Hepatocyte Growth Factor and Its Receptor Are Expressed in Cardiac Myocytes During Early Cardiogenesis

In the mouse, the heart primordium arises when mesodermis set aside during gastrulation, is induced by pharyngeal endoderm, migrates ventrally to the midline of the embryo, forms a tube, and begins beating. Little is known of the molecular mechanisms that mediate the determination, mitosis, differentiation, and migration that lead to the beating heart. Transcripts for hepatocyte growth factor/scatter factor (HGF) and its receptor are coexpressed transiently and dynamically in the premyocardium but not in other heart progenitor cells. Transcripts for HGF ligand and receptor are first detected before cardiac function and looping and persist through the first looping stage, when heart morphology begins to elaborate. HGF ligand and receptor mRNA are detectable after the putative heart transcription factor, Csx/Nkx2-5, and concomitantly with the heart structural gene, cardiac actin. HGF receptor mRNA is detected in the mesoderm of the headfold stage and persists in myocardial precursors of the ventricles and atria (but not in the outflow-tract smooth muscle cells) through the 14- somite stage at 8.75 days after fertilization (day E8.75). At the headfold stage, between E7.5 and E8.0, HGF receptor mRNA was detected in myocardial cells before fusion at the ventral midline. HGF ligand and receptor mRNA transcripts are coexpressed in the embryo, except in the headfold stage (when only the HGF receptor can be detected) and in the heart at the 14- to 18-somite stage (when only HGF ligand can be detected). The dynamic pattern of coexpression suggests an autoregulatory role for HGF and its receptor in early heart development

Expression and function of FGF-4 in peri-implantation development in mouse embryos

One of the earliest events in mammalian embryogenesis is the formation of the inner cell mass (ICM) and the subse- quent delamination of primitive endoderm. We have found that mRNA for fibroblast growth factor (FGF)-4, but not FGF-3, is expressed in preimplantation mouse blastocysts and that the FGF-4 polypeptide is present in ICM cells. ICM-like embryonal carcinoma cells and embryonic stem cells also express FGF-4. Conversely, differentiated embryonal carcinoma cells in the endoderm lineage express FGF-3, but not FGF-4 mRNA. Although mouse embryos expressed FGF-4 mRNA from the 1-cell stage, embryos cultured from the 2-cell through the blastocyst stage in the presence of recombinant FGF-4 did not respond mitogenically. However, when ICMs that were isolated by immunosurgery were cultured with FGF- 4, the number of morphologically distinct, differentiated parietal endoderm cells growing out onto the coverslip increased, without an increase in the number of undiffer- entiated ICM cells. ICM outgrowths cultured with FGF-4 increased their secretion of 92×103 Mrgelatinase and tissue plasminogen activator, a hallmark of migrating cells. Receptors for FGF-4 (FGFR-3 and FGFR-4) are expressed in all cells of the mouse blastocyst. These findings indicate that FGF-4 produced by undifferentiated ICM cells acts in the peri-implantation period of embryogenesis to influence the production and behavior of endoderm cells derived from them. Key words: fibroblast growth factor, mouse embryogenesis, metall

Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos

We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages, mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal–placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~ 2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4) enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2 \u3e 0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation

Two alkaline phosphatase genes are expressed during early development in the mouse embryo

Alkaline phosphatase (AP) activity is stage specific in mouse embryos and may be associated with compaction and separation of trophectoderm from inner cell mass in preimplantation development. We previously sequenced a cDNA and two mouse AP genes that could contribute to the AP activity in embryos. Oligonucleotide primers were constructed from the three sequences and used in the reverse transcription-polymerase chain reaction technique to establish that two of the three AP isozymes are transcribed during preimplantation development. The predominant transcript (E-AP) is from a gene highly homologous to the human tissue-specific APs, but different from the mouse intestinal AP. Tissue non- specific (TN) AP also is transcribed, but there is approximately 10 times less TN-AP than E-AP tran- script. The TN-AP isozyme is the predominant tran- script of 7 to 14 day embryos and primordial germ cells. A switch in predominance from E-AP to TN-AP must occur during early postimplantation development. This study establishes a framework for experiments to determine the functions of the two isozymes during preimplantation development

Serine-threonine kinases and transcription factors active in signal transduction are detected at high levels of phosphorylation during mitosis in preimplantation embryos and trophoblast stem cells

