Cellular changes during placental and fetal growth.
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Animals↗
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Residual proliferative capacity in F9 teratocarcinoma stem cell cultures treated with alpha-difluoromethylornithine, an inducer of parietal endoderm differentiation.
We have previously shown that inhibition of polyamine biosynthesis by treatment with 5 mM alpha-difluoromethylornithine (DFMO) causes growth arrest and induces differentiation of F9 teratocarcinoma stem cells. The resulting phenotype is similar to that of parietal endoderm, and these differentiated cells possess no apparent proliferative capacity. In the present study, however, it is demonstrated that some of the DFMO-treated cells are not terminally differentiated. Upon a change to DFMO-free growth medium these cells eventually start to proliferate. Using a colony forming efficiency assay, it is estimated that less than 1 out of 200,000 cells retains its proliferative capacity after 6-10 days of DFMO treatment. These cells exhibit no apparent resistance to DFMO, and their population doubling time is similar to that of untreated control F9 cells. Consequently, the possible existence of a small, quiescent, cell population possessing proliferative potential must be taken into account when designing therapeutic protocols for DFMO.
Cell Differentiation↗
New functions for gap junctions.
The most significant finding of the past year in gap junction research has been the association of connexin defects with human diseases. Connexin32 mutations cause X-linked Charcot-Marie-Tooth disease, a demyelinating peripheral neuropathy. Mutations in connexin43 may underlie cardiac malformations in visceroatrial heterotaxia syndromes. Genetic approaches and gene targeting have provided new insights, but also raise new questions concerning connexin function, the significance of connexin diversity and the regulation of intercellular communication.
Animals↗
Esophageal/pyloric ligation enhances development of the murine fetal stomach in organ culture.
PURPOSE: The authors hypothesized that increased intraluminal pressure in the fetal stomach would enhance development in a murine organ culture model. METHODS: Gestation day 14 (Gd14) fetal stomachs from time-dated pregnant CD-1 mice (term, 20 days) were maintained in organ culture for 7 days. Some stomachs were ligated at the gastroesophageal (GE) and pyloroduodenal (PD) junctions. Others were left unligated. Gd14, Gd16, and Gd18 stomachs were taken as well to compare organogenesis in vivo. Tissues were processed for histological, morphometric, and immunohistochemical analysis, as well as total protein and DNA determination. RESULTS: The ligated stomachs were visibly distended compared with unligated stomachs in organ culture after 7 days. The length and width of the 7-day in vitro ligated stomachs were significantly increased compared with unligated (2.97+/-0.04 mm v 2.48+/-0.05 mm and 2.14+/-0.04 mm v 1.57+/-0.08 mm, respectively, P < .05). Mucosal epithelial cells showed nuclear polarization, and there was a distinct outer muscle layer in the ligated stomachs, but not in the unligated stomachs, which demonstrated pseudostratified epithelial cells in the mucosa. The ligated stomachs had increased in mucosal thickness compared with unligated (31.4+/-1.3 microm vs 24.9+/-0.9 microm, p < 0.05). The ligated stomachs also had significantly increased protein and DNA content when compared with unligated stomachs (65.8+/-3.1 microg and 23.3+/-1.2 microg v 55.0+/-2.7 microg and 19.0+/-1.2 microg, respectively, P < .05). However, there were no significant differences noted between the protein to DNA ratios. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, demonstrated increased proliferative activity of the mucosal epithelial cells in the ligated stomachs. CONCLUSIONS: Esophageal and pyloric ligation enhanced the development of the fetal stomach in vitro in comparison with unligated stomachs cultured under similar conditions. Developmental characteristics of the ligated stomachs paralleled that of Gd16 stomachs in vivo.
Animals↗
Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva.
To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.
Aging↗
Cellular growth in intrauterine malnutrition.
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Animals↗
Changing genetic world of IVF, stem cells and PGD. B. Polarities and gene expression in differentiating embryo cells and stem cells.
