Tooth Morphogenesis and FGF4 Expression During Development of Molar Tooth in Three Muroid Rodents: Calomyscus elburzensis (Calomyscidae), Mesocricetus auratus (Cricetidae) and Mus musculus (Muridae).

To date, no studies have examined the tooth formation during developmental stages of brush-tailed mice (Calomyscidae) and true hamsters (Cricetidae). Herein, we compared the timing of tooth morphogenesis and FGF4 expression pattern during development of the first lower molar in Goodwin's brush-tailed mouse, Calomyscus elburzensis with two other muroid rodents; the house mouse, Mus musculus (Muridae), model organism for tooth morphogenesis, and the golden hamster, Mesocricetus auratus which shares great similarities in cusp pattern with brush-tailed mice. All three species were bred in captivity and developing embryos were isolated at different embryonic days (E). Histological evaluation of lower molars was performed and spatiotemporal pattern of FGF4 expression was determined by immunohistochemistry. Results indicated that morphogenesis of the tooth cusps starts at the beginning of the cap stage of the first lower molar (E14 in house mouse, about E11.5 in golden hamster and E22 in Goodwin's brush-tailed mouse). During the cap to bell stage (E15 in house mouse, E12 in golden hamster and at about E24 in Goodwin's brush-tailed mouse), a decrease in the expression of FGF4 was observed in the mesenchyme, except for the cusp tips. According to our observations, the developmental process of the first lower molar formation in Goodwin's brush-tailed mouse began much later as compared with the other two species. Despite the differences in the temporal pattern of molar development between these three members of the same superfamily (Muroidea), the correlation in the expression of FGF4 with specific stages of tooth morphogenesis supported its regulatory function. Anat Rec, 300:2138-2149, 2017. © 2017 Wiley Periodicals, Inc.

Generation of aminoterminally truncated, stable types of bioactive bovine and porcine fibroblast growth factor 4 in Escherichia coli.

Fibroblast growth factor 4 (FGF4) is a crucial growth factor for the development of mammalian embryos. We previously produced hexahistidine-tagged, bovine and porcine FGF4 (Pro(32) to Leu(206) ) proteins without a secretory signal peptide at the aminoterminus in Escherichia coli. Here, we found that these were unstable; site-specific cleavage between Ser(54) and Leu(55) in both FGF4 derivatives was identified. In order to generate stable FGF4 derivatives and to investigate their biological activities, aminoterminally truncated and hexahistidine-tagged bovine and porcine FGF4 (Leu(55) to Leu(206) ) proteins, termed HisbFGF4L and HispFGF4L, respectively, were produced in E. coli. These FGF4 derivatives were sufficiently stable and exerted mitogenic activities in fibroblasts. Treatment with the FGF4 derivatives promoted the phosphorylation of ERK1/2, which are crucial kinases in the FGF signaling pathway. In the presence of PD173074, an FGF receptor inhibitor, the phosphorylation of ERK1/2 was inhibited and resulted in abolition of the growth-promoting activity of FGF4 derivatives. Taken together, we demonstrate that HisbFGF4L and HispFGF4L are capable of promoting the proliferation of bovine- and porcine-derived cells, respectively, via an authentic FGF signaling pathway. These FGF4 derivatives may be applicable for dissecting the roles of FGF4 during embryogenesis in cattle and pigs.

ALK5-mediated transforming growth factor ß signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.

The development of the craniofacial muscles requires reciprocal interactions with surrounding craniofacial tissues that originate from cranial neural crest cells (CNCCs). However, the molecular mechanism involved in the tissue-tissue interactions between CNCCs and muscle progenitors during craniofacial muscle development is largely unknown. In the current study, we address how CNCCs regulate the development of the tongue and other craniofacial muscles using Wnt1-Cre; Alk5(fl/fl) mice, in which loss of Alk5 in CNCCs results in severely disrupted muscle formation. We found that Bmp4 is responsible for reduced proliferation of the myogenic progenitor cells in Wnt1-Cre; Alk5(fl/fl) mice during early myogenesis. In addition, Fgf4 and Fgf6 ligands were reduced in Wnt1-Cre; Alk5(fl/fl) mice and are critical for differentiation of the myogenic cells. Addition of Bmp4 or Fgf ligands rescues the proliferation and differentiation defects in the craniofacial muscles of Alk5 mutant mice in vitro. Taken together, our results indicate that CNCCs play critical roles in controlling craniofacial myogenic proliferation and differentiation through tissue-tissue interactions.

Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli.

In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.

Allocation of inner cells to epiblast vs primitive endoderm in the mouse embryo is biased but not determined by the round of asymmetric divisions (8→16- and 16→32-cells).

The epiblast (EPI) and the primitive endoderm (PE), which constitute foundations for the future embryo body and yolk sac, build respectively deep and surface layers of the inner cell mass (ICM) of the blastocyst. Before reaching their target localization within the ICM, the PE and EPI precursor cells, which display distinct lineage-specific markers, are intermingled randomly. Since the ICM cells are produced in two successive rounds of asymmetric divisions at the 8→16 (primary inner cells) and 16→32 cell stage (secondary inner cells) it has been suggested that the fate of inner cells (decision to become EPI or PE) may depend on the time of their origin. Our method of dual labeling of embryos allowed us to distinguish between primary and secondary inner cells contributing ultimately to ICM. Our results show that the presence of two generations of inner cells in the 32-cell stage embryo is the source of heterogeneity within the ICM. We found some bias concerning the level of Fgf4 and Fgfr2 expression between primary and secondary inner cells, resulting from the distinct number of cells expressing these genes. Analysis of experimental aggregates constructed using different ratios of inner cells surrounded by outer cells revealed that the fate of cells does not depend exclusively on the timing of their generation, but also on the number of cells generated in each wave of asymmetric division. Taking together, the observed regulatory mechanism adjusting the proportion of outer to inner cells within the embryo may be mediated by FGF signaling.

