Growth factors and their receptors in differentiation and early murine development.
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A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis.
The distribution of c-myc and N-myc transcripts during mouse organogenesis was investigated by in situ hybridization and compared to proliferation in several tissues. Only c-myc expression was found during the formation of cartilage, brown adipose tissue, glandula submandibularis, thymus and liver. There was a temporally and spatially ordered expression of N-myc only during the organogenesis of brain, retina and eye lens. In some organs (e.g., in lung and tooth bud), c-myc and N-myc were expressed in a striking complementary pattern that reflected the ontogenic origins of different tissue components. Transcripts of both genes were found in the early gut epithelium, but as formation of villi began, the spatial expression pattern of N-myc and c-myc diverged. The results suggest a link between the proliferative state of cell types and the differential expression of N-myc vs. c-myc. Specifically, c-myc is only expressed in rapidly proliferating tissues, while N-myc expression often persists through cytodifferentiation, e.g., during development of eye lens, retina, telencephalon and gut epithelium. Thus, in spite of the structural similarities of N-myc and c-myc genes and proteins their developmental expression patterns suggest different functional roles.
Animals↗
Muscle differentiation.
Recent studies indicate that vertebrate skeletal muscle originates from two distinct populations of muscle precursor cells formed in early embryogenesis. Divergent patterns of expression for the myoD family of myogenic regulatory genes in different vertebrate embryos suggest some functional redundancy amongst the myogenic factors. Initial gene knockout experiments in mice support this view. Systematic mutagenesis has identified a conserved motif in the basic region of these proteins that is necessary for their biological activity. Direct interaction of both MyoD and myogenin with the transcription factor Jun indicates a mechanism for integrating the control of myoblast growth and differentiation.
Animals↗
Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1.
The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.
Aging↗
Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors.
In zebrafish, skeletal muscle precursors can adopt at least three distinct fates: fast, non-pioneer slow, or pioneer slow muscle fibers. Slow muscle fibers develop from adaxial cells and depend on Hedgehog signaling. We analyzed when precursors become committed to their fates and the step(s) along their differentiation pathway affected by Hedgehog. Unexpectedly, we find that embryos deficient in Hedgehog signaling still contain postmitotic adaxial cells that differentiate into fast muscle fibers instead of slow. We show that by the onset of gastrulation, slow and fast muscle precursors are already spatially segregated but uncommitted to their fates until much later, in the segmental plate when slow precursors become independent of Hedgehog. In contrast, pioneer and non-pioneer slow muscle precursors share a common lineage from the onset of gastrulation. Our results demonstrate that slow muscle precursors form independently of Hedgehog signaling and further provide direct evidence for a multipotent muscle precursor population whose commitment to the slow fate depends on Hedgehog at a late stage of development when postmitotic adaxial cells differentiate into slow muscle fibers.
Animals↗
WAVE1 intranuclear trafficking is essential for genomic and cytoskeletal dynamics during fertilization: cell-cycle-dependent shuttling between M-phase and interphase nuclei.
A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. Here we test the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores.
Active Transport, Cell Nucleus↗
Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs.
Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh-/- mutants, the pharyngeal pouches and arches formed by E9.5 and marker expression showed that initial patterning was normal. However, by E10.5-E11.0, the first arch had atrophied and the first pouch was missing. Although small, the second, third, and fourth arches and pouches were present. The expression patterns of Fgf8, Pax1, and Bmp4 suggested that pouch identity was abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers were expressed normally. Our data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh-/- mutants suggested that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. Our data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs.
Animals↗
The chitin synthase genes chs-1 and chs-2 are essential for C. elegans development and responsible for chitin deposition in the eggshell and pharynx, respectively.
It is widely accepted that chitin is present in nematodes. However, its precise role in embryogenesis is unclear and it is unknown if chitin is necessary in other nematode tissues. Here, we determined the roles of chitin and the two predicted chitin synthase genes in Caenorhabditis elegans by chitin localization and gene disruption. Using a novel probe, we detected chitin in the eggshell and discovered elaborate chitin localization patterns in the pharyngeal lumen walls. Chitin deposition in these two sites is likely regulated by the activities of chs-1 (T25G3.2) and chs-2 (F48A11.1), respectively. Reducing chs-1 gene activity by RNAi led to eggs that were fragile and permeable to small molecules, and in the most severe case, absence of embryonic cell division. Complete loss of function in a chs-1 deletion resulted in embryos that lacked chitin in their eggshells and failed to divide. These results showed that eggshell chitin provides both mechanical support and chemical impermeability essential to developing embryos. Knocking down chs-2 by RNAi caused a defect in the pharynx and led to L1 larval arrest, indicating that chitin is involved in the development and function of the pharynx.
Animals↗
Hsp70A and GlsA interact as partner chaperones to regulate asymmetric division in Volvox.
