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Fig. 8 Anal development in mutants with disrupted notochords. Brightfield images of live embryos (A,B,E,F,H,I,L,M) and confocal scans of embryos stained with α-actin-phalloidin (green) and propidium iodide (red) at 120 hpf (C,G,Q). Live embryos were injected with fluorescein salt at 72 hpf,. Brightfield/fluorecent image merge with the fluorescent channel only shown in the inset (D,H,R). The overlay in (B,F,J,L,N,P) shows the approximate identity of the relevant tissues, with the posterior gut in green and pronephric ducts in blue. (A-D) ntl embryos have normal pronephric ducts that open adjacent to the posterior gut. The posterior gut is much thicker in both ntl alleles, but still opens with a normal sized orifice (91% perforate, n11/12). (E-H) flh mutant embryos develop a perforate anus and pronephric duct, which open independently of one another. (87% perforate, n20/23). (I-L) The presence or absence of a notochord near the urogenital opening does not affect its development in bal and doc embryos, which develop a functional anus. (M,N) ctd embryos develop a normal anus and pronephric opening despite undulations in the notochord close to the anus. (O-R) spt embryos have varying pronephric defects, and in some cases lack them altogether; even in the absence of the pronephros (as shown here) the development of the posterior gut is relatively unperturbed (95% perforate, 40/42), although the anal sphincter appears to be absent in most embryos (A,B,E,F,I,J,Q,R) black arrows mark the visible ejection of faecal matter from the anus.