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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02454

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Brachyury is required for elongation and vasculogenesis in the murine allantois

Department of Anatomy, University of Wisconsin-Madison School of Medicine and Public Health, 1300 University Avenue, Madison, WI 53706, USA.

View larger version (77K): [in a new window]   Fig. 1. TC/TC homozygous mutants produce a single but persistent mRNA transcript and transient protein. (A) RT-PCR reactions for +/+ (LHF to 8-s), TC/+ (LHF to 8-s) and TC/TC (LHF, 5-s) conceptuses. A single T transcript was detected in +/+ samples, a strong T and a faint TC band were detected at all stages in TC/+ samples, and a single TC transcript was detected in TC/TC samples. Minus reverse-transcriptase (-RT) control was negative for all reactions. M indicates marker, a 100 bp gene ruler. ß-actin RT-PCR product was used as a loading control for each corresponding T RT-PCR reaction. (B) Immunohistochemical detection of T and TC (brown) in +/+ (a,b) and TC/TC (c,d) conceptuses in sagittally oriented histological sections. T (a) and TC (c) were localized to the primitive streak (ps) at the EB stage. At LHF, T (b) was localized to the allantoic core and primitive streak, and, with the exception of the ectoplacental endoderm (not shown), TC (d) was not detected in the TC/TC conceptus. The slanted black line in b indicates the previously defined boundary between the allantois and posterior primitive streak (Downs and Harmann, 1997). (e-g) Transverse immunohistochemical sections (6 µm) through a 2-s allantois to highlight the T-defined core domain. The total length of this allantois was 258 µm after fixation. (e) The distalmost T(+) section at 102 µm (39.5% of the total fixed allantoic length). (f) A mid-region section at 48 µm. (g) The most proximal section at 6 µm. ac, amniotic cavity; al, allantois; am, amnion; bi, yolk sac blood island; eve, embryonic visceral endoderm; ps, primitive streak; x, exocoelomic cavity; xve, extraembryonic visceral endoderm. Scale bars: in d, 100 µm for a-d; in g, 100 µm for e-g.

View larger version (118K): [in a new window]   Fig. 2. Allantoic dysmorphogenesis and absence of endothelialization in TC/TC allantoises. (A-F) Hematoxylin and eosinstained sections of allantoises (al) in +/+ (A-C) and TC/TC (D-F) conceptuses. Mesothelial cells (arrowheads) were observed in +/+ and TC/TC allantoises. The dense core of +/+ allantoises (black outline, A-C) was not clearly established or maintained in TC/TC. By 1-s, +/+ allantoises exhibited signs of endothelialization in distal regions (inset, C, arrow), a feature that was not readily apparent in TC/TC. Abbreviations as in Fig. 1. Scale bar in F: 100 µm for A-F.

View larger version (60K): [in a new window]   Fig. 3. Migration of mesoderm into TC/TC allantoises from the primitive streak is normal, but proliferation, cell number and allantoic length are reduced. (A) Whole-mount images of the allantois (al) and primitive streak (asterisk) at the time of (a,d) and 12 hours after (b,c,e,f) injection of DiI (red) into the primitive streak of +/+ (a-c) and TC/TC (d-f) EHF conceptuses. (a,b,d,e) Live images of the posterior end (up) through the yolk sac; (c,f) live images of the dorsal allantoic surfaces after the yolk sac was peeled away. The green line in a,b,d,e represents the boundary between the allantois and primitive streak; the yellow line in c,f outlines the extent of the allantois. Scale bar in d: 500 µm for a-e, 250 µm for f. al, allantois; ch, chorion. (B) Mitotic index (MI) calculated for the allantois (a) and primitive streak (b) for +/+, TC/+ and TC/TC genotypes over the 6-hour window during which allantoic length and cell number were reduced in TC/TC. Error bars represent s.e.m.; P-values for each mutant genotype with respect to +/+ are reported below each data point. (C) Bar graph of allantoic nuclei counts in +/+, TC/+ and TC/TC genotypes for the interval LB to 1-s. (D) Bar graph of allantoic lengths (in µm) in +/+, TC/+ and TC/TC genotypes for the interval EHF to 1-s. In C and D, error bars represent the s.e.m.; P-values for the mutant genotypes with respect to +/+ are reported above each bar.

