Specific Bmp4 knock-down by RNAi induces a range of developmental defects post-implantation. (A) RT-PCR of single embryos cultured in vitro for 24 h following electroporation, and embryos recovered at E5.25 and E5.75 following development in utero. Glyceraldehyde 3-phosphate dehydrogenase (GAP3DH) was used as an internal control for cDNA loading. (B) Average Bmp4 mRNA levels (Bmp4/GAP3DH ratio) of embryos electroporated with Bmp4 dsRNA (dsBmp4) shown as a percentage of those of GFP dsRNA (dsGFP)-treated controls. Pale blue, 20 dsBmp4-electroporated embryos cultured in vitro for 24 h (control embryos: 20, dark blue). Light blue, 10 dsBmp4-electroporated embryos recovered at E5.25 and E5.75, respectively, following in utero development (control embryos: 10). Standard deviation bars are indicated. (C) Morphological phenotypes of dsBmp4 (B-E, G-J) and control dsGFP -electroporated embryos (a, f) recovered at E6.5 and E7.5 (DIC micrographs). Anterior faces left when it can be identified. E6.5 and E7.5 represent the embryonic time at collection; actual embryonic stages may vary due to Bmp4 RNAi. dsBmp4-treated embryos are generally reduced size and acquire an abnormal round shape (c-e, i, j); or present gross abnormalities in (g) or lack recognizable embryonic structures (j). Some embryos show an underdeveloped epiblast relative to the ExE (h). The well developed ExM-derived allantois as seen in the control (f) is not normally observed in dsBmp4-treated embryos. Bar, 200 μm. (D) Distribution of morphological phenotypes observed past gastrulation. Emb. day, embryonic day; Nor, morphologically normal; Def, defective; Dl'd, delayed, Art'd, arrested. (E) Whole-mount in situ hybridization with anti-sense probes of indicated genes carried out on control dsGFP RNA (a-l) and dsBmp4 RNA -electroporated embryos (a'-q'). The developmental stages shown represent the embryonic time at collection; actual embryonic stages may vary due to Bmp4 RNAi. Over 50% of the dsBmp4-electroporated embryos [developmentally delayed but otherwise normal and morphologically normal (group1)] express much reduced amounts of mesodermal Brachyury (E6.5 n = 8/15; E7.5 n = 8/16), Eomes (E6.5 n = 5/9; E7.5 n = 4/7) and Lhx1 (E6.5 n = 3/5; E7.5 n = 3/6), suggesting that gastrulation was initiated but failed to continue further (a'-c', g'-h'). In contrast, all defective embryos (group2) lack expression of these genes altogether (E6.5 n = 2, 2, 1; E7.5 n = 3, 3, 2, respectively), indicating that they failed to gastrulate (m'). The expression of visceral endoderm Cer-l, Lefty-1 and Lhx1 markers was also abnormal in the dsBmp4-treated embryos. While Lhx1 expression was strongly downregulated in the VE of 55% of group 1 embryos (g', h') (E6.5 n = 3/5; E7.5 n = 3/6), it was completely absent in defective embryos (n = 3, not shown). Similarly, Lefty-1 transcripts were absent (E6.5 n = 1; E7.5 n = 2, not shown) or restricted to the DVE (E6.5 n = 1; E7.5 n = 2) in defective embryos (O'). In 55% of group 1 embryos (n = 11), Lefty-1 expression was found at or close to the DVE (f') (E6.5 n = 3/6; E7.5 n = 3/5). Accordingly, the expression of Cer-l was also found to be restricted to the DVE in all defective embryos essayed (n') (n = 5). In over 50% (16/31) of the embryos of group 1, the expression was either distally restricted and/or mislocalized, with ectopic transcripts scattered around the embryonic region in the lateral VE at both E6.5 (d') (n = 9) and E7.5 (e') (n = 7), denoting a lack of migration of the DVE cells and ectopic expression of Cer-l. As early as E5.5 and E5.75 the Cer-l expression domain was extended towards both anterior and posterior regions of the VE overlying the epiblast (k', p') (n = 8); restricted distally (l') (n = 2) or absent q' (n = 3) in a total of 16 embryos. Red arrows show the midline of the anterior-posterior axis of the embryo; black arrows delimitate the boundaries of Cer-l expression in the VE. Bmp4 expression in the ExE was already detectable by E6.5 in dsBmp4-electroporated embryos (i'). Epiblast Oct4 expression was normal at both E6.5 (j') and E7.5 (not shown). Anterior faces left when it can be identified, except in d' where both lateral (L) and anterior (A) views are presented. Bar, 200 μm.