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First published online 30 August 2006
doi: 10.1242/dev.02552

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BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

¹ Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, 111 T. W. Alexander Drive, PO Box 12233, MD C4-10, C458, Research Triangle Park, NC 27709, USA.
² Department of Cell Biology, Duke University Medical Center, Durham NC 27710, USA.

View larger version (71K): [in a new window]   Fig. 1. Multiple defects of mesoderm development in Bmpr-MORE embryos. (A-E') Whole-mount views. (A,A') The no allantoic bud stage. Lateral view. Amnion and chorion were formed in control embryos at this stage (A, arrowheads). These tissues were not formed in Bmpr-MORE embryos (A', arrowhead). An acute curvature was observed (A', arrow). (B-C') The head-fold stage. (B,B') Lateral views. (C,C') Frontal views. Amnion and chorion were not formed in Bmpr-MORE embryos (B', arrowheads). Control embryos developed a typical heart crescent (C, arrowheads), whereas Bmpr-MORE embryos did not (C', arrowheads). (D-E') E8.5. (D) Dorsal view. Control embryos developed one column of somites on each side of the neural tube (arrowheads). (D') Ventral view. Mutant embryos developed multiple columns of somites on each side (D', arrowheads). White lines show approximate level of section for F,F'. (E,E') Lateral view. White lines show approximate level of section for G or G'. (F-H') Histological analyses (Hematoxylin and Eosin staining). (F,F') Frontal section. In control embryos, lateral plate mesoderm (LPM) developed between somites (so) (yellow arrowhead) and visceral yolk sac (vy) (black arrowhead). In mutant embryos, mesenchymal cells (blue asterisks) accumulated between somites (yellow arrowheads) and visceral yolk sac (black arrowhead) in mutants (E', see inset). Multiple somites developed to the lateral edge of the embryo (F', yellow arrowheads). (G,G') Transverse section. Between the somites (yellow arrowhead) and the visceral yolk sac (black arrowhead), LPM was observed in control embryos (blue arrowhead) (G). In Bmpr-MORE embryos, mesenchymal cell masses existed between the somites and the visceral yolk sac (G', blue asterisks). Somites developed as multiple rows (G', yellow arrowheads and inset). Heart (he) developed in the anterior region of control embryos (G, red arrowhead) but not in Bmpr-MORE embryos (G', red arrowhead). The anterior half of the mutant embryo consists of neural tissues. Somites develop only in the posterior half of the mutant embryo. (H,H') Sagittal section. Amnion (am) and allantois (al) were formed at this stage in mutant embryos (H', orange arrowheads). (I-L) Transverse section of control or Bmpr-MORE embryos carrying R26R loci after staining for ß-galactosidase activity. K and L are magnified images of I and J, respectively. Recombined (mutant) cells (blue) and heterozygous cells (pink) were distributed evenly among tissues such as somites (yellow arrowheads), LPM (blue arrowhead) or neural tube (nt). (M-P) Transverse section of control and Sox2Cre; Bmpr1aflox/null embryos carrying R26R loci after staining for ß-galactosidase activity. Complete recombination of R26R was observed in the germ layers, namely in somites (O,P). Sox2Cre; Bmpr1aflox/null embryos developed multiple columns of somites (N,P, arrowheads). Scale bars: 170 µm for A; 200 µm for A'; 250 µm for B-C',I,I',M,N; 500 µm for D-E',F-H',K,L,O,P.

View larger version (65K): [in a new window]   Fig. 2. Correct patterning of ectopic somites and improper patterning of LPM in Bmpr-MORE embryos. (A-F') E8.5. Upper side is anterior. Dorsal or ventral view is shown for control or Bmpr-MORE embryos, respectively. Uncx4.1, a caudal marker of each somite, was expressed in a column on each side of control embryos (A, arrowheads). Three or more irregular columns of somites occurred and expressed Uncx4.1 on each side of mutant embryos (A', arrowheads). Double in situ analyses for Uncx4.1 (black) and Mox1 (yellow) (B,B'). Mox1, a marker of somites, was expressed in the entire region of each somite (red arrow) and Uncx4.1 was expressed in the caudal part of each somite in both control and mutant embryos (black arrow). The bracket shows one somite. Epha4 is a rostral presomitic mesoderm marker (C, arrowheads). The expression domain was expanded in Bmpr-MORE embryos (C', arrowheads). Mox1 was expressed in somites (D,E, arrows) but not in LPM in control embryos. In mutants, expression of Mox1 was observed in somites (D',E', arrows) and in LPM (D',E', red arrowheads, see inset). (E,E') Sections of D,D'. Pax1 is expressed in sclerotome in control embryos (F, arrowheads). Pax1 is expressed in expanded somites of Bmpr-MORE embryos (F', arrowheads). (G-I') Expression of LPM markers. (G,G') Ventral view at E8.5. Upper side is anterior. Foxf1 was expressed in LPM of control embryos (G, arrow). The expression of Foxf1 was patchy and weak in Bmpr-MORE embryos (G', arrows). (H-I') Posterior view. In control embryos, Bmp4 was expressed in LPM (arrow), proximal primitive streak (black arrowhead) and allantois (red arrowhead) (H). Bmp4 was not expressed in mutant LPM (H', arrow). Black and red arrowheads show the posterior primitive streak and allantois, respectively (H'). The expression of Lim1 (I,I', arrowhead) in migrating LPM. The Lim1 was not expressed (I') or was decreased in mutant embryos. Scale bars: 300 µm for A-A',C,C',D,D',F,F'; 600 µm for B,B'; 250 µm for G-H'; 125 µm for I,I',E,E'.