View larger version (78K): [in a new window]   Fig. 4. Loss of T results in loss of the allantoic core but maintenance of the allantoic mesothelium and chorio-adhesive cells. (A) Cell death in +/+, TC/+ and TC/TC whole-mount allantoises assessed by LysoTracker Red (pink signal) at 1-s (a-e), 3-s (f,g) and 6-s (h-j). Nuclei (blue) were labeled with Hoechst. White line in each panel outlines the allantois; green bar marks the base of the allantois (which in panel h lies just beneath the field of view); `ys' indicates remnants of the yolk sac after dissection to expose the allantois. Scale bar in j: 100 µm. (B) Immunohistochemical analysis in sections showing that Bmp4 (brown) was excluded from the dense proximal region of +/+ (a) and TC/+ (b) allantoises, but was detected in mesothelial and several underlying cell layers. By contrast, all of the cells in the TC/TC allantois in distal (c) and proximal (d) regions of a single allantois were Bmp4 positive. Scale bar in d: 100 µm for c,d; 200 µm for a,b. (C) Immunohistochemical detection of Vcam1 in +/+ (a,c) and TC/TC (b,d) allantoises. (a) Vcam1 localized to distal mesothelial and core cells (arrowheads) of +/+ allantoises at 5-s, but Vcam1 was not detected in TC/TC (b) allantoises, although it was detected in the developing TC/TC heart (ht, inset in b). (c) At 16-s (9.5 dpc), +/+ allantoises united with the chorion (ch) and contained Vcam1 in the distal mesothelial and core cells (arrowheads). (d) TC/TC allantoises were not fused with the chorion, but Vcam1 was observed in the distal-most mesothelium and some core cells (arrowheads). Black lines distinguish the boundary between the posterior streak and the allantois in a,b and d. Scale bar in d: 100 µm for a,b,d; 200 µm for c and inset in b. (D) Bar graph of allantoic length in Vcam1-stained sections reveals that TC/TC allantoises attain lengths >220 µm (red horizontal line), all of which contained appropriately localized Vcam1, similar to that shown in C,d. (E) Bar graph of Vcam1-negative regions in the same histological sections showing maintenance of the 220 µm Vcam1-negative region in TC/TC specimens. Error bars represent s.e.m. in D and E; P-values are reported above each bar. Abbreviations as in Fig. 1.

View larger version (81K): [in a new window]   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), TC/+ (b) and TC/TC (c) allantoises (arrowheads). (d) Bar graph showing reduced percentage of Flk1-positive cells in TC/TC 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 TC/+ 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.

View larger version (62K): [in a new window]   Fig. 6. Pecam1 identifies a nascent vascular scaffolding that is missing in TC/TC allantoises. (A) Whole-mount Pecam1 (brown) and Flk1 (X-gal, blue) in FlklacZ conceptuses (see Materials and methods). (a) Pecam1 (arrows) was detected in a small cluster as early as the LB stage (inset), whereas lacZ-positive cells were dispersed in the allantois (arrowheads in this and all panels in A). By the EHF stage, Pecam1 defined a central vessel that extended distally and proximally (a-c), and ultimately converged upon the developing embryonic dorsal aortae (da; c). Flk1-positive cells were not localized to a patterned central vessel until 6-s (d). (B) Pecam1 detection in +/+ (a) (inset shows localization on the cell surface), TC/+ (b,c), and TC/TC (d) 2-s conceptuses. Formation of the Pecam1-positive central vessel (arrows in a-c) was variable in TC/+ allantoises. A single Pecam1-positive cell was detected in the TC/TC allantois (arrow in d). Pecam1 was not detected in the posterior embryonic region of TC/+ or TC/TC conceptuses (arrowheads mark the site where continuity with the embryonic dorsal aortae was expected). Scale bar in B, part d: 100 µm for A, parts a,b, B parts a-d; 250 µm for A, parts c,d; 25 µm for inset in A, part a; 50 µm for inset in B, part a.

View larger version (84K): [in a new window]   Fig. 7. TC/TC conceptuses exhibit defects in heart and yolk sac. (A) Equivalent sections (insets in a and b) of +/+ (a) and TC/TC (b) 6-s hearts show a reduction in the pericardial cavity (pc) and both myocardial (my) and endocardial (ec) layers in TC/TC. A few Flk1-positive cells (arrowheads) were observed in the TC/TC mutant endocardium. Scale bar in b: 100 µm for a,b. (B) Yolk sacs were isolated from EHF-stage conceptuses (a) and cultured for 24 hours to produce yolk sac spheres. 1 and 2 indicate the level of cuts made in the conceptus to isolate the yolk sac (see Materials and methods). (b) Histological section through a +/+ yolk sac sphere. (c-g) Higher magnification of yolk sac vasculature in +/+ (c), TC/TC (d,e) and TC/+ (f,g) spheres. Flk1-positive endothelium (arrows) was discontinuous and the endoderm layer was not uniformly thick in TC/TC yolk sacs (compare d and e with wild type in c). Presumptive hematopoietic precursor cells (black arrowheads) were present between TC/TC yolk sac layers, but remained in a tight cluster and contained pyknotic cells (red arrowheads). (f,g) Two different regions of the same TC/+ sphere reveal an intermediate phenotype (see text). Abbreviations as in Fig. 1. ht, heart; en, endoderm of foregut; se, surface ectoderm; mt, mesothelium; `x', former exocoelomic cavity of the intact conceptus. Scale bar in d: 100 µm for c-g; 400 µm for b.

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