Transgenic rescue of aganglionosis and piebaldism in lethal spotted mice.

Complete colonization of the gut by enteric neural precursors depends on activation of ednrB and Ret receptors by their respective ligands, edn3 and gdnf. Mutations that eliminate expression of either ligand or either receptor produce intestinal aganglionosis in rodents and humans. Embryos homozygous for the lethal spotted (ls) allele, a loss of function mutation in the edn3 gene, have no ganglion cells in their terminal large intestines and are spotted, due to incomplete colonization of the skin by melanocyte precursors. Expression of edn3 in enteric neural precursors of transgenic mice compensates fully for deficient endogenous edn3 in ls/ls embryos. The effects of the edn3 transgene are dose-dependent, as lower levels of expression in one line prevent aganglionosis in only a subset of animals and reduce, but fail to eliminate, piebaldism. In contrast, expression of neither constitutively active Ret nor activated ras in enteric neural progenitors alters the severity of aganglionosis or piebaldism in ls/ls mice. Given the spatial and temporal pattern of edn3-transgene expression, our results suggest that edn3/ednrB signals are not required prior to the arrival of crest cells in the gut and endrB stimulation elicits distinct cellular responses from Ret or ras activation. Dev Dyn 2000;217:120-132.

Sympathoadrenal hyperplasia causes renal malformations in Ret(MEN2B)-transgenic mice.

The tyrosine kinase receptor Ret is expressed in the ureteric bud and is required for normal renal development. Constitutive loss of Ret, its co-receptor gfralpha-1, or the ligand glial cell line-derived neurotrophic factor results in renal agenesis. Transgenic embryos that express a constitutively active form of Ret (Ret(MEN2B)) under the control of the dopamine-beta-hydroxylase (DbetaH) promoter develop profound neuroglial hyperplasia of their sympathetic ganglia and adrenal medullae. Embryos from two independent DbetaH-Ret(MEN2B)-transgenic lines exhibit renal malformations. In contrast with ret-/- embryos, renal maldevelopment in DbetaH-Ret(MEN2B)-transgenic embryos results from primary changes in sympathoadrenal organs extrinsic to the kidney. The ureteric bud invades the metanephric mesenchyme normally, but subsequent bud branching and nephrogenesis are retarded, resulting in severe renal hypoplasia. Ablation of sympathoadrenal precursors restores normal renal growth in vivo and in vitro. We postulate that disruption of renal development results because Ret(MEN2B) derived from the hyperplastic nervous tissue competes with endogenous renal Ret for gfralpha-1 or other signaling components. This hypothesis is supported by the observation that renal malformations, which do not normally occur in a transgenic line with low levels of DbetaH-Ret(MEN2B) expression, arise in a gdnf+/- background. However, renal maldevelopment was not recapitulated in kidneys that were co-cultured with explanted transgenic ganglia in vitro. Our observations illustrate a novel pathogenic mechanism for renal dysgenesis that may explain how putative activating mutations of the RET gene can produce a phenotype usually associated with RET deficiency.

Methotrexate-resistant form of dihydrofolate reductase protects transgenic murine embryos from teratogenic effects of methotrexate.

Methotrexate, a potent inhibitor of the ubiquitously expressed enzyme dihydrofolate reductase, induces limb and facial anomalies that resemble vascular disruptions in their evolution and final outcome. Previous studies suggest that inhibition of dihydrofolate reductase is responsible for methotrexate-induced embryopathy, although specific sites of methotrexate activity have not been well defined. In this report, we show that constitutive expression of a methotrexate-resistant form of dihydrofolate reductase in transgenic embryos and their placentas ameliorates methotrexate teratogenicity. However, expression of the transgene in maternal tissues had no significant protective effect. The results confirm the role of dihydrofolate reductase inhibition in the pathogenesis of methotrexate-induced birth defects and provide a foundation for future studies of targeted transgene expression in select embryonic or placental cell populations.

Abnormal microenvironmental signals underlie intestinal aganglionosis in Dominant megacolon mutant mice.

