QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 28 February 2007
doi: 10.1242/dev.02815

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chambers, D. Articles by Lumsden, A. Search for Related Content PubMed PubMed Citation Articles by Chambers, D. Articles by Lumsden, A.

RALDH-independent generation of retinoic acid during vertebrate embryogenesis by CYP1B1

¹ Wellcome Trust Functional Genomics Development Initiative, MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK.
² MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's College London, Guy's Campus, London SE1 1UL, UK.

View larger version (90K): [in this window] [in a new window]   Fig. 1. Expression of Cyp1B1 in the chick embryo. (A,B) Expression in the ectoderm and mesoderm (black arrowheads) of the posterior primitive streak of a HH4 embryo. Black lines denote plane of sections. (C-F) 4s: transcripts are localised to all of the newly formed somites (D, black arrow), as well as to the mediolateral ectoderm of the sinus rhomboidalis (F, white arrow). Black lines denote planes of section shown in D-F. (G) 6s: at HH9-, expression persists in the somites but is lost from the posterior ectoderm. (H) HH10: transcripts are abundant in the neuroepithelium of the mid-hindbrain boundary (MHB) region (white arrowhead), in addition to weak expression in r2 (blue arrow) and the paraxial mesoderm adjacent to the developing hindbrain (black arrow). (I) HH10: flat-mount preparation showing that the expression extends across the DV axis of the MHB and r2. (J,K) HH10: transverse sections though the hindbrain showing strong expression in both ectoderm and mesoderm, possibly including neural crest-derived cells immediately adjacent to the MHB (blue arrow). (L) HH12+: MHB expression has narrowed to a thin band in the posterior midbrain, and an additional band of neuroepithelial expression is seen at the anterior midbrain (blue arrowhead). Expression is evident in the ectoderm immediately anterior to the otic vesicle (white arrowhead). Black lines denote planes of section shown in N-P. (M) Expression is enriched in the dermomyotome (white arrow). (N,O) Transverse sections showing strong expression of Cyp1B1 in the ectoderm abutting the entire mesencephalic vesicle (white arrows). (P,Q) Cyp1B1 message in the mesoderm adjacent to the hindbrain persists at HH12+ (blue arrow), as does expression in the somites. Cyp1B1 expression is also strong in the notochord (black arrows). The section in Q is more posterior than can be shown in L. (R) 30s: new sites of Cyp1B1 expression emerge, including a highly localised expression pattern in the developing eye (white arrowhead) and the epiphysis (blue arrow). (S) Longitudinal sections show expression in lens vesicle (black arrow) and neural retina (blue arrows). There is continued expression in all of the somites. (T) Transverse section at the level indicated in R shows expression throughout the notochord (black arrow) and somites. (U-W) 41s: other sites of expression include a pocket of mesodermal cells lateral to r2 (blue arrow), the sinus venosus of the heart and asymmetrically in the endoderm of the pharyngeal arches (V, blue arrows). Cyp1B1 is also expressed in the mesoderm of the forelimb bud (W, white arrow). (X) Dorsal view of Raldh2 expression in a 12s embryo. (Y) Cyp26C1 is expressed in r2 (black arrow) in a 12s embryo.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Determining the role of CYP1B1 in the biochemistry of t-RA synthesis and breakdown. (A) Reverse phase HPLC profile of the metabolism of t-retinol by CYP1B1 (0.025 nM CYP1B1 in 0.1 M potassium phosphate buffer pH 7.4, plus NADPH-cytochrome p450 reductase; 20 minutes at 37°C). Black arrow, input retinol; blue arrow, output retinal. (B) Reverse phase HPLC profile of the metabolism of t-retinal by CYP1B1 under the same conditions for differing lengths of time (20, 60 and 240 minutes). Black arrow, input retinal; blue arrow, output RA. The rates of synthesis of RA are given in Table 2. (C) Reverse phase HPLC profile of the metabolism of t-RA by CYP1B1 under the same conditions. All incubations were repeated in the absence of NADPH or CYP1B1, and no metabolic conversion of the input substrate was recorded in either case (data not shown). The data support a model in which CYP1B1 can actively convert retinol to retinal and then to RA, but cannot participate in the breakdown of either the newly formed RA or RA from other anabolic sources.