Novel genetic techniques in the later twentieth century led to new analytical methods for assessing the growth of embryos and stem cells and improve preimplantation diagnosis. Increasing attention to the nature of polarities in mouse and human embryos revealed the existence of an animal-vegetal axis in human oocytes and embryos. Combinations of meridional and transverse cleavage divisions, the latter due to spindle rotation, determined the unequal division of ooplasm to embryonic blastomeres. Blastomeres with differing functions were accordingly formed in 4-cell embryos, including founders of inner cell mass and trophectoderm. New forms of gene analysis led to the polymerase chain reaction, while fluorescence in-situ hybridization revealed astonishingly high degrees of heteroploidy in human embryos. Developmental genetics gained immense analytical power as cDNA libraries, microarrays, transcriptomes RNAi and other methods clarified the roles of hundreds of genes in pre- and early post-implantation embryos and stem cells.
Animals↗
Pathologic classification of rhabdomyosarcomas and correlations with molecular studies.
Rhabdomyosarcoma, the most common soft-tissue malignancy of childhood and adolescence, comprises a group of differing pathobiologic entities linked by their common propensity for formation of neoplastic skeletal muscle, a feature that results from biological forces related to aberrant transcription signals and the resultant production of myogenic proteins. At a molecular level, however, rhabdomyosarcomas form a heterogeneous group that can be subdivided into distinct clinicopathologic entities based on morphologic appearance and genetic makeup. These differing morphologic features were recognized in the mid-1900s by Horn and Enterline with their division of rhabdomyosarcomas into embryonal, alveolar, botryoid, and pleomorphic subtypes. More recent histologic and biologic studies have resulted in description of additional entities, such as spindle cell and anaplastic rhabdomyosarcoma, and refinements in recognition of the original entities, such as solid-alveolar rhabdomyosarcoma. Familiarity with newer classifications and their relationship to molecular aberrations is key to stratifying patients on current therapeutic protocols and proposed innovative genetic therapies.
Adolescent↗
p53 in human embryonal carcinoma: identification of a transferable, transcriptional repression domain in the N-terminal region of p53.
Testicular germ cell tumors (TGCTs) arise despite possessing high levels of wild-type p53, suggesting p53 latency. We have previously shown that p53 repression in TGCT-derived human embryonal carcinoma (EC) is relieved upon treatment with all-trans retinoic acid (RA), resulting in enhanced p53 transactivation activity. To further investigate p53 repression in EC, a series of gal4-p53 truncation constructs were generated. Deletion of the core DNA-binding region, residues 117-274, had no effect on basal or RA-induced p53 activity. Progressively, larger truncations were made in the C- or N-terminal direction. Deletion of residues toward the C-terminus of p53 as far as residue 354 did not affect either the basal or RA-inducible activity of gal4-p53. When a small region in the N-terminus was deleted (residues 105-116), relief of the basal repression of p53 activity characteristic of EC was observed. Fusion of this region to the VP16 activation domain (VPAD) resulted in a 10-20-fold repression of VPAD activity in NT2/D1 human EC cells, indicating that this region acts as a heterologous repressor. Owing to its location in the N-terminal half of p53, we have named this region the p53 N-terminal Repression Domain (p53-NRD). The p53-NRD mediated repression in a variety of cell lines, with the most prominent repression observed in human EC cells. While RA alone had no effect on p53-NRD activity, cotreatment with RA and the histone deacetylase inhibitor trichostatin-A (TSA) completely relieved p53-NRD-mediated repression. In contrast, NRD-mediated repression was not sensitive to RA and TSA in a derived RA-resistant cell line with a retinoic acid receptor gamma (RARgamma) defect, but sensitivity could be restored with transfection of RARgamma. These data indicate that a unique repressor domain resides in p53 at residues 90-116 whose activity can be modulated in the presence of 'differentiation therapy' and 'transcription therapy' agents.
Amino Acid Sequence↗
The molecular control of renal branching morphogenesis: current knowledge and emerging insights.
Mammalian kidney development requires the formation of a patterned, branched network of collecting ducts, a process termed renal branching morphogenesis. Disruption of renal branching morphogenesis during human kidney development results in renal dysplasia, the major cause of renal failure in young children. Genetic evidence, combined with in vitro data, have implicated transcription factors, secreted growth factors, and cell surface signaling peptides as critical regulators of renal branching morphogenesis. This review discusses the current knowledge regarding the regulation of renal branching morphogenesis in vivo provided by the analysis of genetic mutations in mice and humans which disrupt collecting duct system development. In addition, in vivo and in vitro evidence regarding the functions of several other gene families are considered, rendering new insight into emerging regulatory roles for these molecules in renal branching morphogenesis.