Dominant megacolon (Dom) is a mutation in an uncharacterized murine gene which is associated with intestinal aganglionosis and the focal absence of melanocytes in heterozygous animals. The phenotype of Dom/+ heterozygotes is similar to the lethal spotted and piebald lethal mutations, which are due to defects in endothelin-mediated intercellular signals. In this study, the DbetaH-nlacZ transgenic marker for enteric neural crest cells is used to study the distribution of enteric neurons and their precursors in Dom/+ mice and embryos. Vagal neural crest-derived cells in wild-type embryos colonize the gut in a cranial-to-caudal progression. In Dom/+ embryos, colonization was retarded from the earliest stages examined (embryonic Day 11.0), including progression through the small intestine. The early onset of this defect contrasts with impaired neural crest colonization associated with the lethal spotted and piebald lethal mutations which manifest only in the large intestine. Analysis of Dom/+ - +/+ aggregation chimeras indicated that defective colonization is not an autonomous (intrinsic) property of Dom/+ neuroblasts, but like lethal spotted and piebald lethal, the Dominant megacolon mutation directly or indirectly affects microenvironmental signals which influence the migration, proliferation, and/or survival of enteric neural crest cells.

Intercellular signals downstream of endothelin receptor-B mediate colonization of the large intestine by enteric neuroblasts.

Mice homozygous for the piebald lethal (sl) mutation, which have a complete deletion of endothelin receptor-B, fail to form ganglion cells in the distal large intestine and are nearly devoid of cutaneous melanocytes. These phenotypic features stem from incomplete colonization of the hindgut and skin by neural crest-derived neuroblasts and melanoblasts, respectively. We have used expression of a transgene, dopamine-beta-hydroxylase-nlacZ, to study colonization of the enteric nervous system in sl/sl embryos and sl/sl <--> wild-type chimeric mice. Enteric neuroblasts derived from the vagal neural crest colonize the developing foregut, midgut and distal small intestine of sl/sl embryos in a cranial-to-caudal manner indistinguishable from sl/+ or +/+ embryos. However, colonization of the large intestine is retarded and the distal large intestine is never colonized, a developmental defect identical to that observed in lethal spotted (endothelin-3 deficient) embryos. The coat pigmentation and relative distributions of mutant and wild-type ganglion cells in sl/sl <--> wild-type chimeras indicate that the defect associated with endothelin receptor-B gene deletion is not strictly neuroblast autonomous (independent of environmental factors). Instead, intercellular interactions downstream of the endothelin receptor-B mediate complete colonization of the skin and gut by neural crest cells.

Induction of a heat shock response (HSP 72) in rat embryos exposed to selected chemical teratogens.

A monoclonal antibody to the 72 kD heat shock protein (HSP 72), Western blot analysis and 2-D gel electrophoresis/autoradiography were used to determine whether selected chemical teratogens induced the synthesis and accumulation of HSP 72 in postimplantation rat embryos exposed in vitro. The chemical teratogens studied include N-Acetoxy-2-acetylaminofluorene (N-Ac-AAF), cadmium chloride (CAD), cyclophosphamide (CP), sodium arsenite (AS), and sodium salicylate (SAL). Exposures to test chemicals were selected that produced obvious embryotoxicity characterized by abnormal development and growth retardation. Of the five chemical teratogens studied, AS and SAL induced the synthesis and accumulation of HSP 72 in day 10 rat embryos. The kinetics of HSP 72 accumulation, however, differed between AS- and SAL-treated embryos. Maximal levels of HSP 72 were observed 24 hours after AS exposure and 10 hours after SAL exposure. N-Ac-AAF, CD, and CP induced obvious embryotoxicity; however, none of these chemical teratogens induced HSP 72 at any of the timepoints assayed. Although only a small sample of chemical teratogens was studied, our results suggest that the heat shock response, characterized by the synthesis and accumulation of HSP 72, is not a general biomarker for chemical teratogens.

Accumulation of heat shock protein 72 (hsp 72) in postimplantation rat embryos after exposure to various periods of hyperthermia (40 degrees -43 degrees C) in vitro: evidence that heat shock protein 72 is a biomarker of heat-induced embryotoxicity.

A monoclonal antibody to the 72 kDa heat shock protein and Western blot analysis were used to determine the induction, accumulation and turnover of hsp 72 after day 10 rat embryos were exposed to elevated temperatures (40 degrees-43 degrees C) for various lengths of time (2.5 minutes to 18 hours). Embryos exposed to temperatures that exceed the normal culture temperature (37 degrees C) by 4 degrees C or more for as little as 2.5 minutes (43 degrees C) or 15 minutes (41, 42 degrees C) synthesized and accumulated detectable amounts of heat-inducible hsp 72. Hsp 72 could not be detected by Western blot analysis of proteins from embryos cultured at 40 degrees C or below. Once induced, hsp 72 can be detected in embryos for 24-48 hours after they are removed from the hyperthermic conditions and returned to normothermic conditions. Our results also indicate that hsp 72 is induced by all hyperthermic exposures that induce alterations in rat embryo growth and development; therefore, hsp 72 is a potential biomarker for heat-induced embryotoxicity.