In situ hybridization showing expression patterns of the main mesodermal markers.

A – d’. Expression of somatic LPM marker Irx3 (A, B) and splanchnic LPM marker Foxf1 (C, D) in control (A, C) and Tgfbr1−/− (B, D) E9.5 embryos. Next to the images of the whole mount embryos shown transversal sections through trunk (a – d) and tail (a’ – d’) regions. Red arrowhead indicates ectopic expression of Irx3 in splanchnic LPM, black arrowhead – ectopic expression of Foxf1 in somatic LPM. c – coelomic cavity, cl – cloaca, DA – dorsal aorta, g – gut, g – gut, rDA – recurved dorsal aorta, V – ventral, D – dorsal.

Main vascular tree of the Tgfr1−/− embryos.

A, a, C, c. Wholemount immunostaining for Pecam1 (red) labelling endothelial cells in E9.5 control (A, a) and mutant (C, c) embryos. Nuclei shown in cyan. Transversal sections through regions marked by the dashed lines in A and C are shown in (a1-5) and (c1-5). B, b, D, d. 3D reconstruction of the main vascular tree (red) and the gut (cyan) of the immunostaining shown in (A, a, C, c). Connection between the umbilical artery (ua) and recurved dorsal aortae (rDA) is marked by the arrowhead. Turn of dorsal aortae (DA) where it is connected to rDA is labelled by the arrow. In the mutant this region is enlarged while rDA is short (compare A, a3 and B to C, c2-5, and D). D – dorsal, L – lateral, V – ventral, c – coelomic cavity, g – gut.

Posterior primitive streak contributes to the pericloacal mesenchyme and gut endoderm.

A, B. Whole mount in situ hybridization showing expression of Foxf1 in E8.5 Tgfbr1−/− (A) and control (B) embryos. al – allantois, g – gut, PS – primitive streak. L – lateral view, V – ventral view, D – dorsal view. While arrow indicates PS/allantois junction. C-d. β-galactosidase cell tracing showing descendance of the primitive streak in the E10.0 (C, c) and E9.5 (D, d) embryos. c and d show transversal sections through regions marked by the dashed lines in C and D. Black arrowhead shows β-galactosidase staining in the pericloacal mesenchyme. Black arrow in d shows β-galactosidase+ cells in the tail gut endoderm. The asterisk in c indicates the cloaca. The asterisk in d indicates the tail gut.

Effects of Tgfbr1 in the LPM, but not in the tail bud, are mediated by Isl1.

A-F”. Whole mount in situ hybridization showing expression of Sox2 (A-C) and Uncx4.1/Tbxt (D-F) in the E9.5 control (A, D), Isl1−/− (B, E), and Tgfbr1−/− (C, F) embryos. Isl1−/− embryos form tail bud (black arrows), unlike Tgfbr1−/− embryos. Insets in the right top corners show dorsal view of the tail bud region. G, h”. Whole mount in situ hybridization showing expression of Foxf1 in the E9.5 control (G) and Isl1−/− (H) embryos. g-g” and h-h” show transversal sections through the regions marked by the dashed line in G and H. Foxf1 is ectopically expressed in the splanchnopleure of the posterior region of the Isl1−/− (black arrowhead in h’ and h”). I-J’. Whole mount in situ hybridization showing expression of Uncx4.1/Tbx4 in E9,5 control (I, I’) and Isl1−/−(J, J’) embryos. Tbx4 is not expressed in pericloacal mesenchyme (yellow arow) and hindlimb buds (yellow arrowheads) of Isl1−/− mutants. da – dorsal aorta, rda – recurved dorsal aorta, c – coelomic cavity, D – dorsal, V – ventral.

Main vascular tree of the Isl1−/− embryos.

A, C. Wholemount immunostaining for Pecam1 (red) labelling endothelial cells in E9.5 control (A) and mutant (C) embryos. a, c. Optical transversal sections through regions marked by the dashed lines in A and C. B, b, D, d. 3D reconstruction of the main vascular tree (red) and the gut (cyan) of the immunostaining shown in (A, a, C, c). In the mutant recurved dorsal aorta (rDA) is underdeveloped and connection between dorsal aortae (DA) and the umbilical artery (ua) is established by a small vessel (white arrowhead in c1 and c2). Branches of DA are enlarged in the Isl1−/− and merge together at the posterior end and ventral to the gut (c3-5, d). D – dorsal, L – lateral, V – ventral, c – coelomic cavity, g – gut.

Endoderm of the Tgfbr1 KO.