Animals↗
Cisplatin-induced in vivo differentiation of human embryonal carcinoma.
OBJECTIVE: To investigate the differentiation of embryonal carcinoma (EC) by cisplatin, and the underlying mechanism, as untreated metastases of nonseminomatous germ cell tumours rarely consist of fully differentiated mature somatic tissues, but such mature metastases are more common after various treatments, including chemotherapy. MATERIALS AND METHODS: The TTSC-3 human testicular EC line heterotransplanted into nude mice was used as a target. After treating tumour-bearing mice with intraperitoneal injections of varying doses of cisplatin, the histopathology of the tumours was assessed and various gene expressions in the tumours determined by cDNA-array technology. RESULTS: When cisplatin at 1 mg/kg/week was injected intraperitoneally into TTSC-3-bearing mice, there was no effect on tumour growth. However, injecting cisplatin at 5 mg/kg/week induced a marked regression of the tumour. In contrast, cisplatin at 3 mg/kg/week had a modest inhibitory effect on tumour growth and induced tumour dormancy. Histological examination showed that 5 weeks after injecting cisplatin (3 mg/kg/week), primitive mesenchymal-like cells increased, and 10 weeks afterward cartilage and well-developed glands (teratoma) were apparent; at > 15 weeks afterward there were no EC cells visible. cDNA probes from reverse-transcribed mRNAs of TTSC-3 treated with cisplatin or saline for 10 weeks were compared to identify genes differentially expressed in cisplatin-treated TTSC-3. Of 1176 different human cDNA transcripts in cisplatin-treated TTSC-3, three genes (tumour necrosis factor receptor 1, caspase 8 and Apaf1), which are associated with apoptosis, were expressed markedly more than after saline injection. CONCLUSIONS: The intermediate dose of cisplatin inhibited tumour growth of EC by inducing differentiation and enhancing apoptosis-related gene expression. These findings suggest that cisplatin may play a significant role in the differentiation of EC in vivo.
Animals↗
Obstruction of ductus venosus stimulates cell proliferation in organs of fetal sheep.
In growth restricted fetuses, hepatic blood flow is reduced. This suggests the hypothesis that liver blood flow controls fetal growth. In 11 near term sheep the ductus venosus was blocked with an embolization coil in one fetus (experimental) and left patent in the twin (control). Arterial catheters were placed in both fetuses. After termination [mean (s.d.) 5 days (2) after surgery] the fetal body and organs were weighed. The cell proliferation rate (pKi-67) was determined in tissue samples of the liver, heart, skeletal muscle, kidneys and placenta (n=6). Blood flow through the umbilical vein measured by Doppler ultrasound did not differ in control and experimental fetuses [experimental: 600 (101) ml/min; control: 626 (89) ml/min]. In experimental fetuses, blood flow through the ductus venosus was negligible (colour Doppler), and thus hepatic blood flow was increased. Absolute and relative (percentage of body weight) liver weights were increased in experimental fetuses [liver weight: 119 (34) g versus 84 (17) g; relative liver weight: 4.3 (0.8) per cent versus 3.4 (0.8) per cent;P=0.002, n=11]. The cell proliferation rate was increased significantly (twofold) in heart muscle, skeletal muscle and kidneys, and sixfold in liver. It is concluded that increases of hepatic blood flow stimulate cell proliferation in major organs of the ovine fetus.
Animals↗
Liver blood perfusion as a possible instrument for fetal growth regulation.