GlsA, a J-protein chaperone, is required for the asymmetric divisions that set aside germ and somatic cell precursors during embryogenesis in Volvox carteri, and previous evidence indicated that this function requires an intact Hsp70-binding site. To determine if Hsp70A, the only known cytoplasmic Hsp70 in V. carteri, is the chaperone partner of GlsA, we investigated the localization of the two proteins during critical stages of embryogenesis and tested their capacity to interact. We found that a substantial fraction of Hsp70A co-localizes with GlsA, both in interphase and mitotic blastomeres. In addition, Hsp70A coimmunoprecipitated with GlsA, and co-expression of GlsA and Hsp70A variants partially rescued the Gls phenotype of a glsA mutant, whereas neither variant by itself rescued the mutant phenotype. Immunofluorescence analysis demonstrated that GlsA is about equally abundant in all blastomeres at all cleavage stages examined but that Hsp70A is more abundant in anterior (asymmetrically dividing) blastomeres than in posterior (symmetrically dividing) blastomeres during the period of asymmetric division. We conclude that Hsp70A and GlsA function as chaperone partners that regulate asymmetric division and that the relative abundance of Hsp70A in asymmetrically dividing embryos may determine which blastomeres divide asymmetrically and which do not.
Algal Proteins↗
Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.
The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.
Aging↗
Parathyroid hormone receptor type 1/Indian hedgehog expression is preserved in the growth plate of human fetuses affected with fibroblast growth factor receptor type 3 activating mutations.
The fibroblast growth factor receptor type 3 (FGFR3) and Indian hedgehog (IHH)/parathyroid hormone (PTH)/PTH-related peptide receptor type 1 (PTHR1) systems are both essential regulators of endochondral ossification. Based on mouse models, activation of the FGFR3 system is suggested to regulate the IHH/PTHR1 pathway. To challenge this possible interaction in humans, we analyzed the femoral growth plates from fetuses carrying activating FGFR3 mutations (9 achondroplasia, 21 and 8 thanatophoric dysplasia types 1 and 2, respectively) and 14 age-matched controls by histological techniques and in situ hybridization using riboprobes for human IHH, PTHR1, type 10 and type 1 collagen transcripts. We show that bone-perichondrial ring enlargement and growth plate increased vascularization in FGFR3-mutated fetuses correlate with the phenotypic severity of the disease. PTHR1 and IHH expression in growth plates, bone-perichondrial rings and vascular canals is not affected by FGFR3 mutations, irrespective of the mutant genotype and age, and is in keeping with cell phenotypes. These results indicate that in humans, FGFR3 signaling does not down-regulate the main players of the IHH/PTHR1 pathway. Furthermore, we show that cells within the bone-perichondrial ring in controls and patients express IHH, PTHR1, and type 10 and type 1 collagen transcripts, suggesting that bone-perichondrial ring formation involves cells of both chondrocytic and osteoblastic phenotypes.
Achondroplasia↗
Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.
Pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains a major therapeutic problem. Moreover, the pathogenesis of pulmonary hypoplasia in case of CDH is controversial. In particular, little is known about early lung development in this anomaly. To investigate lung development separate from diaphragm development we used an in vitro modification of the 2, 4-dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH. This enabled us to investigate the direct effects of Nitrofen on early lung development and branching morphogenesis in an organotypic explant system without the influence of impaired diaphragm development. Epithelial cell differentiation of the lung explants was assessed using surfactant protein-C and Clara cell secretory protein-10 mRNA expression as markers. Furthermore, cell proliferation and apoptosis were investigated. Our results indicate that Nitrofen negatively influences branching morphogenesis of the lung. Initial lung anlage formation is not affected. In addition, epithelial cell differentiation and cell proliferation are attenuated in lungs exposed to Nitrofen. These data indicate that Nitrofen interferes with early lung development before and separate from (aberrant) diaphragm development. Therefore, we postulate the dual-hit hypothesis, which explains pulmonary hypoplasia in CDH by two insults, one affecting both lungs before diaphragm development and one affecting the ipsilateral lung after defective diaphragm development.
Animals↗
Beta1C integrin in epithelial cells correlates with a nonproliferative phenotype: forced expression of beta1C inhibits prostate epithelial cell proliferation.
The expression of the beta1C integrin, an alternatively spliced variant of the beta1 subunit, was investigated in human adult and fetal tissues. In the adult, beta1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, beta1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed beta1C in vivo and in vitro, but no beta1C was seen in basal cells, which are proliferating cells. Fetal prostate expressed beta1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that beta1C is a marker of prostate epithelium differentiation. The beta1C and the common beta1A variants are differentially distributed: beta1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, beta1C and beta1A were coexpressed in biliary epithelium, whereas vascular cells expressed only beta1A. Because we found beta1C in nonproliferative and differentiated epithelium, we investigated whether beta1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a beta1C, but not of a beta1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, beta1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of beta1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.
