Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm

Abstract

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, we now show that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, we find that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources.

Figure 1

Bmp4^LacZ expression in the mouse embryo and explant cultures. (A) At the time of hepatic specification, ∼8 somite stage, Bmp4^LacZ is expressed in the cardiac mesoderm (cm) and more strongly in the septum transversum mesenchyme (stm), but is absent in the ventral foregut endoderm (fe). By 9.5 d of gestation, the liver bud has migrated away from the cm, and into the stm, which continues to express Bmp4^LacZ (B). (C) In situ hybridization with an antisense probe to Mrg1 (D, sense control). Mrg1 is expressed in explant cultures from 8.25 d mouse embryos, indicating that stm tissue is present in the cultures (beating cardiac cells in the culture are circled with a broken line). BMP4 is expressed in the cultures as well, both within the beating cardiac cells and the mesenchyme, as indicated by in situ hybridization (E). The same Bmp4 expression pattern was observed within beating cardiac tissue and mesenchyme in Bmp4^LacZ mouse explants (F) and in the mesenchyme of cultures that do not contain beating cells (G).

Figure 2

Delay in liver bud morphology in BMP4 homozygous null embryos. Transverse paraffin sections of Bmp4^LacZ heterozygous embryos (A–C) and homozygous null embryos (D,E) showing the developing liver bud region. All sections at the same magnification. Twelve somite stage BMP4 heterozygous null embryos display a thickening of the hepatic endoderm (A, arrowhead), and this tissue begins to move into the septum transversum mesenchyme (arrow) by the 17 somite stage (B). At 22 somites, a liver bud (LB) is distinguishable embedded within the stm (C). By contrast, 14–17 somite Bmp4 null embryos do not display the thickening of the foregut endoderm (arrowhead) evident in Bmp4 heterozygotes of comparable stage (D), but the foregut endoderm (arrowhead) does begin to thicken by the 22 somite stage (E). Therefore, there is a marked delay in formation of the liver bud of BMP4 homozygous null embryos. (F) RT–PCR analysis of hepatic regions (liver bud plus surrounding mesenchyme, lanes marked LB) from mid-teen somite stage embryos indicates the presence of albumin in both Bmp4 heterozygous and homozygous null embryos. Neural head tissue (lanes marked H), which does not express BMP4, isolated from the same embryos is used as a negative control.

Figure 3

BMP signaling is required for hepatic specification in vitro. Foregut endoderm, cardiac mesoderm, and septum transversum mesenchyme tissue was dissected from 2–6 somite stage mouse embryos and cultured for 48 h in the presence of CHO cell supernatant containing noggin protein or control supernatant. (A) RT–PCR for albumin gene expression was performed on RNA isolated from the explants. Treatment with noggin (lanes 1–3) resulted in diminished albumin levels, compared with an actin internal control. By contrast, explants cultured in the presence of control CHO supernatant (lanes 4,5) expressed albumin in all cases, usually at levels higher than an actin internal control. (B) The data represented graphically illustrate a summary of multiple experiments; N, number of explants expressing no, low, medium, or high levels of albumin mRNA, relative to actin. Low, markedly lower levels of albumin than actin; medium, equal levels; high, higher levels of actin than albumin. (C) RT–PCR analysis of explants of foregut endoderm and cardiac mesoderm were cultured 48 h in CHO supernatant, noggin, or control, with the addition of exogenous factors.

Figure 4

BMPs are necessary for primary hepatic induction. (A) Explants of foregut endoderm and cardiac mesoderm were isolated at the designated time points and assayed for albumin expression by RT–PCR. (B) RT–PCR data demonstrate the elevation of pdx-1 expression simultaneously with the suppression of albumin expression, in noggin-treated cultures, compared with the control. (C) RT–PCR data of endoderm cultured alone demonstrate that BMP and TGFβ2 were not efficient inducers of albumin gene expression in cultures of ventral foregut endoderm in the absence of cardiac mesoderm, whereas FGF2 was efficient.

Figure 5

GATA4 expression dependent on BMP signaling. (A) In situ hybridization with a GATA4 antisense probe detects significant GATA4 expression in explant colonies (arrows to purple patches). (B) When colonies are cultured in the presence of noggin, there is a drastic reduction of GATA4 expression, to the background level seen in the sense control (C).

Figure 6

BMP signaling required for morphogenetic growth in vitro. β-Galactosidase staining for Bmp4 expression at 24 h (A) and 48 h (B), indicates that by 48 h the BMP4-expressing domains of the explant have moved away (arrows) from the beating cardiac cells (circled), similar to the movement of nascent hepatocytes and the septum transversum mesenchyme in embryos. The thickest tissue areas appear brown. β-Galactosidase staining of explant colonies indicates that wild-type (C) explants treated with noggin and Bmp4 heterozygous explants treated with control supernatant (F) exhibit a typical spreading morphology. Heterozygous Bmp4 explants cultured in noggin (E), or Bmp4 null explants in noggin (D) do not flatten and remain rounded, indicating a dependency on BMP signaling for morphogenetic growth.

Figure 7

Model for the roles of BMPs in hepatogenesis. Our data suggest that BMPs affect various phases of early of liver development. The septum transversum mesenchyme is in close contact with the presumptive hepatic endoderm, prior to hepatic induction by cardiac mesoderm (left). BMPs induce or maintain Gata4 expression in the foregut endoderm, which may impart hepatic competence. BMPs then act in combination with FGFs to promote liver gene expression and inhibit pancreas gene expression during endodermal patterning (center). Finally, BMPs expressed in the septum transversum mesenchyme are required for proper movement of the hepatoblasts into that tissue (right).