Chen Y et al. (2001), Increased XRALDH2 activity has a posteriorizing...

XB-ART-9472

Mech Dev 2001 Mar 01;1011-2:91-103. doi: 10.1016/s0925-4773(00)00558-x.

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Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos.

Chen Y , Pollet N , Niehrs C , Pieler T .

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Retinoic acid (RA) metabolizing enzymes play important roles in RA signaling during vertebrate embryogenesis. We have previously reported on a RA degrading enzyme, XCYP26, which appears to be critical for the anteroposterior patterning of the central nervous system (EMBO J. 17 (1998) 7361). Here, we report on the sequence, expression and function of its counterpart, XRALDH2, a RA generating enzyme in Xenopus. During gastrulation and neurulation, XRALDH2 and XCYP26 show non-overlapping, complementary expression domains. Upon misexpression, XRALDH2 is found to reduce the forebrain territory and to posteriorize the molecular identity of midbrain and individual hindbrain rhombomeres in Xenopus embryos. Furthermore, ectopic XRALDH2, in combination with its substrate, all-trans-retinal (ATR), can mimic the RA phenotype to result in microcephalic embryos. Taken together, our data support the notion that XRALDH2 plays an important role in RA homeostasis by the creation of a critical RA concentration gradient along the anteroposterior axis of early embryos, which is essential for proper patterning of the central nervous system in Xenopus.

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Species referenced: Xenopus
Genes referenced: aldh1a2 atr cyp26a1 egr2 en2 gal.2 hoxb9 otx2 pc.1

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	Fig. 2. XRALDH2 is differentially expressed during Xenopus development. (A) XRALDH2 mRNA levels during Xenopus embryogenesis and in adult tissues revealed by RT-PCR. RT-PCR analysis with RNA preparations from staged embryos (embryonic stages indicated according to Nieuwkoop and Faber (1967)) and from adult tissues. Histone H4 was used as an RNA loading control. (B) Whole-mount in situ hybridization analysis using XRALDH2 antisense RNA. (1–3) Posterior view (top, dorsal; bottom, ventral); (4), dorsal view (top, anterior; bottom, posterior); (5,6), dorsal and ventral views, respectively (left, anterior; right, posterior); (7–10), anterior view; (11), lateral view; (2a), sagittal section of a stage 11 embryo; (4a,11a,11b), transverse sections (top, dorsal; bottom, ventral); (10a), the section plane is indicated by the red rectangle in panel 10. (C) Double staining whole-mount in situ hybridization of XRALDH2 (red) and XCYP26 (blue). (1–3), posterior view (top, dorsal; bottom, ventral); (4), dorsal view (top, posterior; bottom, anterior); (5), anterior view; (6), lateral view; (2a), sagittal section; (6a), higher magnification of the head region of 6; (6b, 6c), frontal section (top, dorsal; bottom, ventral); (6d), horizontal section (top, anterior; bottom, posterior). Red dashed lines and rectangles in (B) and (C) indicate the locations for vibratome sections. Nieuwkoop–Faber stages of embryogenesis are indicated in the lower right corner of each picture ( Nieuwkoop and Faber, 1967). (B,C) ar, Archenteron; cg, cement gland; dc, diencephalon; dz, deep zone of involuting marginal zone; ec, ectoderm; el, epithelial layer of involuting marginal zone; ele, epithelial layer of ectoderm; ls, lens; m, mesoderm; mc, mesencephalon; md, developing mesonephric duct; mg, midgut; m/h, midbrain/hindbrain boundary expression of XCYP26; no, notochord; oe, oral epithelia; op, olfactory placode; pc, prosencephalon; pe, pigment epithelium of retina; r3, rhombomere 3 domain of XCYP26; rc, rhombencephalon; re, retina; sc, spinal cord; sh, stomodeal–hypophyseal anlage; sl, sensorial layer of ectoderm; zii, zone of internal involution.
	Fig. 5. Anterior shift of midbrain and hindbrain marker gene expression upon XRALDH2 overexpression. Roughly 2 ng XRALDH2 encoding mRNA was injected into one blastomere of two-cell stage embryos. β-Gal encoding mRNA was co-injected as a lineage tracer (light blue staining). The expression of the various marker genes (as indicated) was analyzed by whole-mount in situ hybridization analysis (purple staining). (a–d) Embryos were simultaneously probed with Otx2, Krox20 and Hoxb9. (a) Control. (b,c) Anterior view of injected embryos. The midbrain domain of Otx2 and two stripes of Krox20 are anteriorly shifted by one or two rhombomeric units in the injected (right) side. (d) Dorsal view of an injected embryo. Red triangles indicate that the anterior boundary of Hoxb9 expression was not altered. (e–h) Embryos were simultaneously probed with Otx2, En2, Krox20 and Hoxb9. (e) Control. (f–h) Anterior view of injected embryos. The midbrain domain of Otx2 and the stripes of En2 and Krox20 are shifted anteriorly in the injected side, however, the anterior boundary of Hoxb9 expression is not shifted (red triangles).
	Fig. 3. ATRA down-regulates XRALDH2 expression. (a,c) Control; and (b,d), 0.18 μM ATRA treated embryos were probed with XRALDH2 antisense RNA. Nieuwkoop–Faber stages of embryogenesis are indicated in the lower right corner of each picture (Nieuwkoop and Faber, 1967). (a,b) Anterior view (top, dorsal; bottom, ventral), the red arrow indicates the ablation of the XRALDH2 expression domain in stomodeal–hypophyseal, olfactory and eye anlagen; (c,d), lateral view (left, head; right, tail).
	Fig. 4. Misexpression of XRALDH2 mimics the effects of RA in combination with all-trans-retinal. (a) Control embryos at stage 41; (b), embryos which are either mostly unaffected or with partial eye defect or slightly cyclopic (from left to right sequentially) upon 2 μM ATR treatment; (c), embryos without any morphological alterations after 2 ng XRALDH2 overexpression; (d), in the combination of 2 ng XRALDH2 injection and 2 μM ATR treatment, most embryos became microcephalic, and only a few showed a cyclopic eye or traces of eye pigment (white arrows).