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The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest

¹ Laboratoire de Biologie Moleculaire et Cellulaire, CNRS-UMR 49, Ecole Normale Supérieure de Lyon, 46, Allée d’Italie, 69364 Lyon CEDEX 07, France
² Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202, USA
³ IGBMC, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France

View larger version (30K): [in a new window]   Fig. 1. Transcriptional activities of AmphiRAR and AmphiTR2/4 on the consensus RARE (DR5) and IR7 elements expressed as the amount of chloramphenicol acetyl transferase (CAT) activity (A) or luciferase (Luc) activity (B-E) relative to control. Values are averages of three to five independent transfections in ROS17.2/8 cells. Error bars indicate ±one s.d. (A) Dose-dependent repression by AmphiTR2/4 (5-300 ng) of RA-induced transactivation and by AmphiRAR-AmphiRXR (10-500 ng each) on the DR5 element. (B) Dose-dependent repression by AmphiTR2/4 of RA-induced transactivation on the IR7 element (as in A, except for co-transfection with the IR7-containing pGL2 plasmid instead of pBLCAT). (C) Transactivation induced by AmphiTR2/4 on the IR7 element without RA or AmphiRAR-AmphiRXR. Conditions as in B. (D) Inhibition by AmphiRAR-AmphiRXR of transactivation induced by TR2/4 (100 ng) on the IR7 element without RA. AmphiRXR, 300 ng. AmphiRAR, 10-500 ng. (E) Antagonism by BMS009 on transcription induced by RA. Dark bars on left represent activation by RAR-RXR±RA. Light gray bars represent activity with added BMS009. White bars represent activity with BMS009, no RA.

View larger version (47K): [in a new window]   Fig. 2. AmphiRAR and AmphiTR2/4 expression in normal amphioxus embryos. Anterior is towards the left. At 4.5 hours (mid-gastrula), the expression of the two genes considerably overlaps. However, by 9 hours (early neurula), their expression patterns begin to become complementary. AmphiRAR is expressed posteriorly in the neural plate (NP), weakly in the somites (S) and throughout the mesendoderm, with expression strongest posteriorly, whereas AmphiTR2/4 is most strongly expressed in the anterior neural plate and underlying mesendoderm. By 16 hours, complementarity is more pronounced. AmphiRAR is downregulated in posterior and anterior tissues. It is most strongly expressed in the middle third of the neural tube (NT), somites and endoderm but not in the cerebral vesicle (CV) or notochord (N). By contrast, AmphiTR2/4 is most strongly expressed in the cerebral vesicle, Hatschek’s anterior left diverticulum (HD) and the anterior and posterior endoderm. At 24 hours, endodermal expression of AmphiRAR is downregulated except in a small ventromedial area. Expression of AmphiTR2/4 remains high in the cerebral vesicle, Hatschek’s diverticulum, the chordoneural hinge (CNH) and anterior and posterior endoderm. By 30 hours, the primordia of the mouth (M) and first two gill slits (GS1; GS2) have formed. Expression of AmphiRAR is restricted to the middle third of the nerve cord and weakly in the middle third of the somites and endoderm while that of AmphiTR2/4 is strongest in the cerebral vesicle, Hatschek’s diverticulum, the tailbud, mouth and gill slits. NP, neural plate; SO, somite; CV, cerebral vesicle; HD, Hatschek’s left anterior diverticulum; NT, neural tube; CNH, cordoneural hinge; M, mouth; GS1 and GS2, gill slits.

View larger version (107K): [in a new window]   Fig. 3. Expression of AmphiRAR (A-N) in amphioxus embryos in the absence (A-K) and presence (L-N) of 1x10–6 M RA and of AmphiPax1 in the presence of 1.5x10–6 M BMS009 (O-Q). Whole mounts and frontal sections (counterstained pink) with anterior towards the left. Cross sections viewed from posterior end. (A) No expression in the blastula. (B) Gastrula with ubiquitous expression. (C) Early neurula (15 hours). Expression downregulated in the cerebral vesicle (arrow), anterior endoderm and non-neural ectoderm. (D) Frontal section through x-x in C. Transcripts abundant in posterior mesoderm, somites and neural plate posterior to the cerebral vesicle. (E) Frontal section through y-y in C. Transcripts most abundant in the posterior three quarters of the endoderm. (F) Eighteen hour neurula. Expression is downregulated in the anterior third of the nerve cord and upregulated in the middle third. (G) Cross section through x in F. Expression throughout the nerve cord and very weakly in the somites adjacent the notochord. (H) Cross section through y in F. Expression strong in the nerve cord, somites and endoderm. (I) Twenty hour neurula. Expression downregulated in the pharyngeal endoderm. (J) Twenty-four hour embryo. Expression strong in the nerve cord posterior to the cerebral vesicle and a small region of endoderm, but largely downregulated elsewhere. (K) Two day larva. Expression most pronounced in middle of nerve cord. No expression in posterior quarter of the embryo or in forming gill slits. (L) Twenty-two hour embryo (RA treated). Expression generally upregulated extending into the dorsal part of the cerebral vesicle (arrow). (M) Twenty-six hour embryo (RA treated). Expression in the cerebral vesicle and upregulated in the pharynx. (N) Forty hour larva (RA treated). Gill slits and mouth absent. Expression anteriorized and upregulated in the pharynx. (O) Expression of AmphiPax1/9 in the pharynx in a normal 26 hour embryo (arrow indicates first pigment spot in nerve cord; arrowhead indicates posterior limit of pharynx marked by the posterior limit of Pax1/9 expression). (P) AmphiPax1/9 expression is expanded posteriorly in a 24 hour embryo treated with BMS009 (arrow and arrowhead as in O). (Q) AmphiPax1/9 expression remains posteriorly expanded in a 36 hour larva treated with BMS009 (arrow and arrowhead as in O). Scale bars: 50 µm for whole mounts; 25 µm for sections. n, notochord.

