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Fig. 5. The T-maintained allantoic core is required for expansion and coalescence of Flk1-positive angioblasts. (A) Immunohistochemical detection and quantification of Flk1-positive angioblasts (brown) in sections of the allantois. Flk1-positive angioblasts were prominent in the EHF distal core of +/+ (a), T^C/+ (b) and T^C/T^C (c) allantoises (arrowheads). (d) Bar graph showing reduced percentage of Flk1-positive cells in T^C/T^C allantoises. Error bars represent s.e.m.; P-values with respect to +/+ are reported above each bar. Scale bar in c: 100 µm for a-c. (B) Genotypes of allantoic explants are indicated at the lower left of panels a-f. Other indications at lower left (g-m) are WT (wild-type F2, see Materials and methods), transversely (T1/2) or longitudinally bisected wild-type allantoises along the presumptive dorsoventral axis (DV1/2), or axial (A1/2) plane. All explants were immunostained for Flk1 (brown) and counterstained in hematoxylin. (a-c) 24-hour allantoic explant cultures from T^C/+ intercross matings of EHF conceptuses; (d-f) 48-hour allantoic explants from EHF conceptuses. Schematic drawings above g-j demonstrate undivided allantoises (g) and the orientation of each plane of bisection (h-j); each corresponds to the panel directly below (g-j) and, with the exception of the DV1/2, to panels k-m. `1' and `2', and the red lines, indicate the sequence of cuts made by the glass scalpels. The tissue beneath `2' is the posterior primitive streak region. (g,k) Undivided whole allantois explant from EHF (g) or 1-s (k) wild-type conceptus. (h,l) 24-hour proximal T1/2 explants from transversely-bisected EHF (h) and 1-s (l) allantoises. (i) 24-hour EHF longitudinally bisected dorsoventral DV1/2 explants; cut `1' was made at 90° to cut `1' above j. (j,m) 24-hour explants of longitudinally bisected EHF (j) and 1-s (m) axial A1/2 allantoises. The inset in j shows vascularization in an isolated central core of an EHF-stage allantois. Scale bar in m: 500 µm (all panels in B, except for inset in j: 1,250 µm). Abbreviations as in Fig. 1. m, mesoderm; mt, mesothelial cells.

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