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First published online 2 December 2004
doi: 10.1242/dev.01570

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Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung

¹ Department of Pathology, University of Colorado Health Sciences Center, The Children's Hospital, 1056 East 19th Avenue, Denver, CO 80218, USA
² Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA
³ Division of Pathology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

View larger version (52K): [in a new window]   Fig. 1. NKX2.1 activation in foregut endoderm occurs in close proximity to liver specification. (A) Sagittal view of a mouse embryo at the 6-somite stage, demonstrating tissue regions isolated for RT-PCR analysis and used for explant assays. Broken line indicates plane of embryo sections in C-F. (B) RT-PCR analysis of total RNA from designated tissue regions at different somite stages. Tissue microdissected from day 10 embryos (E10), with and without reverse transcriptase (-RT), used as positive and negative controls, respectively. Actin probe used as control for total RNA integrity; FOXA2 probe is used to ensure actin levels reflected isolated endoderm. (C-J) Embryo sections at designated somite stages demonstrating expression of albumin (green) and NKX2.1 (red) in mouse foregut endoderm by double immunofluorescence. (C-F) Transverse embryo section at the 10-somite stage encompassing foregut and region of cardiac mesoderm. (C) Boxed area in DAPI image (to detect nuclei) is magnified in D-F. Yellow signal in merged image (F) reflects nonspecific (present in all filters) fluorescent staining of acellular material frequently seen in the foregut pocket (also seen in Fig. 2B) (Kaufman, 1992; Jung et al., 1999). (D,E) More cells in the foregut endoderm express albumin than NKX2.1 protein. Markers (boxed magnification) are colocalized in merged image (F). (G-J) Albumin and NKX2.1 expression in E9.5 sagittal embryo section. Boxed area in G (DAPI image) is magnified in H-J and encompasses the cardiac and presumptive lung (LuR) and liver (LiR) regions. (K-T) Immunostaining of single cells generated from ventral endoderm isolated at 10- and 14-somite stages. Endoderm cells were dissected away from adjacent mesoderm, dispersed, spun down onto a slide, double stained with antibodies for albumin and NKX2.1, and counterstained with DAPI. Positive cells tended to occur in aggregates. K-N reflect the same cells at the 10-somite sage; (O-R) same cells at 14-somite stage double-stained with albumin and NKX2.1. Arrows indicate cells that express both albumin and NKX2.1. Merged image with DAPI reflects expected cytoplasmic localization of albumin (O) and nuclear localization of NKX2.1 (R). The granular nature of the fluorescent signal was also seen in control adult tissues treated identically. (S) Quantification of albumin-positive, NKX2.1-positive and albumin/NKX2.1-positive ventral endoderm cells relative to total cells on cytospin slides at 8-, 10- and 14-somite stages.

View larger version (67K): [in a new window]   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.

View larger version (72K): [in a new window]   Fig. 5. FGFs are sufficient to activate proximal and distal lung markers. Ventral endoderm explants cultured alone (A-C) or in the presence of 500 ng/ml FGF2 (D-F) for 1 day. Adjacent sections were immunostained with the designated markers.

View larger version (68K): [in a new window]   Fig. 7. Selective inhibition of FGFR1 and FGFR4 diminishes induction of NKX2.1 in ventral endoderm co-cultured with cardiac mesoderm. (A-C) Sagittal sections of mouse embryos at five- (B) and seven- (C) somite stages, demonstrating the onset and expansion of FGFR4 protein expression (arrows) in the foregut (fg) relative to the cardiac domain (cm); (A) adjacent section at five-somite stage, stained with FGFR1. FGFR4 antibody staining of foregut endoderm cultured alone (D) or in the presence of FGF2 100 ng/ml (E), or FGFR4 antibody staining of foregut endoderm cultured alone (D) or in the presence of FGF2 100 ng/ml (E) or cardiac mesoderm (G). (F-Q) Ventral endoderm/cardiac mesoderm explants cultured for 2 days after a single treatment of FGFR1-IgG or FGFR4-IgG, compared with rabbit IgG control (each 1 µg/ml). Same or adjacent sections (F-I;J-L;MO) labeled for designated marker by double immunofluorescence. Frequent colocalization of FGFR4 and NKX2.1 is shown in a merged image (I), with boxed inset demonstrating membranous staining for FGFR4 (nuclear staining is for NKX2.1). Explants in K,L,O have limited or no appreciable staining with albumin (green) or NKX2.1 (red). (P) Influence of FGFR1 and FGFR4 receptor inhibition on protein expression of albumin and NKX2.1 expression in ventral endoderm co-cultured with cardiac mesoderm. For graphic representation, four out of 10 explants in the respective groups were cryosectioned, immunostained for albumin and NKX2.1, and the number of positive cells out of total cells present in the section was determined. Results expressed as mean±s.e.m. (%). Explants treated with FGFR4-IgG exhibit a significant difference in inhibiting NKX2.1 expression (t-test: P<0.0001) but not albumin expression (t-test: P<0.4). FGFR1-IgG treatment effectively diminishes expression of both albumin and NKX2.1 (t-test; P<0.0001). (Q) RT-PCR analysis of RNA from ventral endoderm co-cultured with cardiac mesoderm and exposed to a single dose of soluble FGFR1/IgG-Fc (sFR1-IgG), FGFR4/IgG-Fc (sFR4-IgG) or SU5402; control explants for SU5402 assay cultured in the presence of DMSO. Expression for cardiac troponin and FOXA2 reflect integrity of the cardiac mesoderm and endoderm, respectively.

