Role of Sonic hedgehog in the development of the trachea and oesophagus

Patterns of Shh expression and complementary Ptc1 patterns, in saline-treated E10.5 mouse embryos. In situ hybridisation for Shh (a,c-h) and Ptc1 (b,i,j,k): whole embryos and vibratome sections from whole-mount hybridisation experiments on E10.5 saline-treated embryos. (c-h, i-k) form 2 series of progressively more caudal sections. (a) Shh expression is seen in ventral areas of the brain, the floor plate (Fp), the notochord (arrowhead in a), the foregut (Fo) and hindgut (Hg) endoderm, including the lung buds (arrow in a) and the limb buds (Lb). (b) Ptc1 expression flanks the ventral neural tube (arrow in b) and ventral midline in the brain and also is found in the somites (S), the limb buds, and the mesoderm surrounding the foregut and hindgut. (c-k) Shh is expressed strongly in the floor plate of the neural tube (Fp) and the notochord (Nt), whereas Ptc1 is expressed in the mesoderm adjacent to the ventral neural tube and around the notochord (arrowhead in i). (c,i) At the level of the pharyngeal foregut (Ph), Shh is expressed in both the dorsal and ventral endoderm (arrows in c) but is specifically absent from the endoderm of the pharyngeal pouches (arrowheads in c). Ptc1 is expressed next to the Shh-expressing endoderm (arrows in i). (d,e,j,k) Shh is expressed in the ventral endoderm of the laryngotracheal groove (Lt) with a sharp boundary between ventral expressing and dorsal nonexpressing cells (arrowheads in d,e). Mesodermal expression of Ptc1 also is restricted to the ventral foregut (arrowheads in j,k) although the boundary is not as distinct because of adjacent perinotochordal expression. (f,g) Further caudally, Shh expression is restricted to the endoderm of the prospective trachea (Tr) (arrowheads). (h) At the level of the lung buds (Lb), the Shh dorso-ventral boundary still exists (arrow) but appears less distinct. D, diencephalon; T, telencephalon. Scale bar: a,b, 500 μm; c-k, 100 μm.

Changing patterns of Shh expression and complementary Ptc1 patterns in E11.5 saline-treated embryos. Slide in situ hybridisation for Shh (a-f) and vibratome sections from whole-mount hybridisation experiments for Ptc1 (g-l). (a-f and g-l) represent corresponding levels in progressively more caudal sections through 2 comparable embryos. (a,g) In the pharyngeal foregut (Ph), Shh is expressed ventrally and dorsally but is absent laterally from the endoderm of the pharyngeal pouches (arrowheads in a mark boundary). Ptc1 is expressed adjacent to the ventral (but not dorsal) Shh expressing endoderm (arrow in g). (b,h) At the level of the laryngotracheal groove (Lt), Shh is expressed in a well-defined ventral domain (arrows in b) with Ptc1 expression in the adjacent mesoderm (arrow in h). (c,i) At a slightly more caudal level, Shh is still ventrally expressed (arrowhead in c), but the expression appears to be shifting dorsally. This is reflected by a dorsalward shift in mesodermal Ptc1 expression (arrow in i). (d,j) At an even more caudal level, the pattern of Shh expression has reversed with a well-defined dorsal Shh expression domain (arrowheads in d). Ptc1 is now expressed in the mesoderm adjacent to the dorsal foregut (Fo) (arrow in j). (e,k) Shh is expressed in the oesophagus (Oe), and Ptc1 is expressed in the perioesophageal mesoderm. (f,l) Shh expression in the oesophagus and lung buds (Lb). Ptc1 expression around the lung buds is stronger than perioesophageal expression at this level (asterisk in l). Fp, floor plate; Nt, notochord; Tr, trachea. Scale bar: 100 μm.

Dorso-ventral pattern of Shh expression in the oesophagotrachea of E12.5 Adriamycin-treated embryos reestablishes a similar pattern to saline-treated control embryos. In situ hybridisation for Shh on transverse sections from saline- (a,c,e) and Adriamycin-treated (b,d,f) E12.5 embryos. (a,b) At E12.5, the cranial-most part of the laryngotracheal groove (Lt) has yet to separate from the oesophageal foregut. Both uniformly express Shh in saline- and Adriamycin-treated embryos. (c,d) At a slightly more caudal level, the separated trachea (Tr) is completely negative for Shh, whereas the oesophagus (Oe) retains a strong signal (c). In the Adriamycin-treated embryo (d), the undivided oesophagotrachea (Oet) reflects this dorso-ventral pattern in expression with a sharp boundary between dorsal expressing and ventral nonexpressing cells (arrows). (e,f) This pattern persists further caudally and is unchanged as far as the level of the trifurcation to form lung buds and fistula to the stomach. Scale bar: 100 μm.

The ventral-to-dorsal switch in Shh expression is disturbed in Adriamycin-treated embryos. In situ hybridisation for Shh on saline- (a,c) and Adriamycin-treated (b,d) embryos. Vibratome sections, at the level of the atrial chamber of the heart (a,b) and the level of the atria (c,d), from whole-mount experiments. (a,c) In normal development, Shh expression shifts completely from the ventral, tracheal-prospective epithelium at E10.5 (arrowheads in a mark boundaries), to the oesophageal epithelium at E11.5 (after tracheo-oesophageal separation). (b) In 2 of 5 E10.5 Adriamycin-treated embryos, the undivided foregut (Fo) expresses Shh uniformly along its dorso-ventral axis. (d) All E11.5 Adriamycin-treated embryos with an undivided oesophagotrachea exhibit relatively diffuse staining along the dorso-ventral axis of the common structure. In this embryo, the signal appears stronger at the 2 poles and contrasts sharply with the pattern seen in saline-controls (c). Scale bar: 100 μm.