View larger version (94K): [in a new window]   Fig. 3. Recruitment of prospective PXM is delayed during gastrulation in Bmpr-MORE embryos. (A,A') No allantoic bud stage. Brachyury was expressed in the primitive streak (A, arrow), node and notochord of control embryos. The rostral end of the notochord was at the anterior of the embryo (A, arrowhead). The expression of Brachyury did not extend to the anterior in Bmpr-MORE embryos (A', arrow and arrowhead). (B-C') The early to late allantoic bud stage. Foxa2 was expressed in the node (B, lower arrowhead) and notochord of control embryos. Upper arrowheads indicate embryonic/extra-embryonic border. As the node is formed at the distal region of the primitive streak, the length of the primitive streak corresponds to the length between two arrowheads. The length of the primitive streak of Bmpr-MORE embryos was shorter compared with that of control embryos (B', arrowheads). Definitive endoderm develops in the mutant embryo (B', arrow). (C,C') Noggin was expressed in the node (lower arrowheads). The length of the primitive streak was shorter in mutant embryos (distance between arrowheads). (D,D') The head-fold stage. (D) Tbx6 was expressed in the mesoderm except axial mesoderm (between arrowheads). (D') The length of the primitive streak appeared to be similar between control and Bmpr-MORE embryos (distance between arrowheads). (E,E') The late streak stage. Lefty2 was expressed in LPM and PXM (between arrowheads). (E) In Bmpr-MORE, Lefty2 expression was decreased (E', between arrowheads). (F-G') The early to late allantoic bud stage. Lefty2 was expressed in migrating LPM and PXM (F, black and red arrowheads, respectively). In Bmpr-MORE, the migrating LPM expressed Lefty2 (F', black arrowhead). More recently recruited mesoderm expressing Lefty2 was observed at the middle and distal regions of the primitive streak (F', red arrow and arrowhead, respectively). Pcdh8 (PAPC on figure) was also expressed in LPM and PXM (G, black and red arrowheads, respectively). In Bmpr-MORE, the migrating LPM expressed Pcdh8 (G', black arrowhead). More recently recruited mesoderm expressing Pcdh8 was observed at middle and distal regions of the primitive streak (G', red arrow and arrowhead, respectively). Lefty2 was not expressed at the head-fold stage in control embryos (H). In Bmpr-MORE embryos, Lefty2 was still strongly expressed at distal and middle embryonic region (H', arrowhead and arrow, respectively). Scale bars: 100 µm for A,A',E,E'; 250 µm for B-D'; 200 µm for F-H'.

View larger version (47K): [in a new window]   Fig. 4. Cell lineage analyses of control and mutant mesoderm cells. DiI was applied to the primitive streak mesoderm (A-F). After culture, embryos were observed under visible light (A'-F') or ultra violet light (A''-F''). (A-B'') DiI was injected into the proximal region of the primitive streak (arrowheads) of control (A) or mutant embryos (B) at the late streak stage. Arrows indicate the embryonic/extra-embryonic border. Embryos injected with DiI were cultured for around 30 hours (A',A',B,B', ventral view). Mesoderm cells derived from proximal primitive streak of control embryos contributed to lateral plate (A', arrowhead and yolk sac, A'', arrow), but not to somites (A',A'', asterisk). In Bmpr-MORE embryos, mesoderm cells derived from proximal primitive streak contributed to regions anterior and lateral to paraxial region (B',B'', arrowheads and arrow, respectively) but apparently not to the paraxial region (B',B'', asterisks). (C-D'') DiI was injected into the anterior-to-distal region of the primitive streak (arrowheads) of control (C) or mutant (D) embryos at the late streak stage. Embryos injected with DiI were cultured for around 30 hours (C' and C'', dorsal view; D' and D'', ventral view). (C',C'') Mesoderm cells derived from the anterior- to-distal primitive streak of control embryos contributed to somites (asterisks) and to an axial structure (red arrows and arrowheads, respectively), but not to lateral plate (blue arrows) (see inset). (D',D'') In Bmpr-MORE embryos, mesoderm cells derived from the anterior-to-distal primitive streak contributed to a region anterior to somites (arrowhead) and an axial structure (arrow). (E-F'') DiI was injected into the middle region of the primitive streak (E,F, arrowhead) of control and mutant embryos of the head-fold stage. Arrows indicate the length of the primitive streak. Embryos injected with DiI were cultured for around 24 hours. (E',E'') Dorsal views. (F',F'') Ventral view. (E',E'') In control embryos, mesoderm cells derived from middle primitive streak contributed to lateral plate mesoderm (arrowheads), but not to somites (asterisks). (F',F'') In Bmpr-MORE embryos, mesoderm cells derived from middle primitive streak mainly contributed to lateral row of somites (arrowhead) and fewer mesoderm cells contributed to medial row of somites (arrow). Scale bars: 100 µm for A-D; 250 µm for and E,F; 300 µm for A-B'',D-F''; 600 µm for C',C''.

