increases in identifying in vitro alternatives to whole-animal testing, there is a need to characterize the responses of in vitro systems to defined chemical agents. In this paper we have utilized the medaka (Oryzias latipes) embryo culture system to examine the developmental toxicity of a series of chemically defined model alkylating agents. The medaka embryo culture system was used because it offers numerous advantages over other in vitro embryo systems. This study has examined the developmental toxicity of four model alkylating agents: methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). These alkylating agents represent chemical models for compounds that are environmental and occupational pollutants. The embryotoxic parameters that were monitored include embryolethality and specific malformations. Medaka embryos in early organogenesis (stage 20) were exposed for 2 h to the test compound at concentrations ranging from 0.75 to 120 mM. The embryos were cultured at 20°C in Petri dishes and were assessed daily for viability and gross malformations until hatching. All agents tested produced concentration-dependent decreases in viability and increases in malformations. The developmental toxicity of the four alkylating agents examined is, in order of decreasing potency, MNU > ENU > MMS > EMS. A similar spectrum of malformations was produced following exposure to each alkylating agent; however, agents undergoing first-order nucleophilic substitution reactions were more potent developmental toxins than those agents which undergo second-order nucleophilic substitution reactions. Observed abnormalities included flexure, cardiac, optic, and cephalic malformations.

Embryotoxic action of beta-aminopropionitrile during early organogenesis in rats.

The lathyrogen β-aminopropionitrile (β-APN) has been shown to be teratogenic in rats after administration to the dam at the late organogenesis stage of gestation, commonly inducing cleft palate and a variety of skeletal malformations in fetuses (Wilk et al. 1972; Barrow and Steffek 1974). Unusually among teratogens, however, β-APN has been reported not to affect embryos during the early organogenesis stage of development, when sensitivity to teratogenic insult is often maximal (Barrow and Steffek 1974). This observation is in contrast to that made in the hamster, in which administration of β-APN to the dam during early organogenesis resulted in the induction of neural tube defects (Wiley and Joneja 1976). Furthermore, it has been demonstrated (Steele and Marlow 1985; Simpson et al. 1986) that the cultured early organogenesis stage rat embryo is susceptible to dysmorphogenesis when β-APN is present in the culture medium.

Effects of momorcharins on the mouse embryo at the early organogenesis stage

Previous studies showed that the two forms of momorcharins which are isolated from seeds of Momordica charantia L. are effective in inducing early and midterm abortions in the mouse. Momorcharins were found to be teratogenic to the cultured mouse embryos at the early organogenesis stage. Morphological abnormalities were seen in the head, trunk and limbs. Ultrastructural studies on the visceral yolk sac of the momorcharin-treated embryos showed that the endodermal layer was deranged, membrane invaginations at the apical surface were decreased and intercellular space became distended. It is likely that the teratogenic action of momorcharins on mouse embryos in vitro is mediated through the deleterious effects on the visceral yolk sac, which functions as a vital transport organ for the conceptus at the immediate post-implantation period.

Gonadal development and fertility of mice treated prenatally with cadmium during the early organogenesis stages.

Gonadal development was studied in mouse embryos that were exposed to cadmium during the early organogenesis stages. At 13.5 days, both the male and the female embryos had small genital ridges. Fewer primordial germ cells were found in the male embryos. In both sexes, many primordial germ cells were left outside the genital ridges, presumably as a result of retarded cell migration. In 16.5-day embryos, the size of the testes and ovaries and the number of differentiating germ cells were reduced. Many germ cells degenerated during the differentiation to spermatogonia and meiotic oocytes. The perturbed gonadal development was less likely to be caused primarily by a defective hypothalamopituitary axis but was more a part of the general cadmium-induced damage. The fertility of the male offspring was impaired by the prenatal cadmium insult, but the females were apparently fertile. The epididymal spermatozoa of the cadmium-affected offspring showed a lower fertilizing capacity in vitro. The impaired fertility of the cadmium-affected mice was the result of poor gonadal growth, paucity of germ cells, and defective maturation of the gametes.

Modification and standardization of the culture of early postimplantation embryos for toxicological studies

The method of culturing "whole" rat embryos (days 9.5-11.5 of gestation, i.e. at the early stage of organogenesis) as modified and standardized in our laboratory is presented; We have succeeded in using bovine serum as culture medium instead of rat serum as recommended in the original procedure. Experimental conditions are described for obtaining reproducible results; An improved scoring system was developed which, in connection with a computerized documentation, greatly facilitates the evaluation of the data.

Cellular necrosis in zinc-deficient rat embryos.

Abnormal cellular necrosis was studied in 9.5-11.5-day embryos obtained from zinc-deficient rats. At periods of low maternal zinc status induced by a high intake of a zinc-deficient diet, cell death was observed in those regions of the embryo that were most sensitive to teratogenic insult at that time. As the maternal serum zinc level increased during the fasting phase of the feeding cycle, the degree of necrosis decreased, leaving the tissue histologically more normal even though the embryos were grossly malformed. The mitotic index of cells in the neural epithelium and limb buds of zinc-deficient, non-necrotic embryos was found to be elevated, but there was no evidence of blockage at any particular stage of mitosis. It can be hypothesised that during the early stages of organogenesis, periods of low maternal zinc status initiate unscheduled cell death by some as yet undefined mechanism that in turn, gives rise to the morphological anomalies observed later.

Glucocorticoid teratogenesis in mouse whole embryo culture.

Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.