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Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns

Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (46K): [in a new window]   Fig. 1. Sequence analysis of amphioxus and lamprey AP-2 proteins. (A) Clustal alignment of amphioxus AP-2, lamprey AP-2 and mouse AP-2. Identical residues are shaded black, biochemically similar residues are boxed. Underlined regions of mouse AP-2 represent the proline-rich transactivation domain (N-terminal) and the DNA-binding/dimerization domain (C-terminal). The regions of highest homology between the three sequences are within these functionally important domains. (B) Neighbor-joining phylogenetic tree of vertebrate and amphioxus AP-2 protein sequences. Drosophila serves as an outgroup. Numbers at branch points are confidence values derived from 1000 bootstrap resamplings of the alignment data. Sequence distance is indicated to the bottom left as substitutions per base. Lamprey AP-2 (Pm) is an outgroup to the gnathostome AP-2s and has no affinity for any one AP-2 family member, consistent with there being a single lamprey AP-2 gene. Amphioxus AP-2 (Bf) falls outside the vertebrate clade. The divergent mouse AP-2 groups with amphioxus AP-2 at low bootstrap values, and its phylogenetic relationship to the other vertebrate AP-2 family members is unclear. Dm, Drosophila melanogaster; Bf, Branchiostoma floridae; Pm, Petromyzon marinus; Hs, Homo sapiens; Mm, Mus Musculus; Gg, Gallus gallus; Xl, Xenopus laevis.

View larger version (65K): [in a new window]   Fig. 2. Reduced stringency Southern blot analysis to estimate AP-2 gene number in the amphioxus and lamprey genomes. (A) Genomic DNA from five individual adult amphioxus was digested with four restriction enzymes and analyzed on the same Southern blot. Lanes from three representative individuals are shown. Interestingly, each animal gives a different banding pattern. For a particular enzyme, an individual has no more than two band types, and most of these bands are shared between individuals. For example, individuals 2 and 3 share lowest ClaI band, while all three individuals share a single ApaI band. Overall, the banding patterns are most consistent with various hetero- and homozygotic combinations of restriction fragment length polymorphisms at a single locus, suggesting there is one amphioxus AP-2 gene. (B) Genomic DNA from a single adult lamprey digested with five restriction enzymes. Four out of five digests yield a single band, consistent with there being a single lamprey AP-2 gene. A, ApaI; C, ClaI; V, EcoRV; H, HindIII; E, EcoRI; N, NcoI; P, PstI; S, StuI.

View larger version (129K): [in a new window]   Fig. 3. AP-2 expression in amphioxus. (A) Cross-section through the 9 hour neurula depicted in B at position a, showing AP-2 transcripts in the non-neural ectoderm. (B) Dorsal anterior view of 9 hour neurula showing exclusion of AP-2 from the neural plate. (C) Dorsal view of 11.5 hour neurula, anterior is towards the left. AP-2 is expressed in the epidermis overgrowing the neural plate. (D) Cross-section through the embryo in C at position d. (E) Side view of 18 hour neurula. AP-2 expression in the epidermis is being extinguished from the anterior- and posterior-most ends of the embryo. (F) Optical horizontal section of 24 hour embryo through the gut with anterior towards the top. A spot of AP-2 positive cells is apparent in the left gut diverticulum, the endodermal portion of the pre-oral pit (arrow). (G) Cross-section of 9 hour neurula in C at position g. At more posterior levels, the AP-2 positive epidermis has completely covered the open neural plate. (H) Side view of 24 hour embryo with anterior to the left. AP-2 expression is seen in the cerebral vesicle and pre-oral pit, but has largely faded from the epidermis. (I) Optical horizontal section of 24 hour embryo in H at the level of the neural tube showing symmetrical AP-2 staining in the cerebral vesicle. Anterior is towards top. (J) Two-day larva with AP-2 expression persisting in the cerebral vesicle and pre-oral pit. (K) 36 hour larva with AP-2 expression in cerebral vesicle and pre-oral pit. (L) Cross-section through 24 hour embryo in H at position 1. AP-2 expression in the cerebral vesicle is limited to ventrolateral levels. ar, archenteron; ect, non-neural ectoderm/epidermis; np, neural plate; n, notochord; cv, cerebral vesicle; pp, pre-oral pit; ld, left gut diverticulum.