Placental and fetal liver blood perfusions are reduced in intrauterine growth-restricted human fetuses. We hypothesized that changes in fetal liver blood supply can alter fetal growth. In nine ewes with twin pregnancies at a gestational age of 119+/-2 days, a stent (4 mm) was placed into the ductus venosus of one twin (DV(stent) group). Alternatively, in 17 near term sheep with twin (n=11) or singleton (n=6) pregnancies, a DV was blocked with an embolization coil (DV(coil) group) for about one week. The cell proliferation rate (pKi-67) was determined in the liver, heart, skeletal muscle, kidneys and placenta. The dilatation or occlusion of the DV did not change placental perfusion on the first day or later after surgery. The liver blood supply was decreased in the DV(stent) group by more than half from 499+/-371 to 278+/-219 ml min(-1) (mean+/-s.d., n=4), and increased two-fold in the DV(coil) group (P< 0.05). The percentage of liver/body weight was decreased from 3.9+/-0.6 per cent in control twin to 3.0+/-0.2 per cent (n=3) in the DV(stent) group. Occlusion of the DV lead to an increase in the percentage of liver/body weight from 3.4+/-0.8 per cent to 4.3+/-0.8 per cent (n=11, P< 0.05). Reduced liver blood supply in the DV(stent) group was associated with a decrease of cell proliferation in the liver from 12.43+/-2.31 to 6.5+/-0.62 (nuclei microm(2) 10(-4), n=3, P=0.058), in heart from 1.14+/-0.03 to 0.93+/-0.02 (nuclei microm(2) 10(-4), P< 0.05), and in skeletal muscle from 0.82+/-0.05 to 0.54+/-0.01 (nuclei microm(2) 10(-4), P< 0.05). The increased liver blood perfusion following occlusion of the DV increased cell proliferation sixfold in the liver, (n=9, P< 0.005) and twofold in heart muscle, skeletal muscle and the kidneys (P< 0.05), whereas no significant difference was seen in the placenta. The expression of mRNA for IGF-I and IGF-II in the liver was increased in the DV(coil) group. In conclusion, these results suggest that liver blood perfusion can regulate fetal growth.
Amino Acids↗
The anaphase promoting complex/cyclosome is required during development for modified cell cycles.
Animals and plants use modified cell cycles to achieve particular developmental strategies. In one common example, most animals and plants have tissues in which the cells become polyploid or polytene by means of an S-G cycle, but the mechanism by which mitosis is inhibited in the endo cycle is not understood. The Drosophila morula (mr) gene regulates variant cell cycles, because in addition to disrupting the archetypal cycle (G1-S-G2-M), mr mutations affect the rapid embryonic (S-M) divisions as well as the endo cycle (S-G) that produces polyploid cells. In dividing cells mr mutations cause a metaphase arrest, and endo cycling nurse cells inappropriately reenter mitosis in mr mutants. We show mr encodes the APC2 subunit of the anaphase promoting complex/cyclosome. This finding demonstrates that anaphase promoting complex/cyclosome is required not only in proliferating cells but also to block mitosis in some endo cycles. The mr mutants further indicate that transient mitotic functions in endo cycles change chromosome morphology from polytene to polyploid.
Amino Acid Sequence↗
Programmed cell death in normal fetal rat lung development.
Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.
Animals↗
Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice.
Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.
Animals↗
Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans.
The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.
Animals↗
Interactions between retinoic acid and 1,25(OH)2D in mouse immortalized osteoblastic C1 cells.
The interactions between 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and retinoic acid (RA) on alkaline phosphatase (ALP) activity and vitamin D receptors (VDR) were determined in the SV40 immortalized osteoblastic cell line C1. Biochemical and cytochemical analysis showed that ALP activity increased with C1 cell density and that 1,25(OH)2D inhibited ALP activity at high density, whereas RA increased ALP activity at low density. The interactions of the two hormones were also cell density dependent, with a predominant stimulatory effect of RA at low density and a predominant inhibitory effect of 1,25(OH)2D at high density. In contrast, the two hormones inhibited C1 cell proliferation independently of cell density. Scatchard analysis and immunocytochemical staining showed that nuclear VDR levels increased with cell density and that RA, 1,25(OH)2D, and their combination upregulated VDR levels mainly at high cell density. Although RA increased VDR levels, RA did not potentiate the effect of 1,25(OH)2D on ALP activity. The results indicate that the effects and interactions of 1,25(OH)2D and RA on ALP activity in osteoblastic C1 cells depend on the state of phenotypic maturation.
Alkaline Phosphatase↗