Derivation of embryonic stem cell lines from parthenogenetically developing rat blastocysts.

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

Expression of FGF4 mRNA is mediated by mating behaviours in mice.

In mammals, breeding is preceded by species-specific mating behaviours. In this study, we investigated whether parthenogenetic embryo quality could be improved by mating behaviours in mice. To investigate this hypothesis, female mice were mated with vasectomized Kunming white male mice after superovulation. Oocytes were collected and counted at 16 h after superovulation. The oocytes were then artificially activated by medium containing 10 mM strontium chloride and 5 µg/ml cytochalasin B. Blastocysts were obtained by cultivating activated oocytes in vitro. Expression levels of reprogramming transcription factors (i.e. Oct4, Sox2, Klf4 and c-Myc) in oocytes, apoptosis-related genes (i.e. Bax, Bcl2 and c-Myc) in cumulus cells and pluripotency-related transcription factors (i.e. Oct4, Nanog and FGF4) in blastocysts were analysed in samples collected from mated and unmated mice. Additionally, developmental competence of parthenogenetic embryos was used to assess following fibroblast growth factor 4 (FGF4) treatment. The results showed that the formation rate of blastocysts in unmated mice was significantly higher than that in mated mice (p < 0.05). Embryo development was primarily blocked at the eight-cell stage in mated mice; however, the blastocyst formation rate did not differ significantly between groups after the addition of 25 ng/ml FGF4 to the medium at the four-cell stage (p > 0.05). Moreover, the expression of the reprogramming factor Sox2 was significantly different in oocytes collected from mated versus unmated mice. Taken together, our results demonstrated that mating behaviours influenced embryonic development in vitro by decreasing FGF4 expression.

Inhibition of cardiomyocytes differentiation of mouse embryonic stem cells by CD38/cADPR/Ca2+ signaling pathway.

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca(2+) mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca(2+) in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca(2+) signaling pathway antagonizes the CM differentiation of mouse ES cells.

Production of mouse fibroblast growth factor 4 in E. coli and its application for isolation and maintenance of mouse trophoblast stem cells in vitro.

Fibroblast growth factor 4 (FGF4) promotes isolation of trophoblast stem (TS) cells from mouse blastocysts and maintenance of TS cells in an undifferentiated state in vitro. To date, commercially available, bacterially expressed human FGF4 (RhFGF4) has been used generally for this purpose. In this study, HismFGF4, a 6x histidine-tagged mouse FGF4, was produced in E. coli and purified using heparin column chromatography. We demonstrated that HismFGF4 (25 ng/ml) more efficiently generates mouse TS cells from a single blastocyst than RhFGF4 (25 ng/ml) and that TS cells isolated and maintained with HismFGF4 retained their ability to differentiate into the trophoblast cell lineage in vitro. In addition, TS cells cultured with HismFGF4 (25 ng/ml) were maintained in an undifferentiated state better than with RhFGF4 (25 ng/ml). To the best of our knowledge, this is the first application of a mouse FGF4 derivative for isolation and maintenance of mouse TS cells.

Transient and stable transfections of mouse myoblasts with genes coding for pro-angiogenic factors.

Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.

Improvement of in vitro oocyte maturation with lectin supplementation and expression analysis of Cx43, GDF-9, FGF-4 and Fibronectin mRNA transcripts in Buffalo (Bubalus bubalis).

PURPOSE: To optimize In vitro maturation (IVM) of quality oocytes for embryo production through IVF and SCNT. METHODS: Buffalo oocytes were in vitro matured in the presence of the pokeweed lectin (Phytolacca americana), a potent lymphocyte mitogen. Lectin was supplemented in TCM + 10% FBS at the doses of 0, 1, 5, 10, 15, 20 and 40 microg/ml and cumulus expansion and gene expression patterns were characterized. RESULTS: The degree of cumulus expansion in different lectin treatment levels improved from 1.1 at 1 Ag/ml level to 3.60 at 10 microg/ml level and then decreased in higher concentration 20 microg/ml (1.66) and 40 microg/ml (0.64). IVF embryos showed highest cleavage rate (88.8%) in 10 microg/ml lectin treatment. Expression of all mRNA transcript studied (Cx43, GDF 9, FGF-4 and Fibronectin) was positively correlated with cumulus expansion and polar body extrusion. CONCLUSIONS: Mitogenic lectin supplemented maturation media improves oocyte quality for in vitro embryo production.

Rearrangement and expression of myeov and hst in NIH/3T3 transfectants: a caveat for the interpretation of DNA transfection analyses.

By means of the tumorigenicity assay applying DNA from a patient with a gastric carcinoma (MA) we have already reported the identification of the putative oncogene myeov. In addition we have shown its involvement in t(11;14)-positive multiple myelomas and amplifications of breast tumours and esophageal carcinomas. The failure of myeov cDNA to induce tumour formation in NIH/3T3 cells prompted us to analyze the human sequences present in our MA-T1a1 tertiary transfectants. Sequence analysis revealed the presence of the human oncogene hst (fgf4) at a distance of approximately 9kb from the myeov gene in our MA-T1a1 tertiary transfectants. Both myeov and hst (fgf4) are normally situated approximately 475-kb apart at band 11q13, a region that is frequently amplified and overexpressed in various tumours. Southern and Northern blot analyses confirmed our sequence data and showed rearrangement of hst sequences during the transfection process and its expression in our MA-T1a1 tertiary transfectants.