Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Abstract

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.

Fig. 1

cyp26 expression in the developing hindbrain. Whole mount in situ hybridizations during the hindbrain patterning period. All embryos are shown as dorsal views. Anterior is to the top in A-I and to the left in J-R. In situ probes are noted in brackets beside the panels, embryonic age is noted in hours post fertilization (hpf). A: schematic of an 80% epiboly (8.5 hpf) embryo. The dotted box is the region shown in the flat-mounted embryos in B-E; the arrowhead indicates the advancing margin of the epiblast. During gastrulation, cyp26a1 (B-D,F,G) is expressed in the ectoderm (bracket in B-D) anterior to the domain of RA synthesis indicated by aldh1a2 expression (bracket in E). C,D: ectodermal cyp26a1 expression expands in the presence of sub-teratogenic concentrations of RA (C), but is established independent of RA (D). F, G: cyp26a1 expression recedes anteriorly at the onset of somitogenesis. Krox20 is shown in r3 and r5. Arrowhead indicates the posterior limit of cyp26a1 expression. Bracket marks weak cyp26a1 expression in the anterior trunk mesoderm. AN; anterior neurectodermal expression; TB, tailbud expression. H,I: aldh1a2 expression during early somitogenesis. Bracket shows expression in trunk mesoderm. J-R: dynamic cyp26b1 (J-M) and cyp26c1 (N-R) expression during somitogenesis. Krox20 expression in r3 and 5 is in red in J-P. In Q and R cyp26c1 is in red while hoxd4 (Q) and vhnf1 (R) are in blue. Insets in Q, R correspond to the dotted boxes. Dotted curve in O indicates the cyp26c1-free domain in ventral r3-r6. Scale bars: 100 μM. Scale bar in B is for B-E; scale bar in F is for F-I; scale bar in J is for J,N,R; scale bar in K is for K,O; scale bar in L is for L,M,P.

Fig. 2

cyp26b1 and cyp26c1 function redundantly with cyp26a1 to pattern the hindbrain. Whole-mount RNA in situ hybridizations at 18 hpf (A-J) and 13 hpf (K,L) and immunostaining at 48 hpf (M,N) in wild-type (left column) and cyp26a1^-/- (right column) embryos injected with MOs as shown on the left. A-H: pax2a (blue) marks the optic stalk (os), posterior midbrain and cerebellum (bracket), and the otic vesicles (ov), while hoxd4 (also blue) marks the r7-8 territory and krox20 (red) marks r3 and r5. MO depletion of Cyp26b1 and/or c1 does not affect this pattern in wild-type embryos (C,E,G), but progressively posteriorizes the hindbrain in cyp26a1^-/- embryos (D,F,H). Arrowhead marks the r6/7 boundary which is shifted to the anterior hindbrain in Cyp26-depleted embryos. I,J: en3 (red) marks the posterior midbrain and cerebellum (bracket), hoxb1a (blue) marks r4 which is shifted anteriorly in Cyp26-depleted embryos. K,L: pax2a (blue) and krox20 (red) are expressed as described above. vhnf1 is expressed in the posterior hindbrain up to the r5/6 boundary (arrowheads) and is also shifted anteriorly in Cyp26-depleted embryos. M,N: the isl1-GFP transgene (green) marks cranial motor neurons (nV: trigeminal motor neurons in r2 and r3; nVII: facial motor neurons in r4-6; nX: vagal motor neurons in r8) while the zn5 antibody (red) marks spinal motor neurons (arrows), pharyngeal arch endoderm (pe, arrowheads mark individual pharyngeal arches) and other structures. The large white arrowhead indicates the mid-hindbrain boundary. In Cyp26-depleted embryos, the motor neurons of the vagus nerve (nX) are expanded anteriorly, as are the spinal motor neurons. Scale bar: 100 μm. Scale bar in A is for A-H,K,L; scale bar in I is for I,J.

