Requirement of mesodermal retinoic acid generated by Raldh2 for posterior neural transformation

Abstract

Studies in amphibian embryos have suggested that retinoic acid (RA) may function as a signal that stimulates posterior differentiation of the nervous system as postulated by the activation-transformation model for anteroposterior patterning of the nervous system. We have tested this hypothesis in retinaldehyde dehydrogenase-2 (Raldh2) null mutant mice lacking RA synthesis in the somitic mesoderm. Raldh2(-/-) embryos exhibited neural induction (activation) as evidenced by expression of Sox1 and Sox2 along the neural plate, but differentiation of spinal cord neuroectodermal progenitor cells (posterior transformation) did not occur as demonstrated by a loss of Pax6 and Olig2 expression along the posterior neural plate. Spinal cord differentiation in Raldh2(-/-) embryos was rescued by maternal RA administration, and during the rescue RA was found to act directly in the neuroectoderm but not the somitic mesoderm. RA generated by Raldh2 in the somitic mesoderm was found to normally travel as a signal throughout the mesoderm and neuroectoderm of the trunk and into tailbud neuroectoderm, but not into tailbud mesoderm. Raldh2(-/-) embryos also exhibited increased Fgf8 expression in the tailbud, and decreased cell proliferation in tailbud neuroectoderm. Our findings demonstrate that RA synthesized in the somitic mesoderm is necessary for posterior neural transformation in the mouse and that Raldh2 provides the only source of RA for posterior development. An important concept to emerge from our studies is that the somitic mesodermal RA signal acts in the neuroectoderm but not mesoderm to generate a spinal cord fate.

Fig. 1

RA is unnecessary for neural induction but needed for neuronal differentiation. (A) Double hybridization to detect Raldh2 and Cyp26a1 mRNAs in an E8.5 wild-type (WT) embryo. (B–C) RARE-lacZ expression (RA activity) in E8.5 wild-type and Raldh2^–/– (–/–) embryos; Raldh2^–/– embryos totally lack RA activity. (D–E) Sox1 and (F–G) Sox2 mRNAs were examined at E8.5 by whole-mount in situ hybridization; these Sox genes are markers of neural induction and were still expressed all along the neural tube of Raldh2^–/– embryos (Raldh2^–/– embryos have reduced neural plate folding in the hindbrain region, but neural tube closure occurs in the spinal cord). (H–I) Pax6 mRNA in wild-type and Raldh2^–/– embryos at E8.5; the Raldh2^–/– embryo lacks Pax6 mRNA in the posterior neural tube, whereas the wild-type embryo exhibits expression throughout the hindbrain and spinal cord. (J–K) Olig2 mRNA is not detected in the posterior neural tube of an E8.5 Raldh2^–/– embryo, but expression is still observed in a small domain of the forebrain. (L–M) Nkx6.1 mRNA is still detected in the brain and posterior neural tube of an E8.5 Raldh2^–/– embryo with a posterior border of expression at the anterior end of the tailbud (in both wild-type and mutant there is a gap of expression in the hindbrain, with the mutant having a larger gap).

Fig. 2

Raldh2 functions cell non-autonomously for generation of RA detected in the spinal cord and tailbud. Wild-type embryos at E8.5 (10 somite stage) were compared for expression of the target gene Olig2 (A–D), for the source of RA synthesis by Raldh2 (E–H), and for the distribution of RA activity detected with RARE-lacZ (I–L). For each, transverse sections are shown at somite 5 (s5), somite 9 (s9), and the tailbud (tb). Raldh2 expression is limited to the somites, anterior presomitic mesoderm, and anterior lateral plate mesoderm. A high level of RA activity (RARE-lacZ) is observed throughout the dorsoventral axis of the neural tube (J,K); the developing motor neuron field (marked by Olig2 expression) is included within this region of high RA activity (B,C). Weaker RA activity is also detected in the tailbud, but is absent from the mesoderm (I, L). ec, ectoderm; en, endoderm; lpm, lateral plate mesoderm; me, mesoderm; mn, motor neuron field; n, neural tube; psm, presomitic mesoderm; s, somite; tb, tailbud.

Fig. 3

Maternal dietary RA treatment rescues spinal cord Olig2 expression in Raldh2^–/– embryos. (A) Olig2 expression in E9.5 wild-type embryo (untreated control). Olig2 expression is also shown in E9.5 Raldh2^–/– embryos following maternal dietary RA treatment from E6.5–E7.75 (B), E6.75–E8.25 (C), or E6.75–E9.5 (D); progressively more Olig2 expression along the anteroposterior axis of the spinal cord is observed in Raldh2^–/– embryos with longer RA treatments. (E) RARE-lacZ expression was examined in an E8.5 Raldh2^–/– embryo treated with RA from E6.75–E8.5; shown is a transverse section through the spinal cord at the level of somite 9 demonstrating that maternally administered RA has reached the neural tube and endoderm, but is absent in the somitic mesoderm. en, endoderm; n, neural tube; s, somite.

Fig. 4

Fgf8 and Cyp26a1 expression in the tailbud of Raldh2^–/– embryos. (A) Double hybridization showing Fgf8 mRNA in the tailbud and Uncx4.1 mRNA in somites; Raldh2^–/– embryos exhibit an expansion of the tailbud along its anteroposterior axis characterized by an anterior shift in the border of Fgf8 expression (wild-type and mutant each have 11 somites with the most posterior somite marked by an asterisk). (B) Double hybridization showing Cyp26a1 mRNA in the tailbud and Uncx4.1 mRNA in somites; the Cyp26a1 tailbud expression domain is not significantly altered in Raldh2^–/– embryos (wild-type has 9 somites and mutant has 8 somites with the most posterior somite marked by an asterisk). (C–F) Transverse sections through the tailbud of embryos stained for Fgf8 or Cyp26a1 mRNA (plane of sections depicted by arrows in panels A and B); expression of these genes in the three germ layers is not significantly altered in the mutant; however, Raldh2^–/– tailbuds have an expanded mesodermal zone. ec, ectoderm; en, endoderm; me, mesoderm.

Fig. 5

RA stimulates cell proliferation in tailbud neuroectoderm. Shown are transverse sections through the tailbud (A–B) and posterior neural tube at the level of somite 9 (C–D) stained with antibodies against phosphohistone 3 (H3P) to detect cells undergoing mitosis; note the decrease in H3P staining in Raldh2^–/– tailbud neuroectoderm, but not neural tube neuroectoderm. These sections are representative of those used to determine mitotic indices. ec, ectoderm; en, endoderm; me, mesoderm.