View larger version (61K): [in a new window]   Fig. 5. Inhibition of FGF signaling by a specific antagonist for FGFR1 in Bmpr-MORE embryos. (A) Control embryos developed to the late streak to allantoic bud stage. Bmpr-MORE embryos showed a poorly developed amniotic fold (red arrowheads) and less extended primitive streak (yellow arrows) in culture, as was observed in vivo. Inset shows genotyping results for these embryos. Embryos positive for Cre and heterozygous for Bmpr1a-null allele are Bmpr-MORE embryos (inset, pink stars). (B) Control embryos developed to the allantoic bud stage. Bmpr-MORE embryos developed amnion, chorion and allantois (red arrowheads), and a well-extended primitive streak (yellow arrow). Embryos positive for Cre and heterozygous for Bmpr1a-null allele are Bmpr-MORE embryos (inset, pink star). (C-F) Cultured embryos stained with phospho-Erk1/2 antibody. (C) Control embryos cultured with DMSO. Ectoplacental cone (EPC) (arrow) and chorion (arrowheads) were strongly stained. (D) Mutant embryos cultured with DMSO. Ectoplacental cone (arrow) and the amniotic fold (orange arrowhead) showed strong expression. The extra-embryonic ectoderm in the ectoplacental cavity was weakly stained (black arrowheads). (E) Control embryos cultured with 20 µM SU5402. Expression in the ectoplacental cone was not affected (arrow), but that of the chorion was significantly decreased. (F) Mutant embryos cultured with 20 µM SU5402. Expression in the ectoplacental cone was not affected (arrow) but that of the chorion was significantly decreased (arrowheads). Scale bars: 500 µm for A,B; 100 µm for C-F.

View larger version (65K): [in a new window]   Fig. 6. Inhibition of FGF signaling partially rescues the development of somites in Bmpr-MORE embryos. (A-D) Control and Bmpr-MORE embryos at E6.5-6.75 grown in culture for 20-24 hours were examined for Lefty2 expression. Control embryos cultured with DMSO normally expressed Lefty2 (A, arrowheads). Mutant embryos cultured with DMSO showed a proximal shift and decreased expression of Lefty2 (3/3) (B, arrowheads). Control embryos cultured with 20 µM SU5402 showed normal expression of Lefty2 (C, arrowheads). Exposure to SU5402 normalized the expression of Lefty2 in Bmpr-MORE embryos (2/2) (D, arrowheads). (E-H) Control and Bmpr-MORE embryos at E6.5-6.75 were cultured for around 48 hours and examined for the expression of Uncx4.1. (E) Control embryos cultured with DMSO normally expressed Uncx4.1 (arrowheads). (F) Mutant embryos cultured with DMSO showed a significant expansion of somites to the lateral edge of the embryos (arrows) (n=9). (G) Control embryos were cultured with 20 µM SU5402 for 12-15 hours, extensively washed and then cultured with DMSO for around 48 hours in total. In this condition, somites developed normally (arrowheads). (H) Mutant embryos were cultured with 20 µM SU5402 for 12-15 hours, extensively washed and then with DMSO for around 48 hours in total. In six mutant embryos out of 10 tested, partial rescue of somite development was observed (arrowheads). Scale bars: 85 µm for A,B; 100 µm for C,D; 600 µm for E-H.

View larger version (25K): [in a new window]   Fig. 7. Models for the role for BMPRIA in mesoderm development. (A) In wild-type embryos, epiblast cells that are recruited to the anterior primitive streak (red arrow) or to the middle primitive streak (green arrow) are fated to form somites (red square) or LPM (green hexagon), respectively, at the late streak stage. (B,C) Mutant embryos. (B) In mutant embryos, epiblast cells recruited to the anterior primitive streak probably form somites as in wild-type embryos (red arrows and red square). However, many epiblast cells passing through the middle primitive streak (green arrow), which should form LPM, change their fate to form ectopic somites (yellow arrow and yellow square); LPM is consequently reduced (green arrow and hexagon). (C) Alternatively, some of the prospective PXM cells are abnormally recruited to the middle primitive streak (yellow arrow) and form ectopic somites (yellow square). LPM is reduced owing to loss of contributing cells (green hexagon) resulting from reduced BMP signaling.