Fig. 2

A Spatial expression of Hau-slit and Hau-robo1 in Helobdella embryos during germinal plate formation by double FWMISH. Anterior is up in all views. Column I shows ventral view of embryos at stage 8 to early 9 expressing Hau-slit as longitudinally iterated dots directly apposed across the leading edges of the left and right germinal bands (white arrowhead), which coalesce zipper-like in anteroposterior progression along the future ventral midline (open arrowhead) to form a germinal plate. Column II shows ventral view of embryos at stage 8 to early 9 expressing Hau-robo1 as longitudinally iterated dots directly apposed across the leading edges of the left and right germinal bands, similar to but broader than the pattern of Hau-slit. Column III shows merged image indicating the overlap of Hau-slit and Hau-robo1 expression during germinal plate formation. The cross-sectional images at the bottom row show the expression region in the germinal plate at the early stage 9 embryos. Dotted lines in Column III indicate the planes of sections for the early stage 9 embryos. Yellow indicates the overlap of Hau-slit and Hau-robo1 expression. Hau-slit expression is at the presumptive visceral mesoderm (open arrowhead), ventral and dorsal segmental ganglia (arrowhead). Hau-robo1 expression consists of most or all cells in the segmental ganglia (open arrowhead). B Cell lineage analysis of Hau-slit and Hau-robo1 expression. a: Schematic of injection strategy to identify lineage specific expression of Hau-slit and Hau-robo1 transcripts (adapted from [40]). In a stage 6a embryo labeled with tracer fluorescein-conjugated dextran amine (FDA)) in the left N, OPQ, and M lineage. b: Pseudo-colored image (combined brightfield and fluorescence imaging) of a stage 9 embryo showing the relative positions and contributions of lineage tracer-labeled M (red), N (blue) and OPQ (green) lineages (adapted from [45]). Cross-section of stage 9 embryos showing the lineage of origin of cells expressing Hau-slit and Hau-robo1 transcripts (c-h). c: Cross-section of an M-labeled (green) stage 9 embryo processed to reveal Hau-slit transcripts (red); three sets of M lineage cells expressing Hau-slit in lateral (open arrowhead), visceral and ventral mesoderm (white arrowhead) appear yellow. D: An embryo similar to that in c, but processed for Hau-robo1, reveals expression of Hau-slit in visceral mesoderm (white arrowhead). E: Cross-section of an N-labeled (green) stage 9 embryo processed to reveal Hau-slit transcripts (red); a set of superficial N lineage cells expressing Hau-slit adjacent to the ventral midline (white arrowhead) appear yellow. F: An embryo similar to that in e, but processed for Hau-robo1, reveals expression of Hau-robo1 through most of the N lineage (white arrowhead). G–h: Cross-section of an OPQ-labeled (green) stage 9 embryos processed to reveal Hau-slit and Hau-robo1 transcripts (red) shows no detectable expression of Hau-slit (g) and a few cells expressing Hau-robo1 (h) in lateral ectoderm (white arrowhead). C Representative images of midline formation in control and slit knockdown embryos. a: Hau-slit transcripts are expressed in anterior to posterior germinal plate (white arrowhead) of control embryos. b: Knockdown of Hau-slit (n = 20) results in decreased expression of Hau-slit transcripts and disturbed coalescence of the germinal plate (yellow arrowhead). c: The relative mRNA expression of Hau-slit is shown as the mean with the standard error of the mean (SEM). The dotted line indicates the level from which cross-sections were taken. Scale bar: 100 μm in Column III; 20 μm in bottom cross-sectional image of Column III; 20 μm in Bb-g; 100 μm in Ca-b