Daniel A. Rappolee

Macrophage-Derived Growth Factors

In the early decades of the twentieth century biologists sought to grow cells in culture. Clotted blood was found to contain molecules that accomplished this purpose (Carrel 1912), but only later did biochemists seek to purify these molecules. By the middle of the century, biochemists and biologists sought to explain neonatal eye opening in mice in molecular terms (Cohen 1987; Levi-Montalcini 1987). Each of these goals ultimately led to the isolation of single species of molecules called growth factors by using in vitro or in vivo bioassays for growth and a biochemical algorithm for isolation. Epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), interleukin-1 (IL-1), and macrophage colony-stimulating factor (M-CSF, or CSF-1) were isolated and directly sequenced or molecularly cloned (based on partial sequences) by these means in the 1970s and early 1980s. The production of transformed foci of cells by introduction of fragments of cloned transcripts or genes from tumors also produced a subclass of oncogenes that turned out to be growth factors [c-sis, or PDGF-B chain, and Kaposi’s sarcoma-fibroblast growth factor (kFGF, or FGF-4)]. Most recently, the formation of tumors in vivo after random integration of a highly active viral promoter upstream of cellular genes has produced the int-1 and int-2 (also known as FGF-3) growth factors. Finally, after the founding member of a growth factor family is identified with a bioassay, low-stringency cDNA library screens and polymerase chain reaction can be used to complete the family (JAKOWLEW et al. 1988; HEBERT et al. 1990). All growth factors are operationally isolated and defined by their ability to cause growth, but may also act as nonmitogenic inflammatory factors.

Platelet alpha-granule fibrinogen, albumin, and immunoglobulin G are not synthesized by rat and mouse megakaryocytes.

It has been assumed that endogenous synthesis by the platelet precursor cell, the bone marrow megakaryocyte, is the major source of platelet alpha-granule protein. To test this hypothesis, we used mRNA phenotyping to detect in megakaryocytes the presence of mRNA transcripts specific for various proteins. Our results indicate that megakaryocytes synthesize platelet factor 4, a protein relatively specific for platelets, but do not express mRNA transcripts for the fibrinogen, albumin, or IgG found in alpha-granules. We have previously shown that megakaryocytes endocytose circulating proteins, including fibrinogen, albumin, and IgG, and incorporate them into alpha-granules. Thus, platelets appear to contain a unique type of secretory granule whose contents originate by both endogenous synthesis and endocytosis from plasma. Under basal conditions, the source of alpha-granule fibrinogen is plasma.

Hepatocyte growth factor and its receptor are expressed in cardiac myocytes during early cardiogenesis.

In the mouse, the heart primordium arises when mesoderm is 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 the 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 approximately 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 state (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 of fibroblast growth factor receptors in peri-implantation mouse embryos

FGF receptor (FGFR) function is essential during peri‐implantation mouse development. To understand which receptors are functioning, we tested for the expression of all four FGF receptors in peri‐implantation blastocysts. By RT‐PCR, FGFR‐3 and FGFR‐4 were detected at high levels, FGFR‐2 at lower levels, and FGFR‐1 was detected at background levels compared to control tissues. Because FGFR‐3 and FGFR‐4 were detected at the highest levels, we studied these in detail. Between 3.5 days after fertilization (E3.5) and E6.0, FGFR‐4 mRNA was detected ubiquitously in the peri‐implantation embryo, restricted to the inner cell mass (ICM) and its derivatives and primitive endoderm by E6.0, and was not detected at E6.5. FGFR‐3 mRNA was detected ubiquitously in the peri‐implantation embryo with a tendency towards extraembryonic cells. We tested blastocyst outgrowths, a model for implantation, for FGFR‐3 and FGFR‐4 protein. FGFR‐3 protein was detected in all cells early during the outgrowth. Later, FGFR‐3 was detected in the extraembryonic endoderm and trophoblast giant cells (TGC), but not in the ICM. FGFR‐4 protein was detected in all cells of the implanting embryo, but was restricted to the ICM/primitive endoderm in later stage outgrowths. The distribution of the receptor proteins in the blastocyst outgrowths is similar to the distribution of the mRNA detected by in situ hybridization of sections of embryos. The data suggest roles for FGFR‐3 and FGFR‐4 in peri‐implantation development. Mol. Reprod. Dev. 51:254–264, 1998.