Fig. 3

a selective antagonist of cyp26 enzymes recapitulates the cyp26a1; b1;c1 phenotype. RNA in situs with the markers described in Fig. 2. Compared to DMSO-treated controls (A,C,E), treatment with 10 μM R115866 (B,D,F) causes an anterior shift of hoxb1a (arrowhead in A,B) and hoxd4 (arrowhead in C,D) toward the presumptive cerebellum marked by en3 (red in A,B) and by pax2a (brackets in C-F). This effect of R115866 is reversed by co-treatment with 10 μM DEAB (E,F). Scale bar: 100 μm. Bar in A is for A and B; Bar in C is for C-F.

Fig. 4

cyp26a1 protects the hindbrain from exogenous RA. Wild-type (left column) and cyp26a1^-/- (right column) embryos treated with DMSO (A,B), 10 μM DEAB (C,D) or 10 μM DEAB + 5 nM RA (E-L). RNA in situs use the markers described in Fig. 2 except for I,J, which is a mix of en3 (bracket), krox20 (r3, r5), dlx2 (cranial neural crest (cnc) and forebrain (fb)) and myoD (somites; s). os: optic stalk; e: eye; p: pronephros. Large bracket in (A) indicates the r7-8 region, which is elongated in cyp26a1 mutants (B). C: In DEAB-treated embryos, posterior rhombomeres (r5-8) are absent (arrow indicates the absence of high hoxd4 expression characteristic of r7-8). D: This phenotype is partially rescued in cyp26a1 mutants, as seen by rescue of r5 but not r7-8. E-L: The DEAB phenotype is fully rescued in wild-type embryos by treatment with 5 nM RA (E, G, I, K), while in cyp26a1 mutants this low dose of RA causes strong posteriorization of the brain (F,H,J,L). This phenotype resembles that of wild-type embryos treated with 200 nM RA (inset in J). Scale bar: 100 μm.

Fig. 5

Exogenous RA disrupts cyp26b1 and cyp26c1 expression in cyp26a1^-/- embryos but not in wild-type. Cyp26b1 (A-D) and cyp26c1 (E-H) expression is established normally in wild-type (A,E) and cyp26a1^-/- (B,F) embryos at the 6-somite stage (12 hpf). Cyp26b1 and cyp26c1 expression is also established normally in wild-type embryos treated with a sub-teratogenic concentration of RA (5 nM; C,G), but not in cyp26a1^-/- embryos treated with 5 nM RA (D,H).

Fig. 6

cyp26a1 protects against teratogenic effects of the RA precursor retinal. Wild-type (A) and cyp26a1^-/- (B) embryos injected with 18 pmol retinal at the 1-cell stage. Wild-type embryos are only mildly affected by approximately triple the normal levels of retinal, while cyp26 mutants are strongly posteriorized, with hoxd4 expression extending throughout the brain. Scale bar: 100 μm.

Fig. 7

A model for hindbrain patterning through regulated RA inactivation by Cyp26 enzymes. A: RA-responsive gene expression in Cyp26-depleted embryos. Embryos depleted of all three cyp26 genes experience unpatterned RA signaling; as a result the three RA-responsive genes examined in this study, hoxb1a (green), vhnf1 (yellow), and hoxd4 (orange) are expressed throughout the transformed hindbrain. Tel: telencephalon; di; diencephalon; mb: midbrain; c: cerebellum. B: A “gradient free” model for hindbrain patterning through regulated RA inactivation. Dynamic patterns of Cyp26 expression in the hindbrain (blue bars) antagonize RA-dependent gene expression by eliminating RA(red bars) first in the anterior hindbrain (6-9 hpf), then in r2-4 (9-11 hpf), then in r2-6 (11-12 hpf). At each point, sequential RA-responsive genes (colored bars) are limited to progressively more posterior rhombomeres. At the same time, Cyp26a1-dependent RA degradation in the trunk mesoderm suppresses global RA levels (black hammers on right side).