Factors Required for Differentiation of Chick Precardiac Mesoderm Cultured in vitro.

Abstract

Abstract:Precardiac mesoderm (PCM) of chick embryo at stage 5 was excised with or without endoderm and ectoderm and cultured in vitro using Eagle's minimum essential medium (MEM) as a basic medium. PCM placed alone in MEM degenerated.When PCM was associated with endoderm, it produced beating tissue in MEM, while it did not show pulsation with ectoderm in the same condition. If MEM was supplemented with 15% horse serum, PCM developed into beating tissue without endoderm. Insulin (25 ug/m1) was effective in differentiation of PCM to myocardial cells.

Precardiac mesoderm is specified during gastrulation in quail

“…2A,B) Although explants maintained for up to 1 week in culture showed no loss in viability, the percentage of contracting explants did not increase (data not shown). These findings are in agreement with previous studies (Le Douarin, 1974;Lough et al, 1990;Sugi et al, 1993;Yamazaki et al, 1991), showing that mesoderm explants will survive and differentiate if grown in a minimal basal medium supplemented with fetal calf serum, embryo extract, or insulin. Since our aim was to determine when PCM was capable of differentiating in isolation from other cell layers and in the absence of potential signaling molecules, we tested additional defined media for their ability to support PCM survival and differentiation.…”

“…Within 12 hr, however, cells became vacuolated and by 24 hr more than half of the cells of most explants were floating in the medium (see also Yamazaki and Hirakow, 1991). Staining with nile blue (Mills and Bellairs, 1989) demonstrated that virtually all floating cells were dead or dying.…”

“…Explant experiments have examined the level of commitment of mesoderm isolated from heart forming regions of chick embryos. When cultured in medium containing serum or embryo extract, mesodermal cells taken from heart forming regions of stage 4-7 chick embryos can differentiate as intact explants (Le Douarin, 1974;Arias et al, 1987;Gonzalez-Sanchez and Bader, 1990;Yamazaki and Hirakow, 1991) or as individual cells grown at clonal density (GonzalezSanchez and Bader, 1990). Micromolar concentrations of insulin plus selenium and transferrin can substitute for serum in supporting the differentiation of mesoderm explants from stage 5 chick embryos (Yamazaki and Hirakow, 1991;Sugi et al, 1993).…”

“…When cultured in medium containing serum or embryo extract, mesodermal cells taken from heart forming regions of stage 4-7 chick embryos can differentiate as intact explants (Le Douarin, 1974;Arias et al, 1987;Gonzalez-Sanchez and Bader, 1990;Yamazaki and Hirakow, 1991) or as individual cells grown at clonal density (GonzalezSanchez and Bader, 1990). Micromolar concentrations of insulin plus selenium and transferrin can substitute for serum in supporting the differentiation of mesoderm explants from stage 5 chick embryos (Yamazaki and Hirakow, 1991;Sugi et al, 1993). With respect to regulatory signals that might influence PCM development, however, these experiments are difficult to interpret since serum and embryo extract contain growth factors that have been implicated in dorsoventral patterning and in the diversification of mesoderm during early embryonic development (Kimelman and Kirschner, 1987;Smith, 1989;Cooke and Wong, 1991;Kimelman et al, 1992).…”

Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative

“…2A). Moreover neither insulin, which can support terminal differentiation of precardiac mesoderm (Yamazaki and Hirakow, 1991), nor LIF, which induces cardiac cell hypertrophy via the cardiotrophin receptor (Sheng et al, 1996), was effective. These findings, plus the inability of some BMP homologues to induce specification, provide strong evidence for BMP family specificity in the induction of cardiac specification.…”