Alternative Splicing↗
Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors.
The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.
Animals↗
Opposite trends of changes in reactive behaviours of macrophages and astrocytes following gamma-irradiation performed at different stages of prenatal development. A study in the injured brain of 6-day-old rat.
Pregnant Wistar rats were exposed to a single 1.0 Gy dose of gamma rays on gestational days 13, 15, 17 or 19 (E13s, E15s, E17s and E19s, respectively). A mechanical injury was made in the cerebral hemisphere of their 6 day-old male offsprings. The injured rats were injected with [3H] thymidine on day 1 or 2 after injury and killed 4 h after the injection. Brain sections were processed for BSI-B4 isolectin histochemistry, subjected to autoradiography and examined microscopically to record numbers of proliferating and unproliferating macrophages located within the region of injury. The total number of macrophages as well as number of their divisions were minimal in E13s then showed a regular increase in E15s and E17s, and reached its maximal level in brains irradiated on E19. The trend of changes was opposite to that showed by changes in the intensity of astrocyte proliferation [Z. Setkowicz, K. Janeczko, Effects of prenatal gamma-irradiation on the astrocyte proliferation in response to injury in the brain of 6-day-old rat, Brain Res. 803 (1998) 122-128.]. The recruitment and proliferation of macrophages and the astrocyte proliferation were regarded as reactive processes occurring under control of different regulatory mechanisms acting within the region of injury.
Analysis of Variance↗
Tracheal ligation increases cell proliferation but decreases surfactant protein in fetal murine lungs in vitro.
Tracheal occlusion affects both fetal lung growth and maturation. The authors used a murine in vitro whole organ culture model to investigate these effects. The authors hypothesized that tracheal ligation would increase lung growth by increasing cell proliferation and would change surfactant protein synthesis in this system. Lungs were removed from day 14 gestation murine fetuses (term, 21 days). Tracheas were ligated and explants cultured in chemically defined, serum-free media for 1, 3, 4, 5, 7, or 14 days. DNA synthesis and cell division were assessed using a 5-bromo-2'-deoxy-uridine (BrdU) incorporation assay. Surfactant proteins A and B, markers of lung maturity, were detected using immunohistochemistry. Ligated lungs showed more BrdU-labeled cells per 1,000 x field (cells/hpf) at every time point. Ligated lungs on day 1 showed 27% more cells/hpf than unligated, on day 3, 21% more, on day 5, 54% more, on day 7, 60% more, and on day 14, 123% more (P < .05). In contrast, ligated lungs showed significantly less staining for surfactant proteins A and B than did unligated lungs. The authors conclude that tracheal ligation increases cell division but decreases surfactant protein in fetal murine lungs in vitro. These data suggest that although tracheal occlusion increases lung growth, it may decrease or delay lung maturation. This model provides a powerful tool for investigating the mechanisms underlying fetal lung development and tracheal occlusion-induced pulmonary hyperplasia.
Animals↗
Hemodynamic alteration by fetal surgery accelerates myocyte proliferation in fetal guinea pig hearts.
BACKGROUND: Fetal heart development occurs by hyperplasia as myocytes lose the capacity to proliferate at birth. This potential for cell division may have application in altering fetal growth patterns in congenital cardiac malformations, but it is not known whether the proliferative activity can be modified by intrauterine surgical manipulation. The purpose of this study was to determine whether hemodynamic alteration by fetal surgery influences myocyte proliferation and myocardial development. METHODS: Six pregnant guinea pigs of 50 to 52 days of gestation (term, 65 days) underwent hysterotomy, and the fetal ascending aorta was banded and narrowed by 50% (AoB). Cesarean section was performed near term, and the heart was assessed for myocyte proliferative activity (Ki-67 monoclonal antibody), apoptosis, and morphologic features. RESULTS: The heart to body weight ratio (1.02% +/- 0.12% versus 0.42% +/- 0.02%, p < 0.01) and left ventricular posterior wall thickness (1.89 +/- 0.25 mm versus 1.31 +/- 0.19 mm, p < 0.01) were significantly higher in the AoB group. The percentage of Ki-67 positive cells was increased in AoB group (29.5% +/- 4.4% versus 15.3% +/- 1.3% in right ventricle, 35.8% +/- 5.1% versus 13.1% +/- 1.7% in interventricular septum, and 39.8% +/- 3.2% versus 12.0% +/- 2.0% in left ventricle (p < 0.01). The apoptotic cell to myocyte ratio was less than 1/1000 in both groups. CONCLUSIONS: Fetal hemodynamic alteration by aortic banding accelerates myocardial cellular proliferation without affecting apoptosis, suggesting that in utero cardiac interventions have a greater influence on myocardial development compared with postnatal intervention.
Animals↗
Role of VEGF-flt receptor system in normal and tumor angiogenesis.
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Animals↗