View larger version (118K): [in a new window]   Fig. 4. AP-2 expression in lamprey. (A) Dorsal anterior view of 4-day-old neurula. AP-2 expression is observed in the non-neural ectoderm at the exclusion of the neural plate. (B) Dorsal anterior view of 6 day neurula showing expression confined to the neural rod. An AP-2-free gap disrupts the neural rod signal anteriorly. (C) Side view of embryo in B with head protruding upwards. Dorsal neural rod expression is indicated by the arrow. (D) Side view of 7-day-old embryo, anterior is towards the right. AP-2 expression is enhanced in the head epidermis and mesenchyme, but is excluded from the otic cup. (E) 7.5-day-old embryo showing AP-2 expression throughout the head. (F) Cross-section through the neurula in A at f. AP-2 transcripts are absent from the neural plate. (G) Cross-section through 6-day-old neurula in C at position g, showing AP-2 expression in the dorsal neural tube (arrow). (H) Cross-section through 7-day-old embryo in D at position h, showing AP-2 staining in dorsal neural tube, head mesenchyme and epidermis (arrows). (I) Cross-section of 7.5-day-old embryo in E through the otic vesicle at position i. AP-2 transcripts are detected in the dorsal neural tube and in mesenchyme above and below the otic vesicles (arrows). No staining is apparent medial to the vesicle where it approximates the neural tube. (J) Cross-section through 8-day-old embryo in K at the level of the first somite (j). AP-2 expression is observed in mesenchyme superficial to the ectoderm and adjacent to the gut (arrows). (K) AP-2 expression in the head of an 8-day-old embryo. Staining is most intense in the region of the forming pharyngeal arches. (L) AP-2 expression in the head at 8.5 days, anterior is towards the right. AP-2-positive cells are localized more ventrally than at 8 days. (M) AP-2 expression in the head at 9 days, showing extensive staining in the pharyngeal region. (N) Side view of 11-day-old embryo showing AP-2 expression in the pharyngeal arches and strong staining in the anterior lip. (O) Cross-section through the 11-day-old embryo in N at position o. AP-2 mRNA is detected in a cluster of neurons in the lateral neural tube and in mesenchyme surrounding the pharynx (arrows). (P) Horizontal section through a 7.5-day-old embryo at the approximate level of p in E, showing expression in the dorsal neural tube, mesenchyme and ectoderm (arrows). (Q) Horizontal section through the head of the embryo in L at position q. Anterior is towards the right. AP-2-expressing cells are seen in the mesenchyme surrounding the pharyngeal mesoderm (arrows). (R) Horizontal section through an 11-day-old embryo at about the level of r in N. Expression is observed in the mesenchyme of the pharynx (arrows). ar, archenteron; np, neural plate; ect, non-neural ectoderm; n, notochord; ot, otic pit/vesicle; nt, neural tube/rod; ph, pharynx; pa, pharyngeal arches; md, mandibular arch; mx, maxillary region (anterior lip); so, somite.

View larger version (101K): [in a new window]   Fig. 6. Comparison of AP-2 expression in the head of lamprey and axolotl. (A) Dorsal anterior view of 7-day-old lamprey embryo showing AP-2 expression in neural tube, early migrating neural crest and ectoderm. AP-2-positive cells are excluded from a discrete region of the neural tube (arrow) and the otic cup. (B) Side view of an 8-day-old lamprey embryo head showing AP-2 staining in cranial neural crest cells. Putative streams of migrating cells are bordered by dotted lines. Rostrally, white dots indicate the anterior edge of the trigeminal stream, which splits into the ophthalmic (rostralmost white line), maxillary and mandibular streams (ventral white lines). Blue dots border the rostral edge of the putative hyoid crest stream, and yellow dots indicate the position of the common branchial stream. (C) Horizontal section through the pharynx of an 11 day lamprey embryo showing AP-2-positive cells in the mesenchyme medial and lateral to the arch mesoderm (arrows). For reference, colored lines outline the three germ layers in a single arch; mesoderm is red, ectoderm is blue and endoderm is yellow. (D) Dorsal anterior view of approximately stage 21 axolotl embryo showing AP-2 staining in early migrating neural crest. AP-2 expression is reduced in rhombomeres 3 and 5 (black arrows) and a region of rhombomere 1 (white arrow). Compared with the lamprey embryo in A, axolotl shows greater patterning of AP-2 expression along the anterior neural tube. (E) Side view of approximately stage 25 axolotl embryo showing AP-2 expression in cranial neural crest streams. Streams are marked with dotted lines as in B. White dots border the trigeminal stream, which splits into the ophthalmic, maxillary and mandibular streams. Blue dots border the hyoid stream, and yellow dots border the first branchial and common 2/3/4 branchial streams. Overall, the distribution of AP-2 positive cells in the axolotl head resembles that of lamprey (B), except ventrally, where the divisions between putative neural crest streams are much less defined in lamprey than in axolotl. (F) Horizontal sections through the pharynx of stage 30 axolotl embryo. Germ layers are marked as in C. AP-2-positive cells are observed in the mesenchyme surrounding the arch mesoderm (arrow). Both lamprey (C) and axolotl have AP-2-positive cells lying medial to the pharyngeal arch mesoderm. ot, otic pit/vesicle; md, mandibular neural crest stream; mx, maxillary neural crest stream; op, ophthalmic neural crest stream; br, branchial neural crest stream; hy, hyoid neural crest stream; tr, trigeminal neural crest stream; ph, pharynx; r3, rhombomere 3; r5, rhombomere 5.

View larger version (87K): [in a new window]   Fig. 5. Conserved aspects of AP-2 expression in amphioxus, lamprey, and gnathostomes. (A) Dorsal anterior view of similarly staged amphioxus (left panel), lamprey (middle panel) and axolotl (right panel) neurulae. In all three embryos, AP-2 is upregulated in the non-neural ectoderm and excluded from the neural plate. (B) Cross-sections through the anterior neural tube of amphioxus (left panel), lamprey (middle panel) and axolotl (right panel). All three embryos show AP-2 staining in the lateral walls of anterior neural tube (arrows) after neurulation. n, notocord.; np, neural plate; etc, non-neural ectoderm; nt, neural tube.