BMP induction of cardiogenesis in P19 cells requires prior cell-cell interaction(s)

Abstract

Mouse P19 embryonal carcinoma cells undergo cardiogenesis in response to high density and DMSO. We have derived a clonal subline that undergoes cardiogenesis in response to high density, but without requiring exposure to DMSO. The new subline retains the capacity to differentiate into skeletal muscle and neuronal cells in response to DMSO and retinoic acid. However, upon aggregation, these Oct 4-positive cells, termed P19-SI because they "self-induce" cardiac muscle, exhibit increased mRNAs encoding the mesodermal factor Brachyury, cardiac transcription factors Nkx 2.5 and GATA 4, the transcriptional repressor Msx-1, and cytokines Wnt 3a, Noggin, and BMP 4. Exposure of aggregated P19-SI cells to BMP 4, a known inducer of cardiogenesis, accelerates cardiogenesis, as determined by rhythmic beating and myosin staining. However, cardiogenesis is severely inhibited when P19-SI cells are aggregated in the presence of BMP 4. These results demonstrate that cell-cell interaction is required before P19-SI cells can undergo a cardiogenic response to BMP 4. A concurrent increase in the expression of Msx-1 suggests one possible process underlying the inhibition of cardiogenesis. The phenotype of P19-SI cells offers an opportunity to explore new aspects of cardiac induction.

Fig. 1

Clonal morphology of “parental” P19 and variant P19-SI cells. Parental P19 cells were plated at clonal density (see Experimental Procedures) and grown for 5 days. The clones were located, marked and observed using an inverted, phase contrast microscope. The majority of clones (>95%) displayed a “tight” morphology (A1 and A2), while the morphology of the minority clone population was less compact (B). When clones of the latter type were expanded, the cells were found to be “self-inducing” to a cardiac phenotype (see text). The images in A1 and B are the same magnification.

Fig. 2

Assessment of the “undifferentiated state” of P19-SI cells. Mass cultures of P19-SI cells were prepared for antibody staining after 15 hrs (A–C) and after 5 days (D) of culture. (A) Oct 3/4. (B) Oct 3/4 and phalloidin. (C) and (D) Oct 3/4 and DAPI.

Fig. 3

Assessment of endoderm formation in 5-day P19-SI aggregates. Cryosectioned P19-SI aggregates were stained for: (A) Sox 17 (green & arrow) and alpha myosin heavy chain (red & arrowhead); (B) composite of (A) and DAPI staining; (C) composite of (A) and phase contrast image.

Fig. 4

BMP 4 effects on P19-SI cell cardiogenesis. P19 parental and P19-SI cells were cultured as hanging drops (50–100 cells per 30ul) as described in Materials and Methods. (□) P19-SI cells cultured with no BMP 4 added; (◆) 10ng/ml BMP 4 added at the time the hanging drop cultures were established; (▲) 10 ng/ml BMP 4 added in 3ul on day 2; (■) “parental” P19 cells cultured with 0.5% DMSO. Hanging drop cultures (32 per experimental condition) were scored daily for beating activity. BMP 4 added prior to cell aggregation (◆) inhibits cardiogenesis; BMP 4 added after the cells have aggregated (▲) enhances cardiogenesis. Control P19-SI aggregates (□) appear to “self-induce” to cardiocytes as efficiently as do “parental” P19 cell aggregates induced with DMSO (■).

Fig. 5

Effect of aggregation on P19-SI cell gene expression patterns. P19-SI cells were grown in monolayer ( ) or mass aggregate ( ) cultures. Samples were harvested from 13–124 hrs and assayed by quantitative RT-PCR to detect expression of the early mesodermal marker, Brachyury T; known cardiac transcription factors (Nkx 2.5, GATA 4, Myocardin); the cardiogenic signaling molecule, BMP 4; Noggin, Wnt 3a and Msx-1. Data in b) through i) are normalized to HPRT (a) expression. Msx-1 mRNA was not measured in monolayer cultures. See text for complete discussion.

Fig. 6

P19-SI cardiocytes form clusters within aggregates. Six – day 27 beating P19-SI aggregates were pooled, fixed and sectioned as described in Experimental Procedures so as to illustrate clusters of cardiomyocytes within several individual aggregates. The sections were stained with DAPI (blue) to display the nuclei, and with alpha-myosin heavy chain mAb (green) to locate the cardiomyocytes.

Fig. 7

BMP 4 effects on P19-SI aggregate morphology. P19-SI aggregates were grown for 5 days and then photographed (see legend to Figure 2). When 10 ng/ml BMP 4 was added at the time hanging drop cultures were established (B), the resulting aggregates were much smaller than Controls (A). When BMP 4 was added after 2 days of aggregate formation (C), the aggregates developed a more pronounced “zone” of peripheral cells. Arrow shows peripheral endoderm-like layer (see Fig. 3).

Fig. 8

P19-SI cell cardiogenesis is inhibited by exposure to BMP 4 at the beginning of aggregation. P19-SI cultures were established in aggregation conditions either with ( ) or without ( )10ng/ml BMP4. Samples were harvested up to 110 hrs after plating and assayed by quantitative RT-PCR to detect expression of the early mesodermal marker, Brachyury T; known cardiac transcription factors (Nkx 2.5, GATA 4, Myocardin); the cardiogenic signaling molecule, BMP 4; Noggin, Wnt 3a and Msx-1. Data in b) through i) are normalized to HPRT expression. Cardiac transcription factors do not increase if BMP 4 is added before the cells have begun to aggregate. Note also the earlier increase in Msx-1 expression when BMP 4 is added at the time the aggregate cultures are established. See text for complete discussion.

Fig. 9

Analysis of markers for both cardiac and non-cardiac gene expression in P19-SI aggregates. Twenty (20) day-old beating aggregates of P19-SI cells were pooled and mRNA was prepared as described in Material and Methods. “Conventional” reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that genes representative of all three germ layers are expressed in these older aggregates (lane 1). Lanes 2 & 3, showing results with mRNA isolated from control tissues, demonstrate the specificity of the primers.

Fig. 10

Assessment of skeletal muscle induction of P19-SI cells by DMSO. P19-SI cells were aggregated in the presence of 0.5% DMSO. Day 4 aggregates were plated onto plastic tissue culture plates (an attachable surface), cultured for 15 days, then fixed for antibody staining (see Materials and Methods). (A) Phase contrast image of an area containing a cohort of bipolar cells. (B) The same area stained with antibody to skeletal myosin heavy chain. (C) Phase contrast image of another aggregate-derived “island”. (D) The same area stained with antibody to Pax 7 or (E) desmin. (F) Merged image of Pax 7 - and desmin-positive cells.

Fig. 11

Assessment of neuronal cell induction of P19-SI cells by retinoic acid. P19-SI cells were aggregated in the presence of 0.2 uM retinoic acid. After 6 days, aggregates were plated onto plastic tissue culture plates, onto which they attached and spread. (A) After 2 days of incubation (10x objective). (B) The same aggregate-derived “island” after 10 days of incubation (20x objective). Eleven (11)-day cultures were fixed for antibody staining. (C) Phase contrast image of an area containing a putative neuronal “cluster”. (D) The same area stained with antibody to neurofilament protein.