Serine-threonine kinases and transcription factors play important roles in the G1-S phase progression of the cell cycle. Assays that use quantitative fluorescence by immunocytochemical means, or that measure band strength during Western blot analysis, may have confused interpretations if the intention is to measure G1-S phase commitment of a small subpopulation of phosphorylated proteins, when a larger conversion of the same population of proteins can occur during late G2 and M phases. In mouse trophoblast stem cells (TSC), a human placental cell line (HTR), and/or mouse preimplantation embryos, 8/19 ser- ine-threonine and tyrosine kinases, 3/8 transcription factors, and 8/14 phospho substrate and miscellaneous proteins were phosphorylated at higher levels in M phase than in interphase. Most phosphoproteins appeared to associate with the spindle complex during M phase, but one (p38MAPK) associated with the spindle pole and five (Cdx2, MEK1, 2, p27, and RSK1) associated with the DNA. Phosphorylation was detected throughout apparent metaphase, anaphase and telophase for some proteins, or for only one of these segments for others. The phosphorylation was from 2.1- to 6.2-fold higher during M phase compared with interphase. These data suggest that, when planning and interpreting quantitative data and perturbation experiments, consideration must be given to the role of serine-threonine kinases and transcription factors during decision making in M phase as well as in G1-S phase

Developmental Expression of PDGF, TGF-a, and TGF-j Genes in Preimplantation Mouse Embryos

Control of growth and differentiation during mammalian embryogenesis may be regulated by growth factors from embryonic or maternal sources. With the use of single-cell messenger RNA phenotyping, the simultaneous expression of growth factor transcripts in single or small numbers of preimplantation mouse embryos was examined. Transcripts for platelet-derived growth factor A chain (PDGF-A), trans- forming growth factor (TGF)-a, and TGF- 1, but not for four other growth factors, were found in whole blastocysts. TGF-cx, TGF-(31, and PDGF antigens were detected in blastocysts by immunocytochemistry. Both PDGF-A and TGF-a were detected as maternal transcripts in the unferilized ovulated oocyte, and again in blastocysts. TGF- P1 transcripts appeared only after fertilization. The expression of a subset of growth factors in mouse blastocysts suggests a role for these factors in the growth and differentiation of early mammalian embryos

Basement Membrane and Repair of Injury to Peripheral Nerve: Defining a Potential Role for Macrophages, Matrix Metalloproteinases, and Tissue Inhibitor of Metalloproteinases-1

Injury to a peripheral nerve is followed by a remodeling process consisting of axonal degenera- tion and regeneration. It is not known how Schwann cell–derived basement membrane is pre- served after injury or what role matrix metalloproteinases (MMPs) and their inhibitors play in axonal degeneration and regeneration. We showed that the MMPs gelatinase B (MMP-9), stromelysin-1 (MMP-3), and the tissue inhibitor of MMPs (TIMP)-1 were induced in crush and distal segments of mouse sciatic nerve after injury. TIMP-1 inhibitor activity was present in excess of proteinase activity in extracts of injured nerve. TIMP-1 protected basement mem- brane type IV collagen from degradation by exogenous gelatinase B in cryostat sections of nerve in vitro. In vivo, during the early phase (1 d after crush) and later phase (4 d after crush) after injury, induction of TNF- a and TGF- B 1 mRNAs, known modulators of TIMP-1 ex- pression, were paralleled by an upregulation of TIMP-1 and gelatinase B mRNAs. At 4 days after injury, TIMP-1, gelatinase B, and TNF- a mRNAs were localized to infiltrating mac- rophages and Schwann cells in the regions of nerve infiltrated by elicited macrophages. TIMP-1 and cytokine mRNA expression was upregulated in undamaged nerve explants incubated with medium conditioned by macrophages or containing the cytokines TGF- B 1, TNF- a , and IL-1 a . These results show that TIMP-1 may protect basement membrane from uncontrolled degrada- tion after injury and that cytokines produced by macrophages may participate in the regulation of TIMP-1 levels during nerve repair

Adaptive and Pathogenic Responses to Stress by Stem Cells during Development

Cellular stress is the basis of a dose-dependent continuum of responses leading to adaptive health or pathogenesis. For all cells, stress leads to reduction in macromolecular synthesis by shared pathways and tissue and stress-specific homeostatic mechanisms. For stem cells during embryonic, fetal, and placental development, higher exposures of stress lead to decreased anabolism, macromolecular synthesis and cell proliferation. Coupled with diminished stem cell proliferation is a stress-induced differentiation which generates minimal necessary function by producing more differentiated product/cell. This compensatory differentiation is accompanied by a second strategy to insure organismal survival as multipotent and pluripotent stem cells differentiate into the lineages in their repertoire. During stressed differentiation, the first lineage in the repertoire is increased and later lineages are suppressed, thus prioritized differentiation occurs. Compensatory and prioritized differentiation is regulated by at least two types of stress enzymes. AMP-activated protein kinase (AMPK) which mediates loss of nuclear potency factors and stress-activated protein kinase (SAPK) that does not. SAPK mediates an increase in the first essential lineage and decreases in later lineages in placental stem cells. The clinical significance of compensatory and prioritized differentiation is that stem cell pools are depleted and imbalanced differentiation leads to gestational diseases and long term postnatal pathologies