Fig. 8. Cyclopamine treatment disrupts formation of the gut and coelomic mesenchyme (see Materials and Methods for details of treatment). (A,B) Dorsal views of the anterior and posterior portions, respectively, of a control embryo at stage 11 (200 hours AZD) showing the extent of the proboscis (double headed white arrow in A) and crop (double headed white arrow in B); note the well-differentiated crop caeca (black arrowheads). (C-E) Combined bright-field and fluorescence views of transverse sections (ventral is down; counterstained with Hoechst 33258, blue) through the proboscis (at level of black arrows in A), esophagus (left black arrows in B) and crop (right black arrows in B), of a comparable embryo, at roughly the levels indicated in A and B. Note that the proboscis (arrow in C) has well-defined inner and outer layers separated by a middle layer containing relatively few nuclei and that the visceral mesoderm has spread to form a thin layer of nuclei surrounding the crop [arrowhead in E; E' is an enlarged view of the box in E showing the ventral blood vessel (arrowhead)]. (F,G) Lateral views of the anterior and posterior portions of a sibling embryo treated with 5 µM cyclopamine. Note the shortened proboscis (double headed white arrow in F) and crop (double headed white arrow in G) and the incomplete differentiation of the crop caeca (white arrowheads in G) relative to those in the control. (H,I) Dorsal views of the anterior and posterior portion of another embryo treated with 5 µM cyclopamine. The proboscis (double headed arrow in H) and crop (double headed arrow in I) are similarly affected, whereas the intestine (box in I) appears normal. (J,K) Lateral views of the anterior and posterior portions of a sibling embryo treated with 10 µM cyclopamine. The proboscis (extent indicated by white double headed arrow in J) is shortened and has failed to invert. The esophagus (extent indicated by black double headed arrow in J) is thin and elongated. The crop lacks even the large posterior caeca, but intestine and rectum are still present (arrowhead in K). (L-O) Views of transverse sections at successively more posterior levels (indicated by black arrows and arrowheads in J) through the proboscis (white arrow in L), esophagus (white arrows in M and N) and crop (white arrow in O), of a comparable cyclopamine-treated embryo. In such embryos, the tri-radiate geometry of the proboscis lumen is less well defined (arrowhead in L) and the middle ring of circumferential muscles is missing (compare L with C). In addition, the coelomic lacunae remain largely devoid of cells (compare N with D) and visceral mesoderm (arrowhead in O) has failed to expand around the crop, but the ventral blood vessel is still present (arrowhead in O'). Scale bar, 100 µm in A, B, F-K; 50 µm in C-E, L-O; 12 µm in E' and O'.

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