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First published online 2 December 2004
doi: 10.1242/dev.01554

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Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus

¹ Ecole Normale Supérieure de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS-UMR5161/INRA-UMR1237, 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, USA

View larger version (48K): [in a new window]   Fig. 1. Right (A) and left (A') side views of 24-hour larvae of amphioxus (Branchiostoma floridae) showing the mouth (M) on the left and the primordium of the first gill slit (1) on the ventral right side. The first pigment spot (arrowhead) in the nerve cord is at the level of the primordium of the future third gill slit. Right (B) and left (B') views of a 3-day larva showing gill slits 1 and 2 opening on the right (gill slit 3 has not yet opened) and the mouth (M) opening on the left. Just anterior to the first gill slit is the club-shaped gland (CSG) and the endostyle (E). Scale bars: A, A'=50 µm; B, B'=100 µm.

View larger version (90K): [in a new window]   Fig. 2. Expression of AmphiHox1 (A-L) and AmphiWnt3 (M-R) in amphioxus embryos treated as follows: DMSO control (A-D,M,N), 1 x 10-6 M RA (E-H,O,P) and 1 x 10-6 M BMS009 (a RA antagonist) (I-L,Q,R). Anterior to left. Whole mounts viewed from the left side and frontal sections in C, G, K viewed from dorsal side. The `x' in B,F,J shows the level of the section in C,G,K, respectively. Arrowheads indicate the anterior limit of expression in the endoderm. Scale bars=50 µm. (A) In 15-hour control neurulae, AmphiHox1 is expressed in the posterior half of the endoderm and overlying ectoderm with the highest expression localizing to the middle third. (B-D) At 20 hours (B,C) and 30 hours (D) of development, the anterior limit of endodermal and ectodermal expression is just posterior to the pharynx. (E-H) RA upregulates expression of AmphiHox1 in endoderm and ectoderm and expands it anteriorly. By 30 hours (H) expression in the endoderm is restricted to the extreme anterior end of the larva. (I-L) Treatments with the RA antagonist BMS009 downregulate AmphiHox1 and shift expression posteriorly in both the ectoderm and endoderm. (M,N) In mid-neurulae and early larvae of amphioxus, AmphiWnt3 is expressed in the ventral endoderm just posterior to the pharynx. (O,P) Treatment with RA initially shifts endodermal expression of AmphiWnt3 anteriorly and subsequently leads to a downregulation of expression by 30 hours of development (P). (Q,R) In BMS009-treated embryos, AmphiWnt3 expression is expanded posteriorly.

View larger version (56K): [in a new window]   Fig. 3. The posterior limit of pharyngeal expression of AmphiPax1/9 (A-F), AmphiPitx (GO) and AmphiNotch (P-U) is shifted anteriorly by RA (C,D,J-L,R,S) and posteriorly by BMS009 (E,F,M O,T,U) compared to expression in control animals treated with DMSO (A,B, G-I,P,Q). Anterior to left. Arrowheads indicate the posterior limit of endodermal expression. Double arrowheads in I,K,L,O point to Hatschek's anterior left diverticulum, the precursor of Hatschek's pit, which is the amphioxus homolog of the vertebrate adenohypophysis. Scale bars=50 µm. Mid-neurula (18 hours)=A,C,E. Late neurula (20 hours)=G,J,M,P,R,T. Early larva (30 hours)= B,D,F,H,K,N,Q,S,U. Mid-larva (48 hours)=I,L,O.

View larger version (82K): [in a new window]   Fig. 4. The posterior limit of expression of AmphiNodal (A-L) and AmphiOtx (M-R) in the endoderm is not affected by altered levels of RA signaling until the late neurula/early larval stage. Anterior to left. Whole mounts viewed from the left side and frontal sections in B,F,J viewed from dorsal side. The `x' in A,E,I shows the level of the section in B,F,J, respectively. Arrowheads indicate the posterior limit of expression in the endoderm. Scale bars=50 µm. (A) At midneurula (16 hours), AmphiNodal is expressed throughout the length of the endoderm. The posterior limit of expression in the endoderm at this stage is not affected by either RA (E) or by BMS009 (I). The frontal sections show that expression of AmphiNodal is restricted to the left side of the pharynx in controls (B), RA-treated (F), and BMS009-treated (J) embryos. By the late (24-hour) neurula, expression of AmphiNodal is restricted to the tail bud and pharyngeal endoderm in DMSO-treated controls (C), and is shifted slightly anteriorly by RA (G) and posteriorly by BMS009 (K). In early (30-hour) larvae, pharyngeal expression of AmphiNodal is reduced in the pharynx of controls (D), eliminated in RA-treated larvae (H) and expanded posteriorly in animals treated with BMS009 (L). (M) At the late (20-hour) neurula, AmphiOtx is expressed throughout the length of the pharynx in DMSO-treated controls. At this stage, the posterior limit of AmphiOtx expression is not affected by either RA (O) or BMS009 (Q). In 30-hour larvae, endodermal expression of AmphiOtx is restricted to the pharyngeal endoderm in DMSO-treated controls (N) and its posterior limit is shifted anteriorly by RA (P) and posteriorly by BMS009 (R).

