Fate of haploid parthenogenetic cells in mouse chimeras during development

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.     

Tissue specific loss of proliferative capacity of parthenogenetic cells in fetal mouse chimeras

Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.     

Systematic elimination of parthenogenetic cells in mouse chimeras

The developmental potential of primitive ectoderm cells lacking paternal chromosomes was investigated by examining the distribution of parthenogenetic cells in chimeras. Using GPI-1 allozymes as marker, parthenogenetic cells were detected in most organs and tissues in adult chimeras. However, these cells were under severe selective pressure compared with cells from normal fertilized embryos. In the majority of chimeras, parthenogenetic cells in individual animals were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. Nevertheless, parthenogenetic cells were detected more consistently in some organs, especially the brain, heart, kidney and spleen. In contrast, there was apparently a systematic selection against parthenogenetic cells in some tissues, most notably in skeletal muscle, liver and pancreas. These results suggest that paternally derived genes are probably required not only for the development of extraembryonic structures but also for subsequent development of embryonic tissues derived from the primitive ectoderm lineage.     

Postnatal development of parthenogenetic in equilibrium with fertilized mouse aggregation chimeras

Chimeras were made from parthenogenetic and fertilized cleavage-stage mouse embryos. The perinatal mortality was high. The parthenogenetic contributions to different tissues at birth ranged from 0 to 50%. No selection of parthenogenetic cells was observed in the pigmentation of the coat, but this does not exclude that such selection could act in other tissues. The weight of chimeras at birth negatively correlated to the average contribution of the parthenogenetic part. The growth rate of chimeras was lower than that of nonchimeric animals. The data presented demonstrated that, although parthenogenetic cells are not cell lethals and they can participate to some degree in normal development of most tissues, their extensive presence reduces the viability of chimeras and retards the postnatal development.     

Distribution of androgenetic cells in fetal mouse chimeras

To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.     

Proliferation and differentiation of androgenetic cells in fetal mouse chimeras

The properties of androgenetic cells and their ability to proliferate and differentiate were examined in post-midgestation chimeras. In several tissues, namely the brain, cardiac muscle, skeletal muscle and intestinal epithelium, the rate of proliferation of androgenetic cells was higher than that of normal cells in day 13 embryos. This higher rate of proliferation was however less pronounced by day 17–18 of development. It is possible that IGF2, a major growth factor regulating fetal growth, could play a role in the increased proliferation of androgenetic cells. Igf2 is also an imprinted gene that is expressed only when inherited paternally. Indeed, in the smooth muscle, cartilage and intestinal epithelium, patches of androgenetic (ag) cells exhibited higher levels of IGF2 mRNA than neighbouring wild-type cells. Surprisingly, we also detected expression of Igf2 in ag cells of ectodermal origin; this gene is not normally expressed in this lineage. This expression was observed in the brain, epidermis and in the epithelium of the tongue. We attempted to confirm the identity and differentiation status of ag cells with the help of cell-type specific antibodies and lectins. Evidence for only one of the cell types analysed, i.e. the goblet cells of the gut, suggests a delay or aberrant differentiation of ag cells.     

Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices

Plasmodesmata provide symplastic continuity linking individual plant cells. However, specialized cells may be isolated, either by the absence of plasmodesmata or by down regulation of the cytoplasmic flux through these channels, resulting in the formation of symplastic domains. Maintenance of these domains may be essential for the co-ordination of growth and development. While cells in the center of the meristem divide slowly and remain undifferentiated, cells on the meristem periphery divide more frequently and respond to signals determining organ fate. Such symplastic domains were visualized within shoot apices of Arabidopsis, by monitoring fluorescent symplastic tracers (HPTS: 8-hydroxypyrene 1,3,6 trisulfonic acid and CF: carboxy fluorescein). Tracers were loaded through cut leaves and distributed throughout the whole plant. Confocal laser scanning microscopy on living Arabidopsis plants indicates that HPTS moves via the vascular tissue from leaves to the apex where the tracer exits the phloem and moves symplastically into surrounding cells. The distribution of HPTS was monitored in vegetative apices, and just prior to, during, and after the switch to production of flowers. The apices of vegetative plants loaded with HPTS had detectable amounts of tracer in the tunica layer of the meristem and in very young primordia, whereas the corpus of the meristem excluded tracer uptake. Fluorescence signal intensity decreased prior to the onset of flowering. Moreover, at approximately the time the plants were committed to flowering, HPTS was undetectable in the inflorescence meristem or young primordia. Later in development, after several secondary inflorescences and mature siliques appeared, inflorescence apices again showed tracer loading at levels comparable to that of vegetative apices. Thus, analysis of fluorescent tracer movement via plasmodesmata reveals there is distinct temporal and spatial regulation of symplastic domains at the apex, dependent on the developmental stage of the plant.     

Temporal and spatial controls of Aspergillus development.

