Embryonic gut anomalies in a mouse model of retinoic Acid-induced caudal regression syndrome: delayed gut looping, rudimentary cecum, and anorectal anomalies

Abstract

Vitamin A and its derivatives such as retinoic acid (RA) are important signaling molecules for morphogenesis of vertebrate embryos. Little is known, however, about morphogenetic factors controlling the development of the gastrointestinal tract and RA is likely to be involved. In the mouse, teratogenic doses of RA cause truncation of the embryonic caudal body axis that parallel the caudal regression syndrome as described in humans. These changes are often associated with anomalies of the lower digestive tract. Overlapping spatiotemporal expression of retinoic acid receptor-beta (RAR beta) and cellular retinol-binding protein I, CRBPI, with Hoxb5 and c-ret in the gut mesoderm imply possible cooperation required for proper neuromuscular development. To determine susceptibility and responsiveness of the developing gut and its neuromusculature to exogenous retinoids we used a mouse model of RA-induced caudal regression syndrome. The results showed that stage-specific RA treatment both in vivo and in vitro affected gut looping/rotation morphogenesis and growth of asymmetrical structures such as the cecum together with delayed differentiation of the gut mesoderm and colonization of the postcecal gut by neural crest-derived enteric neuronal precursors. These observations demonstrate that RA has a direct effect on gut morphogenesis and innervation.

Figure 1.

Morphological changes in affected embryos and gastrointestinal tract after in vivo RA treatment compared with controls. A: Control, vehicle-treated embryo (E10.5) with normally developed tail (arrow) and small proportion of apoptotic cells detected in the prevertebral region and at the caudal extremity of the tail bud. B: Degeneration of the embryonic tail because of apoptosis (dark red color) detected with TUNEL method in the caudal extremity of the tail bud in affected embryo at E10.5. C and D: Difference in the appearance of E12.5 control embryo (C) and embryo affected with RA-induced truncation of caudal body axis (D). E: Caudal extremity of a gut whole mount showing the anorectal region (arrow) and neural crest cell precursors expressing c-ret in control embryo. F: Saggital vibratome section through the caudal extremity of the gut in the anorectal region shown with arrow in D showing normally developed rectum after immunostaining with Hoxb5. Arrow shows mesodermal layer surrounding gut epithelium. G: Ring-like constriction (arrowhead) in the caudal extremity of E16.5 gut whole mount after RA treatment in the corresponding anorectal level as shown in E. H: Saggital vibratome section through the caudal gut extremity shown in G. Arrowhead shows ring-like constriction. I: Saggital vibratome section through the ring-like malformation (arrowhead) obstructing the anorectal canal. J: Saggital vibratome section through the blind-ending caudal gut extremity (arrowhead) at E16.5 after RA treatment. K: Appearance of the cecal bud (asterisk) at E11.5 in gut whole mount in control embryo. L: Poorly formed cecal bud (asterisk) at (E11.5) in RA-treated embryo. M: Well-developed cecal bud(asterisk) in gut whole mount at E12.5 in control, vehicle-treated embryo. N: Gut whole mount (E12.5) after RA treatment showing a poorly formed cecal bud (asterisk). O and P: Ventral view of gut whole mounts (E12.5); control (O) showing well-formed second gut loop, RA-treated (P) showing an absence of the second gut loop (asterisk) resulting in the malrotation of this gut region, which prevents the cecum to be exposed on the ventral site of the gut as seen in the control. Scale bars, 2 mm (C and D), 1 mm (A, B, E, G, and H), 0.5 mm (F, K, L, O, and P), and 0.2 mm (M and N). Abbreviation: e, epithelium.

Figure 2.