A – b. Expression of Foxa2 in E9.5 control (A, a) and Tgfbr1 KO (B, b) embryos. a, b. show sagittal sections though the tail region. C - d. Keratin 8 staining of the cloaca region in the control (C,c) and Tgfbr1−/− (D,d) E10.5 embryos. Tgfbr1−/− do not initiate enlargement of the cloacal cavity. Insets show higher magnification of the cloacal membrane (cm). c and d show transversal optical sections marked by the dashed line in C and D. Yellow arrowhead in d shows keratin 8 staining in expanded LPM of the Tgfbr1 mutant embryo. E – e’. Apela expression in the posterior region of the E9.5 control (E, e) and mutant (E’, e’) embryos. e and e’ show transversal sections of regions marked by the dashed line in E and E’. F–f’. Apela expression in the posterior region of the E10.5 control (F, f) and mutant (F’, f’) embryos. f and f’ show transversal sections of regions marked by the dashed line in F and F’. Black arrow – gut endoderm, black arrowhead – Apela-expressing cells in LPM of the mutant embryo. V–ventral, L–lateral, cl – cloaca, c – coelomic cavity, da – dorsal aorta, g – gut, hg – hindgut.

Analysis of the contribution of the visceral endoderm to the embryonic gut.

GFP expression from the Afp-GFP transgenics was analyzed at E8.5 (A-B’), E9.5 (C-D’’) or E10.5 (E-F’’’) in wild type (A, A’, C-C’’, E,E’) or Tgfbr1−/− (B, B’,D-D’’, F-F’’’) embryos. C’ and D’ show a 3D image of the embryo, and C’’ and D’’ show transversal sections. F’ and F’’ show transversal sections through the caudal part of F’. The embryonic endoderm was labeled by immunofluorescence against Epcam. Arrows in A’, B’ indicate the hindgut; arrows in C’’ and E’ indicate the tail gut; arrows in D’’ and F’ indicate the cloacal membrane. Size bars: A, B: 200 μm; A’, B’: 100 μm; C, D: 300 μm; C’, D’: 200 μm; C’’, D’’: 150 μm; E: 300 μm; F: 200 μm; E’: 150 μm; F’: 100 μm.

Tail gut endoderm have contribution from the posterior pool of tailbud cells.

A. Whole mount in situ hybridization showing expression of Apela in E10.5 wild type embryo. B. Sagittal section through the region marked by rectangle in A shows presence of Apela-stained structure posterior to tail gut endoderm. b. Series of transversal sections though the Apela-expressing region posterior to the gut endoderm (marked by square bracket in B). C-D”. Sagittal optical sections through the tail region of the whole-mount immunostaining for Keratin 8 (white) in E10.5 control (C-C”) and Tgfbr1−/− (D-D”) embryos. Nuclei are shown in blue. Squares in C’ and D’ show the pool of epithelial cells posterior to the tail gut tube. This region coincides with newly formed endodermal cells expressing Apela shown in B, b. C”. D” higher magnification of the region marked by square in C’ and D’. A – anterior, P – posterior, g – gut. E, E’. Whole mount images of embryos injected with DiI in the Apela-positive region of the tail bud posterior to the gut endoderm, just after injection (E) or after 20 hours of incubation (E’). The magnification of the tail bud in the inset shows the absence of label in the gut tube. F-h’. Optical sections from two-photon images of the embryo in E’ to show the presence of DiI cells in the gut tube. F-H show sagittal sections; f-h’ show transverse sections. F-f’’ show the DiI channel; tail, neural tube (nt) and gut (g) are outlined with a dashed line. G-g’ show DiI and DAPI channels together. H-h’ show magnification of DiI labeling in the gut tube (white arrowheads).

Schematic representation of Tgfbr1 activity on the posterior epiblast/PS region during the trunk to tail transition. At E8.5 embryo undergoes turning, associated with anterior relocation of the allantois along the ventral side of the embryo. In wild type embryos (top panel) Tgfbr1 acts upstream of Isl1, which induces hindlimb (HL) formation from the somatic LPM, marking the posterior limit of this mesodermal compartment. Additionally, Isl1 is involved in formation of the pericloacal mesenchyme (PCM), likely from the Foxf1+ region in the posterior PS (shown in green), and in the development of the recurved dorsal aorta (rDA). Tailgut (tg) growth is, at least in part, supplied by the cells located in the tail bud (endodermal Apela+ cells are shown in purple). At E10.5 the trunk to tail transition is completed, resulting in the formation of the posterior trunk structures, including HL, cloaca (Cl), PCM and the connection of embryonic/extraembryonic (umbilical artery - ua) blood circulation via rDA. Tail growth continues generating neural tube (dark grey), presomitic mesoderm (not shown), and tg (blue). In the absence of Tgfbr1 (bottom panel) Isl1 is not activated, hindlimbs are not induced from the LPM, which, instead, keeps extending posteriorly. PCM and tg progenitor cells are misrouted and trapped in the posteriorly extended LPM. The rDA is underdeveloped. Development of Tgfbr1 mutants is halted around E10.5.