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Fig. 2. Close proximity and sufficient quantity of cardiac mesoderm is required to
reconstitute lung specification in vitro. (A,B) Sagittal view of the
seven-somite embryo with foregut endoderm and cardiogenic mesoderm designated
by immunostaining for FOXA2 (HNF3ß) and muscle-specific actin (msa),
respectively. (C-N) Explants containing ventral endoderm (VE) or ventral
endoderm with cardiac mesoderm (CM) were isolated from 2- to 5-somite embryos
and cultured for 2 days. (C) Circled area in boxed inset denotes beating
cardiac cells in captured phase image prior to fixation of colony. Antibody
staining for msa was used to delineate the cardiac domain in sectioned
explants and to confirm the absence of contaminating cardiac mesoderm in
nonbeating endoderm cultures. (D,E) RT-PCR analysis of RNA from cultured
ventral endoderm explants either with (CM) or without (VE) beating cardiac
cells, using designated lung (NKX2.1, SP-C, CC10), liver (albumin) and
pancreas (PDX1) markers. Endoderm or CM mesoderm cultured alone never
expressed albumin or NKX2.1. In contrast to albumin, which is expressed in 95%
of ventral endoderm/cardiac mesoderm co-cultures, lung markers are induced in
56% of explants with cardiac mesoderm. PDX1 is typically expressed in isolated
ventral endoderm. (F-N) Endoderm explants were serially sectioned and the same
section (G,H,J,K) or adjacent sections (M,N) were double-labeled for NKX2.1
and albumin using indirect immunofluorescence. (F,I,L) Merged image for FOXA2
and MSA reflects location of ventral endoderm and cardiac cells, respectively.
Bright signal denotes positive immunoreactivity. NKX2.1-positive cells, when
present, are within endoderm contiguous with the cardiac domain (J), while
albumin-expressing cells were frequently seen in more distal locations (C,N).
No appreciable positive staining is seen in G,H,M. Arrows in I,J indicate
overlapping NKX2.1 and FOXA2 signal; arrow in N indicates albumin-expressing
cells distant from the cardiac domain.
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