Gut whole mounts and transverse vibratome sections after in vivo RA treatment showing α-SMA, Hoxb5 immunostaining, and in situ hybridization for c-ret compared with controls. A: Onset of α-SMA immunoreactivity in rostral, prececal gut in E11.5 control gut (arrow). The rest of the prececal gut shows the reflection of α-SMA-positive vitelline artery attached to the ventral side of the gut. B: Dorsal (top) and ventral (bottom) view of gut whole mounts (E11.5) after RA treatment showing absent α-SMA immunostaining (arrow) in the same region of the prececal gut as shown in A. The immunostaining seen in the prececal gut is the reflection of α-SMA-positive vitelline artery (arrowhead) attached to the dorsal side of the gut as shown at the top. C: Control gut whole mount (E12.5) showing α-SMA expression in the whole prececal gut up to the cecal level (asterisk). D: Gut whole mount (E12.5) after RA treatment showing delayed onset of α-SMA expression in the rostral, prececal gut (arrow). E: Saggital vibratome section through the gut of control embryo (E12.5) showing well-developed circular muscle layer in the peripheral mesoderm of the small intestine (arrow). F: Saggital vibratome section through RA-treated gut at E12.5 showing α-SMA-positive (arrow) poorly differentiated peripheral mesoderm in the rostral small intestine. G: Control gut whole mount (E11.5), in situ hybridization for c-ret showing neural crest-derived enteric neuronal precursors colonizing the whole prececal gut, the cecum (asterisk) and rostral, postcecal gut. H: In situ hybridization to c-ret-expressing neural crest cells present in the prececal gut up to the cecum level (asterisk) at E11.5 after RA treatment. I: Control gut whole mount (E12.5), in situ hybridization for c-ret in migrating neuronal precursors colonizing almost the whole large intestine (arrow). Caudal extremity of the large intestine shown by an asterisk. J: Gut whole mount (E12.5) after RA treatment, in situ hybridization for c-ret in neuronal precursors colonizing a large intestine adjacent to the cecum (arrow). Caudal extremity of the large intestine as in I shown by an asterisk. K: Control gut whole mount (E12.5) immunostained for Hoxb5 in the enteric neuronal precursors in the small intestine up to the cecal bud (arrow). L: Gut whole mount (E12.5) after RA treatment immunostained for Hoxb5 in the enteric neuronal precursors present in the small intestine up to the cecal bud (arrow). Stomach shown with an asterisk. Scale bars, 1 mm (A–D, G, I–L), 0.5 mm (H), 100 μm (E and F).

Figure 3.

Hoxb5 immunohistochemistry and in situ hybridization for c-ret in whole mounts of embryo, gut, and vibratome sections after RA treatment compared with controls. A: Control gut whole mount (E11.5) immunostained for Hoxb5 showing rostral expression boundary in the stomach (asterisk) and caudal expression limit at the cecal bud level (arrow). B: Gut whole mount (E11.5) after RA treatment immunostained for Hoxb5 showing a rostral shift of the rostral expression boundary into the esophagus (asterisk). C: Transverse vibratome section of control gut whole mount (E11.5) at the level of the lung buds showing no Hoxb5 immunostaining in the esophagus (asterisk). D: Transverse vibratome section of a gut whole mount (E11.5) after RA treatment showing the presence of Hoxb5-positive cells in the esophagus (asterisk). E: Control gut whole mount (E11.5) hybridizedfor c-ret (blue) and immunostained for Hoxb5 (red) showing the presenceof c-ret-positive cells in the vagus lateral to the esophagus (arrowhead). No c-ret-positive cells are seen in the esophagus. F: Gut whole mount (E11.5) hybridized for c-ret (blue) and immunostained for Hoxb5 (red) after RA treatments showing the presence of c-ret-positive cells in the esophagus (asterisk). G: Whole-mount control embryo (E10.5) showing the anterior expression boundary of Hoxb5 in the embryonal hindbrain (arrowhead). H: Whole-mount embryo (E10.5) after RA treatment showing a rostral shift of Hoxb5 expression to the preotic hindbrain (arrowhead). I: Whole mount of a control embryo (E10.5) with c-ret expression present in the cranial sensory ganglia (arrow). J: Whole mount of an embryo (E10.5) after RA treatment showing ectopic c-ret expression in the midbrain (arrowhead) and caudally in the dorsal rami of the spinal nerves (asterisk). Arrow indicates the otic vesicle located between rhombomeres 4 and 6 of the rostral and caudal hindbrain. Scale bars: 1 mm (A, E–J), 0.5 mm (B), 200 μm in (C and D).

Figure 4.

Whole-mount in situ hybridization for c-ret, immunostaining for Hoxb5 and α-SMA, and TUNEL method for apoptotic cells in gut explants after 72 hours in culture in controls and after RA treatment. A: α-SMA immunoreactivity in a cultured, control gut explant showing strong expression in the prececal gut (asterisk) close to the well-developed cecal bud (arrow). B: Gut explant treated overnight with RA showing an absence of α-SMA immunoreactivity in the prececal gut including the poorly developed cecum (arrow). C: Control gut explant after 72 hours in culture showing c-ret expression. Note the well-developed gut loops and the cecal bud (arrow). C-ret-expressing neural crest cells colonize the entire gut including postcecal region (asterisk). D: Dorsal side of RA-treated gut explant showing c-ret expression in the esophagus (arrowhead) and the prececal gut but not in the postcecal gut (asterisk). Note the poorly developed gut loops and the rudimentary cecum (arrow). E: Control gut explant showing strong Hoxb5 expression in the prececal gut (arrowhead). F: RA-treated gut explant showing a rostral shift of Hoxb5 expression to the esophagus (arrowhead). Cecum (arrow). G: TUNEL-stained RA-treated gut explant showing no apoptotic cells. Cecum (arrow). H: TUNEL-stained control gut explant showing the presence of apoptotic cells in the prececal gut up to the cecal bud (arrow) after apoptosis induced by anisomycin. Scale bars, 1 mm (A–H).