BMP2b is required for normal ENS and intestinal smooth muscle development. (A,a,c,e;B,a,b,e;C,a,b,e,f,g) wild-type embryos, (A,b,d,f;B,c,d,f;C,c,d,h,i,j) BMP2b MO embryos. (A,b) Dorsal viewing for the vagal region of 36 hpf embryos hybridized with crestin probe showed a defective vagal NCC migration to the anterior end of the intestine in BMP2MO morphants(26/37, 70.3%). (A,d,f) Lateral view of 60 hpf embryos hybridized with phox2b probe showing a failure of precursors migration to the anterior of the intestine in BMP2MO morphants(26/34, 76.5%). (B,c,d) Immunofluorescence showed the developmental defect of intestinal smooth muscle in the intestine of BMP2b MO embryos(22/30, 73.3%). (B,f) Lateral view of the intestine for 72 hpf embryos hybridized with α-SMA showing a reduce of α-SMA expressing in the intestine of BMP2b MO embryos(31/39, 79.5%). (C,c,d,h,i,j) Lateral and transverse view the vagal region of 34 hpf embryos labeled with GFP for foxd3 showed a failure of vagal NCC migration in BMP2MO morphants(29/36, 80.5%). (D) BMP2 overexpression results in an increase in enteric neurons. Lateral views of phox2b in enteric ganglia of 72 hpf control (D,a) and HS embryos (D,b). (D,c) Bar graph summarizes the results from one of three independent BMP2 HS experiments (six zebrafish in different groups). The phox2b expressing cells in the HS group is approximately 1.5 fold of ctrl group (n = 6, t-test, ∗P < 0.05, compared with ctrl group). (E) BMP2b is necessary to maintain the anterior-posterior axis of embryos display and liver development. (a) control embryos(114/120, 95%). (b) BMP 2b MO treated embryos that have the anterior–posterior axis of embryos displayed defect(43/60, 71.7%). (c) control embryos(68/80, 85%). (d) BMP2b MO treated embryos, the liver became smaller on day 3(51/76, 67.1%). Bar, 100 μm. Notice: here “26/37” means 26 out of the 37 embryos had defective NCC migration like in the image showed. The following statistical numbers for embryos like this have the same meaning in the Figure legends. Blue arrows (A) indicate the migrating enteric precursors. White arrowheads (A) indicate phox2b expressing cells in the intestine. Asterisk (B) indicate gut. Yellow arrowheads (C) indicate vagal region. White arrows (C) indicate the migrating enteric precursors.
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