View larger version (91K): [in this window] [in a new window]   Fig. 3. Expression of Cyp1B1 in normal and vitamin A-deficient (VAD) quail embryos. Arrows represent regions where expression of Cyp1B1 is seen in normal embryos but not in the VAD embryos. (Aa) Dorsal view of a normal 10s (HH10) embryo. (Ab,Ac) Equivalent view of a HH10 VAD embryo showing Cyp1B1 expression in the MHB (white arrow), paraxial mesoderm of the hindbrain (blue arrowhead) and all of the somites. Expression is absent from the r2 region as compared with normal embryos (white arrow). (Ba) Cyp1B1 expression in a HH16 embryo and (Bb) in a transverse section in the plane indicated by the white line. (Bc) Dorsal view of 23s VAD quail and (Bd) enlargement of spinal cord region. The location of transverse sections are indicated by black lines. Cyp1B1 is missing from the sinus venosus (red arrow), eye (white arrow) and pharyngeal arch areas (blue arrow). Transverse sections (Be-Bg) reveal that the expression in the notochord (Be, orange arrow) is lost as compared with the normal (Bb, orange arrow). The green arrow in Be indicates mesoderm beside the hindbrain. (Ca-Cc) Sites of altered expression are also observed in a 30s (HH17) VAD embryo compared with its normal counterpart. These include the epiphysis, the eye and heart (Ca compared with Cc, orange, white and pink arrows, respectively). The blue arrows in Ca and Cc indicate loss of expression in the branchial arches. (Da,Db) Rescue of Cyp1B1 expression in the anterior of the embryo and particularly the eye (white arrow) by the exogenous addition of RA.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Overexpression of Cyp1B1 alters DV patterning and motor neuron specification. (Aa) Full-length Cyp1B1 (1629 nt) with a modified Kozak sequence was subcloned into the pCAB IRES-GFP vector (Gilthorpe et al., 2002) to generate Cyp1B1-IRES-GFP. (Ab,Ac) Following electroporation of Cyp1B1-IRES-GFP into HH11 embryos, both a high level of GFP (Ab, white arrowhead) and Cyp1B1 (Ac, blue arrowhead) transcripts are detected in the left-hand side of neuroepithelium. (Ad) Endogenous expression of cCyp1B1 at HH12 in the hindbrain. (B-E) Embryos were electroporated with Cyp1B1-IRES-GFP at HH10-12, cultured for 16-20 hours and analysed by in situ hybridisation for (Ba-Bf) Shh, (Ca-Cf) Nkx6.1, (Da-Dh) Isl1 and (Ea-Ed) Gata2. Wild-type expression of each gene is shown in the `normal' column and embryos electroporated with the pCAB IRES-GFP vector alone in the `control' column. Isolated neural tubes from either the control or Cyp1B1-IRES-GFP-electroporated embryos are shown in Bc,Bd, Cc,Cd and Dc,Dd, respectively. Arrows in Bd and Cd indicate domains of loss of expression. (Be,Bf) Comparison of hindbrain flat-mounts of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Shh showing that expression is reduced in the floorplate cells (Bf, green arrow). (Ce,Cf) Comparison of flat-mount hindbrain preparation of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Nkx6.1 shows alteration of expression domains in the electroporated embryo (Cf, green arrow). The blue arrow in Cf indicates the region of downregulation. (De,Df) Comparison of hindbrain flat-mount preparation of a normal and a Cyp1B1-IRES-GFP-electroporated embryo analysed for Isl1 expression shows downregulation (Df, green arrow). (Dg) Lateral view of the non-electroporated control side of an embryo processed for Isl1 expression (white arrow, placodal expression). (Dh) Expression of Isl1 in the placodes is reduced on the Cyp1B1-IRES-GFP-electroporated side (white arrows). (Ec,Ed) Gata2 expression is reduced in hindbrain of Cyp1B1-IRES-GFP-electroporated embryo. The location of the facial motor nuclei is indicated by VII (green arrow).

