Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone

Abstract

Retinoic acid (RA) synthesized by Raldh3 in the frontonasal surface ectoderm of chick embryos has been suggested to function in early forebrain patterning by regulating Fgf8, Shh, and Meis2 expression. Similar expression of Raldh3 exists in E8.75 mouse embryos, but Raldh2 is also expressed in the optic vesicle at this stage suggesting that both genes may play a role in early forebrain patterning. Furthermore, Raldh3 is expressed later in the forebrain itself (lateral ganglionic eminence; LGE) starting at E12.5, suggesting a later role in forebrain neurogenesis. Here we have analyzed mouse embryos carrying single or double null mutations in Raldh2 and Raldh3 for defects in forebrain development. Raldh2(-/-);Raldh3(-/-) embryos completely lacked RA signaling activity in the early forebrain, but exhibited relatively normal expression of Fgf8, Shh, and Meis2 in the forebrain. Thus, we find no clear requirement for RA in controlling expression of these important forebrain patterning genes, but Raldh3 expression in the frontonasal surface ectoderm was found to be needed for normal Fgf8 expression in the olfactory pit. Our studies revealed that later expression of Raldh3 in the subventricular zone of the LGE is required for RA signaling activity in the ventral forebrain. Importantly, expression of dopamine receptor D2 in E18.5 Raldh3(-/-) embryos was essentially eliminated in the developing nucleus accumbens, a tissue lying close to the source of RA provided by Raldh3. Our results suggest that the role of RA during forebrain development begins late when Raldh3 expression initiates in the ventral subventricular zone.

Fig. 1. Location of rostral RA synthesis and RA activity in the developing mouse embryo

(A-B) Detection of Raldh2 and Raldh3 mRNAs at E8.75. (C-D) RARE-lacZ expression at E9.0 and transverse section demonstrating RA activity throughout the forebrain. (E-F) Detection of Raldh2 and Raldh3 mRNAs at E10.5. e, eye; f, forebrain telencephalic vesicle; ol, olfactory pit; op, optic vesicle.

Fig. 2. Loss of rostral RA activity does not affect Fgf8 or Shh expression except in olfactory pit

All embryos shown are at E8.75 and are unrescued (not treated with RA). (A-D) RARE-lacZ expression demonstrating partial loss of RA activity in Raldh2 null mutant and complete loss of RA activity in Raldh2-Raldh3 double null mutant (R2-/-:R3-/-). (E-H) Fgf8 expression and (I-L) Shh expression are not significantly altered following complete loss of RA; arrows point to the relevant expression domains in the forebrain field. (M-N) Fgf8 mRNA in Raldh3^-/- olfactory pit (ol) exhibits abnormal ventral extension indicated by asterisk.

Fig. 3. Loss of rostral RA activity does not affect expression of Meis2, a marker of forebrain intermediate character

(A-B) Rescue of Raldh2^-/- early lethality by maternal RA treatment from E6.75-E8.5 does not stimulate RA activity in the forebrain or other head tissues. (C-F) Coronal sections through the telencephalon showing that Meis2 expression is not significantly affected in E10.5 single mutants or Raldh2-Raldh3 double null mutants (R2-/-:R3-/-) rescued by maternal RA treatment from E6.75-E8.5 (which does not stimulate RA activity in the forebrain as shown in panel A). f, forebrain telencephalic vesicles.

Fig. 4. Raldh3 is responsible for RA detectable in ventral forebrain at later stages

(A) Coronal section through E14.5 forebrain demonstrates that Raldh3 mRNA is localized to the subventricular zone (SVZ) of the lateral ganglionic eminence (LGE). (B-G) F9-RARE-lacZ RA-reporter cells exposed to supernatants of cultured E14.5 embryonic tissues; in wild-type tissues RA activity is detected in the eye and LGE but not cortex; in Raldh3^-/- tissues RA activity is greatly reduced in the eye and not detected in the LGE.

Fig. 5. RA generated by Raldh3 regulates RARβ but not DARPP expression in LGE

All panels are coronal sections through the forebrain. (A-B) At E18.5, RARβ mRNA is greatly reduced in the striatum (LGE) of an Raldh3^-/- (R3-/) embryo particularly in the ventral region including the nucleus accumbens. (C-D) DARPP-32 immunohistochemistry at E18.5 demonstrating no significant difference between wild-type and Raldh3^-/- forebrain. ac, anterior commissure; cx, cortex; LGE, lateral ganglionic eminence; na, nucleus accumbens; str, striatum.

Fig. 6. Raldh3 functions in a paracrine fashion to provide RA for control of Drd2 expression in the nucleus accumbens

(A-B) Loss of Drd2 expression in an E18.5 Raldh3^-/- forebrain is specific for the nucleus accumbens; coronal sections through the left lobe are shown. (C) Forebrain Raldh3 expression in an E18.5 wild-type coronal section (left lobe) comparable to that shown for Drd2 in panels A-B; Raldh3 is expressed in the subventricular zone of the ventral striatum positioned just outside the nucleus accumbens in the dorsomedial direction. (D) RARβ expression at E18.5 in sections equivalent to those of panels A-C. (E-F) Comparison of Raldh2 and RARβ mRNAs in sagittal sections through the forebrain at E18.5; Raldh3 expression occurs in a small domain along the anteroposterior axis near the anterior end of the ventricle; RARβ expression is also localized to this same anteroposterior domain, but extends further ventrally than Raldh3 into the region where the nucleus accumbens resides. ac, anterior commissure; cx, cortex; LGE, lateral ganglionic eminence; na, nucleus accumbens; ob, olfactory bulb; ot olfactory tubercle; str, striatum; svz, subventricular zone.