The mechanisms regulating elaboration of the multicellular asexual reproductive apparatus, the conidiophore, of the filamentous ascomycete Aspergillus nidulans provide a model for the control of fungal development. Recent advances have been made on three fronts. First, new physical and chemical signals have been discovered that control commitment of cells to the conidiation pathway. Second, positively acting developmental regulatory genes have been cloned and characterized. In addition, evidence has been obtained for the existence of negatively acting regulatory loci. Finally, an anonymous developmentally regulated gene has been used to make a directed mutation that has revealed the morphogenetic function of the gene.     

Temporal and spatial expression of TGF-beta2 in chicken somites during early embryonic development

A multifunctional growth and differentiation factor TGF-beta is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF-beta2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF-betas were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole-mount in situ hybridization. TGF-beta2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF-beta2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29-31 (6-7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF-beta2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF-beta2 were clearly observed in the myotomal somite tips as well as the neural tube. RT-PCR analysis showed that TGF-beta2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF-beta2 may be a regulatory factor participating in the somitogenesis of chicken embryos.     

Temporal and spatial expression patterns of Cdc25 phosphatase isoforms during early Xenopus development

In early animal development, cell proliferation and differentiation are tightly linked and coordinated. It is important, therefore, to know how the cell cycle is controlled during early development. Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell-cycle progression. In Xenopus laevis, three isoforms of cdc25 have been identified, viz. cdc25A, cdc25B and cdc25C. In this study, we isolated a cDNA encoding a novel Xenopus Cdc25 phosphatase (named cdc25D). We investigated the temporal and spatial expression patterns of the four cdc25 isoforms during early Xenopus development, using RT-PCR and whole-mount in situ hybridization. cdc25A and cdc25C were expressed both maternally and zygotically, whereas cdc25B and cdc25D were expressed zygotically. Both cdc25A and cdc25C were expressed mainly in prospective neural regions, whereas cdc25B was expressed preferentially in the central nervous system (CNS), such as the spinal cord and the brain. Interestingly, cdc25D was expressed in the epidermal ectoderm of the late-neurula embryo, and in the liver diverticulum endoderm of the mid-tailbud embryo. In agreement with the spatial expression patterns in whole embryos, inhibition of bone morphoge- netic protein (BMP), a crucial step for neural induction, induced an upregulation of cdc25B, but a downregulation of cdc25D in animal cap assays.These results indicate that different cdc25 isoforms are differently expressed and play different roles during early Xenopus development.     

Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.     

Metabolism and cell allocation during parthenogenetic preimplantation mouse development

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed. © 1996 Wiley-Liss, Inc.     

Temporal and spatial expression of neurotrophins and their receptors during male germ cell development.

Spermatogenesis is a stepwise cellular differentiation process involving proliferation and commitment to differentiate in spermatogonia, meiosis in spermatocytes, and morphological changes in round spermatids. The whole process is regulated by intercellular communication between the germ cells and the supporting cells. In order to investigate whether neurotrophin family and their receptors contribute to the intercellular communication, we examined the expression of neurotrophins and their receptors in testis during spermatogenesis. One of neurotrophin family, NT-3 was expressed in spermatocytes and spermatogonia while its high affinity receptor, TrkC was found mainly in late spermatids and their low affinity receptor, TrkA in spermatocytes and round spermatids. On the other hand, BDNF immunoreactivity was found in Sertoli cells while its high affinity receptor, TrkB was found in spermatogonia. The temporally and spatially regulated expression of neurotrophins, NT-3 and BDNF, and their receptors, TrkC and TrkB, during male germ cell development suggests that neurotrophins play as the paracrine factors in the intercellular communication between the germ cells and the supporting somatic cells to control germ cell development.     

Temporal and spatial regulation of expression of two galectins during kidney development of the chicken

Organogenesis and the establishment of the mature phenotype require an interplay between diverse recognition systems. Concerning protein–carbohydrate interactions, galectins are known to be involved in several extra- and intracellular functions. Due to the occurrence of two avian galectins in liver (chicken galectin-16; CG-16) and intestine (chicken galectin-14; CG-14) with different developmental regulation, the questions addressed are to what extent and where these galectins are present during chicken kidney development. Using Western blot analysis, the presence of both activities in tissue extracts was ascertained. A solid-phase assay showed peak levels at day 12 followed by a decline. A histochemical analysis was carried out in combination with routine staining. Epithelial cells of the mesonephric proximal tubules were immunoreactive in the cytoplasm for CG-14 from day 5 of incubation onwards. Additionally, epithelial cells of the metanephric collecting ducts were stained. For CG-16 a rather similar pattern of staining was seen, additional positivity in early glomerular podocytes being notable. At the electron microscopical level, a diffuse staining for CG-14 was seen in the cytoplasm, whereas immunoreactivity for CG-16 was observed mainly in mitochondria. These results demonstrate quantitative differences in the developmental regulation of the two avian galectins with obvious similarities in the cell-type pattern but with a disparate intracellular localisation profile.     

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.