View larger version (97K): [in this window] [in a new window]   Fig. 5. All-trans-RA, but not all-trans-retinal, phenocopies Cyp1B1 overexpression. Beads soaked in either RAL or RA were implanted into the neural tubes (bead-implantation site marked by a black or white arrow) of HH11 chick embryos and allowed to develop for a further 16-20 hours before being harvested. Beads soaked in retinoid carrier solution were used as negative controls and no changes in target gene expression were recorded (data not shown). (Aa-Ad) Lateral view of embryos. (Aa) Expression of Shh with RAL bead implanted in hindbrain showing no effect on expression levels. (Ab,Ac) RAL bead has no effect on Nkx6.1 and Isl1 expression. (Ad) Shh expression is completely lost (blue arrows) in response to a single RA-soaked bead implanted into spinal cord. These data demonstrate an antagonist role for RA but not RAL in the regulation of Shh expression. (Ba-Bd) Following electroporation, a Raldh2-containing construct downregulates Shh expression (Bc,Bd) when compared with the control electroporated embryos (Ba,Bb). Arrows in Bb mark the normal domain of Shh, and those in Bc and Bd mark a reduction in expression levels. (Ca-Cd) Transverse sections of Shh expression in the hindbrain of a normal (Ca), VAD (Cb,Cc) or Cyp1B1-electroporated (Cd) embryo. Arrows in Cb and Cc mark expansion domains and the arrows in Cd mark reduction domains.

View larger version (66K): [in this window] [in a new window]   Fig. 6. Expression of Cyp1B1 in the pharyngeal arch region and its role in the patterning of the epibranchial placodes. (Aa-Ac) Cyp1B1 is expressed in the endoderm of the pharyngeal pouch (PP) and grooves (Aa,Ab, blue arrows). Longitudinal section through the region shown (Ac, white line) shows highly localised expression to the endoderm of PP2 (white arrows in Ab). (Ba-Bc) At a similar stage, Raldh2 is not expressed in the region of the pharyngeal pouches (Ba, black arrow). Cyp26C1, known to attenuate RA-signalling, is also expressed in the endoderm of PP1-3 (Bb,Bc, white arrows). (C) Electroporation strategy used to overexpress Cyp1B1 in the ectoderm adjacent to the endoderm of the PPs. (Da-Dc) Phox2a expression in the epibranchial placodes of the non-electroporated side of an embryo (Da, black arrows). Ectopic expression of Cyp1B1 represses the expression of Phox2a (Db, white arrows). The observed effects are coincident with ectopic sites of GFP expression (Dc, white arrows).

View larger version (79K): [in this window] [in a new window]   Fig. 7. CYP1B1 contributes to maintaining the appropriate expression of Hoxb1. (Aa-Ac) Hoxb1 expression in a HH17 normal embryo (Aa), the isolated neural tube (Ab) and flat-mount preparation of the hindbrain (Ac). (Ba-Bc) Hoxb1 expression is reduced (n=5/7) by implantation of a bead soaked in 10-4 nM TMS, a specific inhibitor of CYP1B1 (green arrow represents bead position). Expression is less intense in r4, r6 (red arrow) and the anterior spinal cord (blue arrow). (Ca-Cc) A similar reduction was noted with 10-5 nM TMS (n=5/6). In some cases (Cc), the borders of the Hoxb1 r4 expression domain appeared to be smaller in the treated embryos (black arrows). These effects were not seen in either untreated embryos (Aa-Ac) or embryos where a DMSO-soaked bead was implanted into r4 (data not shown). TMS-treated and untreated embryos were processed simultaneously using an in situ protocol in which the Hoxb1 probe was derived from a common mix and colour reactions were performed for the same length of time.

View larger version (76K): [in this window] [in a new window]   Fig. 8. The role of CYP1B1 in RA biochemistry and its potential role in patterning the ventral neural tube and motor neuron differentiation. (A) Oxidative cascade leading to the synthesis and breakdown of RA. CYP1B1 can generate retinal and RA but does not subsequently catabolise RA. The other major components of the pathway are shown. In this model, only the ADHs are thought to be ubiquitously expressed, with the others showing developmentally restricted patterns of expression. (B) Expression domains of Shh, homeodomain and bHLH transcription factors in the ventral spinal cord. Mutual cross-repressive interactions between class I and II proteins establish a motor neuron progenitor domain exclusively in the region of PAX6 and NKX6 that is defined by the expression of OLIG2. Later steps in acquisition of a committed motor neuron phenotype are marked by MNR2 and ISL1. RA is known to play a role in the regulation of this process (reviewed by Jessell, 2000). (C) Following overexpression of Cyp1B1 in the ventral neural tube, the phenotypic effects can be explained by a disruption of the pMN domain via the excess production of RA. (D) RA is known to be involved in many other steps (green arrows) of MN specification/maturation (see references listed). RA produced by CYP1B1 could also contribute to these events.