View larger version (74K): [in a new window]   Fig. 5. The posterior limit of endodermal expression of AmphiIslet (A-I), AmphiFoxA2 (J-R), and AmphiHh (S-X) is not affected by altered levels of RA signaling. Anterior to left. Arrowheads indicate the posterior limit of endodermal expression. Scale bars=50 µm. (A-I) AmphiIslet is expressed in the anterior three-quarters of the endoderm in controls at all stages. Expression is not changed by RA or BMS009. (J-R) The posterior limit of AmphiFoxA2 is at the posterior end of the endoderm in DMSO controls and in RA- and BMS009-treated embryos at all stages. (S-X) AmphiHh is expressed strongly in the extreme anterior endoderm and more weakly throughout the remainder of the endoderm in DMSO controls at all stages (S,T). In mid-neurula embryos, the posterior limit of the region of weak endodermal expression is not affected by RA (U) or BMS009 (W). At this stage, AmphiHh expression in the endoderm of RA-treated embryos is largely downregulated (V), while expression in embryos treated with BMS009 appears to be slightly upregulated. A,D,G,J,M,P,S,U,W=20 hours. B,E,H,K,N,Q=24 hours. C,F,I,T,V,X=30 hours. L,O,R=48 hours.

View larger version (95K): [in a new window]   Fig. 6. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide mimics the effect of BMS009 treatments on the posterior limit of the pharynx in amphioxus. Scale bar=50 µm. (A,C,E,G) Embryos injected with the control morpholino. (B,D,F,H) Embryos injected with the AmphiHox1 antisense morpholino-oligonucleotide. (A,B) AmphiHox1 expression. In controls (A), the anterior limit of AmphiHox1 expression in the endoderm (arrowhead) coincides with the first pigment spot to form in the nerve cord, and is shifted slightly posteriorly in embryos injected with the AmphiHox1 morpholino (B). (C,D) AmphiPax1/9 expression. The posterior limit of AmphiPax1/9 expression in the endoderm (arrowhead) of an amphioxus larva injected with the control morpholino (C) coincides with the first pigment spot to form in the nerve cord (double arrowhead), whereas in larvae injected with the AmphiHox1-specific morpholino (D), the pharynx is expanded posteriorly and the posterior limit of AmphiPax1/9 expression in the pharyngeal endoderm (arrowhead) is posterior to the first pigment spot in the nerve cord (double arrowhead). (E-H) AmphiOtx expression. In control embryos (E,G), the posterior limit of AmphiOtx expression (arrowhead) coincides with the first pigment spot in the nerve cord (double arrowhead). AmphiOtx expression is expanded posteriorly in embryos injected with the AmphiHox1-specific morpholino (F,H). A,B,E,F=30 hours. C,D,G,H=40 hours.

View larger version (26K): [in a new window]   Fig. 7. Summary of expression of endodermal markers in amphioxus at the mid-neurula (20 hours) and early larval (30 hours) stages for (A) controls, (B) RA-treated embryos and (C) BMS009-treated embryos. The thickness of the lines is proportional to the relative level of expression. Changes of expression in the mouth and gill slit primordia are not shown. The position of the pigment spot in the nerve cord at the level of somite 5 is not affected by levels of RA signaling. Markers of the posterior foregut/midgut endoderm (AmphiRAR, AmphiHox1 and AmphiWnt3) are in light blue, those with expression restricted to the pharynx from the early neurula (AmphiPax1/9, AmphiPitx and AmphiNotch) are in purple, markers with expression becoming restricted to the pharynx at the early larval stage (AmphiNodal and AmphiOtx) are in orange, and those expressed throughout the length of the endoderm at all stages (AmphiIslet, AmphiFoxA2 and AmphiHh) are in brown. These groupings in controls (A) are predictive of the changes in expression in response to RA (B) and the RA antagonist BMS009 (C). In controls (A) AmphiRAR, AmphiHox1 and AmphiWnt3 are most strongly expressed in the middle third of the endoderm. RA (B) shifts their expression anteriorly. BMS009 (C) downregulates AmphiRAR and AmphiHox1 and by 30 hours shifts posteriorly their anterior limits of expression and expands posteriorly the domain of AmphiWnt3. In controls (A), expression of genes in the second group (AmphiPax1/9, AmphiPitx and AmphiNotch) is restricted to the pharynx at both stages. RA (B) shifts the posterior limits of their expression anteriorly and by 30 hours downregulates expression of AmphiPitx and AmphiNotch. BMS009 (C) has the opposite effect, expanding the domains of all three posteriorly. In controls (A), expression of genes in the third group (AmphiNodal and AmphiOtx) becomes restricted to the pharynx by 30 hours and is not affected by RA (B) until 30 hours, when expression is truncated posteriorly and/or downregulated. BMS009 (C) keeps expression of both genes high throughout much of the length of the endoderm. In controls (A), genes in the fourth group (AmphiIslet, AmphiFoxA2 and AmphiHh) are expressed throughout most of the length of the endoderm at both stages. The posterior limits of their expression are not markedly affected by either RA (B) or BMS009 (C).

View larger version (11K): [in a new window]   Fig. 8. Model for the mechanism whereby RA signaling establishes the posterior limit of the amphioxus pharynx. RA signaling via heterodimers of RAR/RXR in the posterior fore/midgut endoderm directly activates Hox1. RAR probably autoregulates its own transcription. Hox1 in turn represses expression of the pharyngeal markers Pax1/9 and Otx (black type). Pitx, Notch and Nodal (gray) are also repressed in the posterior fore/midgut by RA signaling and may also be downstream of Hox1.