Fig. 5

A Expression of Hau-slit, Hau-robo1, and Hau-robo2 in Helobdella embryos at late stage 11. FWMISH was carried out before the appearance of pigmentation in the eyespots. All embryos show dorsal view and are oriented anterior to left. Aa’, Ab’ and Ac’ indicate the lateral view of the head region shown in Ba-c. a: Hau-slit is expressed in the middle line of the head and the developing ventral segmental ganglia with greater intensity in the body region. In addition, Hau-slit expression is seen in visceral mesoderm and/or endoderm of the intestine and muscle associated with rectum (white arrowhead). a’: In the magnified lateral view of head region, the expression of Hau-slit is clearly visible as a linear patch in cerebral ganglia (open arrowhead). b: Hau-robo1 is expressed in the nerve cord, visceral mesoderm and/or mesoderm of the intestine, presumptive primordial germline (yellow arrowhead), and the region corresponding to the seven fused segments of the posterior sucker (yellow bracket). b’: In the magnified lateral view of head region, Hau-robo1 is expressed in the eye precursor (yellow open arrowhead) and cerebral ganglia (yellow arrowhead). c–c’: Hau-robo2 is expressed selectively in the developing eyespots (white arrowhead). B Differential expression pattern between Hau-slit and duplicated Hau-robo orthologs in Helobdella embryos at late stage 11 using fluorescent in situ hybridization. DAPI staining was performed to visualize cross sectional morphology by nuclei labeling. White arrows indicate sectional region. a: Dorsal view of head region of late stage 11 embryo showing Hau-slit staining the eyespots (white arrowhead) and in a narrow longitudinal patch of tissue between them. b: Dorsal view of late stage 11 embryo showing Hau-robo1 staining the eyespot (white arrowhead). c: Hau-robo2 transcripts are detected in the eyespots (white arrowhead) and phaosomes (open arrowhead). d: Hau-r opsin was used as a marker to compare the differential expression patterns in eyespots and phaosomes (open arrowhead). Dorsal view of head region of late stage 11 embryo showing Hau-r opsin staining the eyespots (white arrowhead). C Co-localization of Hau-slit and Hau-robo1 transcripts. a”-b: Overlapped expressions of Hau-slit and Hau-robo1 transcripts are shown in eyespots (white arrowhead). Scale bar: 75 ​μm in Aa-c; 50 μm in Aa'-c'; 50 μm in Ba-d; 30 µm in Ba'-d'; 50 μm in Ca-a''; 20 µm in Cb