View larger version (55K): [in a new window]   Fig. 3. Dose-dependent induction of NKX2.1 and albumin by FGF2. Ventral endoderm (VE) explants were initiated with a single dose of FGF2 and cultured for 24 or 48 hours; similar results were obtained from 1 or 2 days of exposure. (A-H) Immunostaining for NKX2.1 and albumin in serial sections (A,B) or the same explant (C-H). Bright signal indicates positive staining. (I) RT-PCR analysis of RNA from cultured ventral endoderm with heparin sulfate (50 ng/ml) and the designated concentration of FGF2. Control explants (embryonic neural tissue, midsection, tail) failed to express albumin or NKX2.1 in the presence of FGF2, at all doses. (J) Number of explants (positive signal/total tested), at indicated concentration of FGF2, which expressed NKX2.1 or albumin protein (immunostaining, I) or mRNA (RT-PCR, R).

View larger version (57K): [in a new window]   Fig. 4. Dose-dependent induction of NKX2.1 and albumin by FGF1. Induction of NKX2.1 or albumin protein (I) or gene (R) expression in ventral endoderm treated with recombinant FGF1, at designated concentrations, within 2 hours of isolation. Double-immunostaining (A-H) or RT-PCR analysis (J) of explants performed after 1-2 days of culture. Arrow indicates focal positive expression. (G-I) Ventral endoderm treated with FGF8B, at similar concentrations, did not consistently induce NKX2.1 or albumin; adjacent section stained with FOXA2 as marker of ventral endoderm. (K) Number of positive explants/total tested, at indicated concentration of FGF1.

View larger version (45K): [in a new window]   Fig. 6. FGF signaling in dorsal midgut endoderm. (A) Midgut dorsal endoderm cultured alone (A, lane 1) or exposed to the designated concentration of FGF (A, lanes 2-4; B-E) for 2 days. Explants were analyzed for the activation of the designated marker by RT-PCR analysis (A) or immunofluorescence (B-E).

View larger version (32K): [in a new window]   Fig. 8. A model for patterning of the foregut endoderm through threshold of FGF activity. Schematic representation of embryo expression data shown in Figs 1 and 7, using sagittal cross-section through cardiac and foregut regions. (A) The onset of FGF2 expression in the five- to six-somite cardiac mesoderm (Jung et al., 1999) (data not shown) occurs just prior to albumin activation in the foregut endoderm, which predominantly expresses FGFR1. This threshold of FGF2 (broken arrow) is sufficient for liver but not lung induction. (B) Coincident with NKX2.1 activation in the seven- to eight-somite ventral endoderm, is the expansion of FGFR4 expression and the addition of FGF1 with FGF2 in the developing heart (Jung et al., 1999). The dose of FGF signaling (broken arrow) is now sufficient to induce lung specification, with frequent co-expression of NKX2.1 and albumin in the same cells. Broken arrows reflect that induction of liver and lung by cardiac signaling may be direct or indirect (see Discussion). Endogenous FGF2 in the adjacent notochord (arrow) may induce albumin expression in dorsal foregut endoderm. High doses of FGF, inhibitory to albumin in vitro, could contribute to refinement of cell commitment (C). Functional data (Fig. 7) indicates that induction of NKX2.1, but not albumin, relies on intact FGFR4 activity. (C) Once cell specification occurs, presumed positive and negative feedback between neighboring germ layers, including the mesenchyme of the septum transversum, maintain and refine cell commitment. Dark red indicates cardiac region.