Use of Hyperosmolar Stress to Measure Stress-Activated Protein Kinase Activation and Function in Human HTR Cells and Mouse Trophoblast Stem Cells

Embryo growth is inversely correlated with hyperosmolar stress-induced stress-activated protein kinase/jun kinase (SAPK/JNK) induction. To examine whether stress has similar effects in stem cells derived from the embryo, the authors test trophoblast stem cells. The stress response of human placental and mouse trophoblast stem cell lines are tested here. Peak phosphorylated SAPK/JNK was induced by 400 mM sorbitol at 0.5 hours. At this dose, there is an SAPK/JNK-dependent decrease in mitogenic, phosphorylated cMyc at 0.5 hours preceding an SAPK/JNK-dependent decrease in cell cycle entrance at 24 hours. At 0.5 hours, SAPK/JNK decreases terminal deoxynucleotidyltransferase dUTP nick end labeling/apoptosis at sorbitol doses from 50 mM to 400 mM and induces phosphorylated cJun prior to an SAPK/JNK-dependent, approximate 8-fold increase in apoptosis by 24 hours at 400 mM. SAPK/JNK phosphorylation peaked at 0.5 to 4 hours and largely subsided by 12 hours. Thus, total SAPK/JNK exists before stress and mediates rapid, homeostatic molecular responses that become biologic consequences after phosphorylated SAPK/JNK ends. This suggests continuity in the homeostatic mechanisms and functions of SAPK/JNK in placental lineage cells during implantation, in which SAPK/JNK is completely responsible for cell cycle arrest and largely responsible for apoptosis.

A Role for Hepatocyte Growth Factor During Early Postimplantation Growth of the Placental Lineage in Mice

Abstract Hepatocyte growth factor (HGF) is implicated in placental development; hgfr and hgf null mutant embryos develop placental insufficiency and lethality at 11.5 days (E11.5) after fertilization. The function of HGF in placentation at implantation (E4.5) has not been studied. Using reverse transcription-polymerase chain reaction, we detected HGF receptor (HGFR) mRNA in preimplantation embryos and in cultured blastocyst outgrowths. HGFR protein was detected in trophoblast cells in blastocyst outgrowths. HGF mRNA was not detected at these stages but was detected in the uterus at E5.5. Using in situ hybridization, we detected HGF mRNA in the mesometrial uterus, near the embryo, from E6.5 through E8.5. At E8.5, HGFR mRNA was detected in the chorionic placenta, and HGF mRNA was detected in the allantois. The expression for HGF and HGFR suggested a maternal-to-embryonic communication before the development of the allantois. To test this, blastocyst outgrowths were cultured with HGF. HGF stimulated the outgrowth of trophoblasts in a time-dependent manner and stimulated the expression of proliferating cell nuclear antigen, but it did not scatter trophoblasts. HGF stimulated an increase in the trophoblast cell number, but caused a decrease in the total number of terminally differentiated trophoblasts expressing placental lactogen-1 protein. These data suggest that HGF stimulates the cell division, but not the differentiation, of trophoblast cells during implantation.

Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo.

Acquisition of essential somatic cell cycle regulatory protein expression and implied activity occurs at the second to third cell division in mouse preimplantation embryos

It is clear that G1–S phase control is exerted after the mouse embryo implants into the uterus 4.5 days after fertilization (E4.5); null mutants of genes that control cell cycle commitment such as max, rb (retinoblastoma), and dp1 are embryonic lethal after implantation with proliferation phenotypes. But, a number of studies of genes mediating proliferation control in the embryo after fertilization‐implantation have yielded confusing results. In order to understand when embryos might first exert G1–S phase regulatory control, we assayed preimplantation mouse embryos for the acquisition of expression of mRNA, protein, and phospho‐protein for max, Rb, and DP‐1, and for the proliferation‐promoting phospho‐protein forms of mycC (thr58/ser62) and Rb (ser795). The key findings are that: (1) DP‐1 protein was present in the nucleus as early as the four‐cell stage onwards, (2) max protein was in the nucleus, suggesting function from the four‐cell stage onwards, (3) both mycC and Rb all form protein was present at increasing quantities in the cytoplasm from the 2 cell and 4/8 cell stage, respectively, (4) the phosphorylated form of mycC phospho was present in the nucleus at high levels from the two‐cell stage through blastocyst‐stage, and (5) the phosphorylated form of Rb was detected at low levels in the two‐cell stage embryo and was highly expressed at the 4/8‐cell stage through the blastocyst stage. Taken together, these data suggest that activation of mycC phospho/max dimer pairs, (E2F)/DP‐1 dimer pairs, and repression of Rb inhibition of cell cycle progression via phosphorylation at ser795 occurs at the earliest stages of embryonic development. In addition, the presence of max, mycC phospho, DP‐1, and Rb phospho in the nuclei of embryonic and placental lineage cells in the blastocyst and in trophoblast stem cells suggests that a similar type of cell cycle regulation is present throughout preimplantation development and in both embryonic and extra‐embryonic cell lineages.

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 24h 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>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.

It's not just baby's babble/Babel: recent progress in understanding the language of early mammalian development: a minireview.

DANIEL A. RAPPOLEE1,2,3,4* 1Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 2Department of Obstetrics and Gynecology, Northwestern University Medical School, Chicago, Illinois 3Lurie Cancer Center, Center for Reproductive Sciences, Northwestern University, Chicago, Illinois 4Feinberg Cardiovascular Research Institute, Center for Reproductive Sciences, Northwestern University, Chicago, Illinois