View larger version (105K): [in a new window]   Fig. 4. Expression of AmphiTr2/4 in untreated (A-M) amphioxus embryos and in embryos treated with 1x10–6 RA (N,O) or BMS009 (P-U). For whole mounts, anterior is towards the left. Cross-sections (counterstained pink) viewed from posterior end. (A) Mid-gastrula. Expression in dorsal hypoblast and epiblast. (B) Thirteen hour neurula. Intense expression in anterior neural plate and endoderm. (C) Dorsal view of embryo in B. (D) Nineteen hour embryo. Transcripts present in the cerebral vesicle (cv) and the endoderm, most strongly in the posterior third and in Hatschek’s anterior left diverticulum (just below the cerebral vesicle). (E) Twenty-four hour neurula. Strong expression in cerebral vesicle and throughout the endoderm, in Hatschek’s anterior left diverticulum (arrow) and primordium of first gill slit (arrowhead). (F) Cross-section through x in E. Strong expression throughout the cerebral vesicle and in Hatschek’s anterior left diverticulum (arrow). (G) Cross-section through y in E. Weak expression throughout pharyngeal endoderm. (H) Twenty-six hour embryo, overstained. Expression in tailbud, cerebral vesicle and pharyngeal endoderm. (I) Higher magnification of an embryo at the same stage as in H. (arrow indicates Hatschek’s diverticulum; arrowhead indicates primordium of first gill slit. (J) Thirty-four hour larva. Expression upregulated in endoderm of first gill slit (arrowhead). (K) Cross-section through x in J. Strong expression in pharyngeal endoderm and ectoderm around the open mouth. (L) Cross-section through y in J. Strong expression in gill slit primordium. (M) Cross-section through z in J. Very weak expression in non-pharyngeal endoderm. (N) Twenty-four hour embryo, RA treated. AmphiTR2/4 expression anteriorized in the pharynx, but unaffected in cerebral vesicle. (O) Thirty-six hour larva, RA treated. Compare with control in J. Expression anteriorized in pharynx and largely downregulated in cerebral vesicle. (P) Twenty-six hour embryo, BMS009 treated, with slightly expanded pharynx. Expression of AmphiTR2/4 is strong in the endoderm (particularly in the anterior and posterior thirds) and posterior mesoderm and moderate in the cerebral vesicle. x, y, z indicate levels of cross sections in Q-S. (Q) Cross-section through level x in P. Expression is strong in endoderm, particularly in Hatschek’s diverticulum, and weaker in the cerebral vesicle. (R) Cross-section through level y in P. Expression is strong in gill slit primordium (arrowhead). (S) Cross-section through level z in P. (T) Forty-eight hour embryo (arrow indicates the mouth; arrowheads indicate the first pigment spot in the nerve cord; 1, 2, 3 indicate expanded non-penetrating gill slit primordia). (U) Cross-section through a 48 hour larva at the level of first pigment spot in the nerve cord. Expression is weak in the expanded pharyngeal endoderm. Scale bar: 50 µm in whole mounts; 25 µm in sections.

View larger version (21K): [in a new window]   Fig. 5. RA treatment inhibits expansion of the pharynx in BMS009-treated larvae. (Top) Body length in larvae treated with 1x10–6M BMS009 and 0-1x10–6M RA is same as in untreated controls. (Bottom) The length of the pharynx is approximately doubled in larvae treated with 1x10–6M BMS009. Pharyngeal length is progressively restored to normal by increasing amounts of RA. Each bar represents the average of 10 larvae. Error bars represent ±one sd.

View larger version (81K): [in a new window]   Fig. 6. Quantification of mRNA of AmphTR2/4 and AmphiRAR by RT-PCR normalized to that of cytoplasmic actin in 15 hour embryos treated with 2x10–6 M RA (RA), in control embryos treated with DMSO alone (0) and with 2x10–6 M BMS009 (BMS009). Even when additional sample was loaded, no amplification of AmphiRAR was detectable in BMS009-treated embryos.

View larger version (43K): [in a new window]   Fig. 7. Diagram of the effects RA and the RA-antagonist BMS009 on pharyngeal morphology and expression of AmphiRAR and AmphiTR2/4 at the late neurula/early larva stage. (Left) In normal embryos, AmphiRAR and AmphiTR2/4 are expressed in approximately complementary patterns: AmphiTR2/4 expression being high in the cerebral vesicle, pharynx and tailbud where AmphiRAR expression is low. (Right) Application of RA shortens the pharynx and shifts it anteriorly (mouth and gill slits never form), while the RA antagonist BMS009 expands the pharynx posteriorly (subsequently an enlarged mouth forms; gill slit primordia shift posteriorly; extra ones are initiated, but none penetrates). AmphiRAR expression is upregulated and shifted anteriorly by RA and completely downregulated by BMS009. By contrast, although the level of AmphiTR2/4 expression is only slightly affected by RA and BMS009, RA shifts pharyngeal expression anteriorly, while BMS-009 expands it posteriorly. Together, these results suggest that levels of RA signaling, mediated in part by competitive inhibition of AmphiRAR by AmphiTR2/4, regulate anterior/posterior patterning in the nerve cord and endoderm.