Fig. 3

A Expression of Hau-slit, Hau-robo1, and Hau-robo2 in Helobdella embryos from stages 9 to late 10 as revealed by FWMISH. Dotted lines in panels Ac and Af indicate the planes of sections for the views shown in Ba-c and Bd,e, respectively. Anterior is to the left in all panels. a: Hau-slit is expressed in the dorsal ganglia (white arrowhead) of the head, the developing ventral segmental ganglia and at the lateral edges of the expanding germinal plate during stage 9, with stronger expression in the anterior portion of the embryo. b: During stage 10, Hau-slit transcripts are detected along all the ventral segmental ganglia, in the anteriormost part of the everted proboscis (white arrowhead), and at the lateral edges of the germinal plate, with increasing intensity in the posterior region. c: At late stage 10, Hau-slit expression persists in the anteriormost proboscis and continues all along the ventral segmental ganglia, with greater intensity in the posterior region. As the expanding edges of the germinal plate begin to fuse dorsally, the lateral domains of Hau-slit expression come to occupy the dorsal midline (white arrowhead). In addition, Hau-slit is expressed in visceral mesoderm and/or endoderm of the intestine (white arrowhead) and muscle associated with rectum (yellow arrowhead). d: During stage 9, Hau-robo1 transcripts levels are higher in the anterior to middle ventral segmental ganglia than the posterior region; expression is also seen in the developing proboscis and dorsal ganglia of the head (open arrowhead). e: At stage 10, Hau-robo1 is expressed in the everted proboscis (open arrowhead) and throughout the segmental ventral ganglia, but is still stronger in the anterior half. f: At late stage 10, Hau-robo1 is expressed more strongly in the posterior half of the nerve cord than in the anterior half, especially the region corresponding to the seven fused segments of the posterior sucker (bracket); also at this stage the expression has become more punctate both within the segmental ganglia and elsewhere, including the foregut (yellow bracket). h-j: In marked contrast to Hau-robo1, Hau-robo2 is expressed selectively and with increasing abundance in the developing eyespots from stage 9 to 10. Arrow indicates eyespots. B Sectioned specimens reveal further details of Hau-slit and Hau-robo1 expression in late stage 10 embryos. Dorsal is up in all panels. a: Anterior cross section shows Hau-slit transcripts at dorsal and ventral midline cells (white arrowhead), in subsets of ganglionic cells and in the outer ring of proboscis (open arrowhead). b: A more posterior cross section shows Hau-slit transcripts at dorsal and ventral midlines and ganglion (white arrowhead), but not in posterior proboscis. c: A tangentially horizontal section through the posterior part of the embryo highlights Hau-slit expression in ganglia, ventral midline of the body wall, and in visceral mesoderm and/or endoderm of the posterior midgut (intestine, white arrowhead). d: Anterior cross section reveals prominent ring of Hau-robo1 expression in the outer portion of the anterior proboscis (yellow arrowhead), and in subsets of ganglionic cells (yellow arrowhead). e: Cross section through reproductive segments shows Hau-robo1 transcripts in a U-shaped pattern corresponding to the reproductive organs (yellow arrowhead), as well as proboscis musculature and ganglia (yellow open arrowhead). Scale bar: 200 μm in A; 20 μm in B