Fang H and Elinson RP (1996), Patterns of distal-less gene expression and ind...

XB-ART-17586

Dev Biol 1996 Oct 10;1791:160-72. doi: 10.1006/dbio.1996.0248.

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Patterns of distal-less gene expression and inductive interactions in the head of the direct developing frog Eleutherodactylus coqui.

Fang H , Elinson RP .

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The direct developing frog Eleutherodactylus coqui exhibits radical changes in its embryogenesis. A frog-like head forms directly with no appearance of a cement gland or several jaw cartilages characteristic of tadpoles, and limbs appear early in development. The numerous differences in the embryogenesis of E. coqui provide an opportunity to examine developmental causes for the evolutionary shift from biphasic to direct development. We have cloned DNA fragments corresponding to four E. coqui genes related to the Drosophila distal-less gene Dll. While the expression patterns of the distal-less genes are generally conserved, there are some spatiotemporal differences when embryos of E. coqui are compared to those of Xenopus laevis. The changes in gene expression are correlated with the embryonic changes in head structures including craniofacial cartilages and in particular, the cement gland. We have then examined inductive interactions involved in cement gland formation by interspecific transplants and recombinants. E. coqui embryos can generate signaling that culminates in cement gland formation, but E. coqui ectoderm appears to be incapable of a cement gland response. These results show here that inductive interactions in the anterior region of the E. coqui embryo have been modified during the evolution of direct development, and that changes in the competence of the E. coqui ectoderm may be responsible for the loss of certain tadpole-specific structures, such as cement gland.

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Species referenced: Xenopus laevis
Genes referenced: dlc dll4 dlx5 emb ncam1

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	FIG. 1. Embryos of E. coqui compared to those of X. laevis. Comparably staged embryos of E. coqui and X. laevis are drawn to scale to illustrate their size differences (left scale bar, 1 mm). An E. coqui embryo at the neural fold stage (S14) is compared to a slightly later neurula of X. laevis (NF 15). A TS 4 E. coqui embryo has not yet formed gill rudiments or eye pigment, similar to a NF 28 X. laevis embryo. The advanced E. coqui embryo (TS 12) on the right is drawn to a smaller scale (right scale bar, 2 mm).
	FIG. 2. Alignment of distal-less nucleotide sequences of EcDix2 vs X-dl14 (A) and EcDix4 vs X-dl13 (B). Underlined primers 1 and 3 were used for PCR amplification of EcDix2 and EcDix4 DNA fragments. The homeodomains are shaded. Dashes represent gaps introduced to optimize alignment.
	FIG. 3. Analysis of spatial expression of EcDlx2 (A, B, C, and E) and EcDlx4 (F, G, H, and I) by whole-mount in situ hybridization. In all panels anterior is to the left. (A) A dorsal view of a stage 15 E. coqui embryo stained with the EcDlx2 probe. Staining is observed in the mandibular streams (m) of neural crest cells. (A, inset) A homologous gene X-dll4 is expressed in the presumptive cement gland (white arrow) in a stage 16/17 X. laevis embryo (side view). This expression domain is not found in E. coqui. No expression of X-dll4 is detected in neural crest cells at stage 16/17. (B, C) Side views of EcDlx2 expression in stage TS4 (B) and TS5 (C) E. coqui embryos. EcDlx2 transcripts were detected in migrating neural crest cells and otic vesicles (Ot). (B, inset) X-dll4 is also expressed in migrating neural crest cells in a stage 24 X. laevis embryo beside the continued expression in the cement gland. (D, E) Frontal view of X-dll4 expression in a stage 24 X. laevis embryo (D) and EcDlx2 expression in a stage TS4 E. coqui embryo (E). Note the difference in the shape of the mandibular neural crest stream. (F) Dorsal view of EcDlx4 expression in a stage 14 E. coqui embryo. Staining was detected in the rim of anterior neural fold (black arrow). (F, inset) The neural fold expression domain (black arrow) is conserved in X. laevis, as indicated by the staining with the homologous gene probe X-dll3 in a stage 13 X. laevis embryo. X-dll3 transcripts were also detected in the presumptive cement gland (white arrow). However, the homologous gene EcDlx4 is not expressed in the corresponding region in E. coqui embryos. (G) Dorsal view of a stage 15 E. coqui embryo shows that EcDlx4 expression (black arrow) is restricted to the anterior region of dorsal midline after closure of the neural tube. No EcDlx4 expression is detected in the region anterior to the closed neural tube. (G, inset) In a stage 16/17 X. laevis embryo, X-dll3 expression is restricted to the anterior region of the closed neural tube (black arrow) and in the cement gland (white arrow). (H) Side view of a stage TS5 E. coqui embryo. EcDlx4 transcripts are detected in migrated neural crest cells in the distal tips of branchial arches. (I) Lateral view of a stage TS6 E. coqui embryo. EcDlx4 is expressed in all branchial arches, in olfactory vesicles (Of), otic vesicles (Ot), and in the distal ridge of the limb buds (AER). In all pictures, white arrows indicate cement gland, black arrows point to the anterior neural fold, and black arrowheads point to mandibular neural crest streams (m).
	FIG. 4. Immunocytochemical staining of embryos of X. laevis, R. pipiens, and E. coqui with an antisera against distal-less homeodomains. (A) Dorsolateral view of a stage 14 X. laevis embryo. (B) Lateral view of a stage 15 R. pipiens embryo. (C) Dorsal view of a stage 14 E. coqui embryo (yolk has been removed). Anterior is to the right in all panels. White arrowheads indicate cement glands. Black arrows indicate anterior neural fold.
	FIG. 5. (A) Transplantation of X. laevis animal cap to E. coqui gastrula. The large white E. coqui gastrula has received a transplant of a dark X. laevis animal cap, dorsal to the dorsal lip (faint groove). Two X. laevis donors with their animal caps removed are shoved against the E. coqui gastrula to illustrate the difference in size of these embryos. (B) E. coqui embryos with X. laevis cement glands. These anterior views of TS 3 E. coqui embryos show the developing head. Transplanted X. laevis animal caps have integrated into the embryos and form distinct cement glands (arrows) anterior to the head. (C) E. coqui transplants in R. pipiens neurulae. These ventral views of R. pipiens neurulae have E. coqui tissue (white) at their anterior ends, in the region where the cement gland will form. Tissues were transplanted the previous day, at the gastrula stage. Some pigmented R. pipiens cells have invaded the graft. (D) E. coqui transplant in the R. pipiens cement gland region. The cement gland (arrows) consists of a flat, round anterior portion connected to a posterior groove. Scale bar in A corresponds to 1 mm (A), 0.67 mm (B, C), and 0.3 mm (D).
	FIG. 6. (A) E. coqui cells near R. pipiens cement gland. E. coqui cells (Ec) lack pigment granules, which allow them to be distinguished from pigmented R. pipiens cells. Cement gland cells (CG) are elongated and intensely stained with PAS. All cement gland cells are derived from R. pipiens, none are from E. coqui. (B) Recombinants made with surface ventral ectoderm of stage 10 X. laevis (pigmented tissue) and anterior neural plate of stage 13 E. coqui (nonpigmented tissue). Arrows indicate cement glands induced by E. coqui neural tissue. (C) Section of tissue recombinant between early gastrula ectoderm from E. coqui (Ec, outer nonpigmented cells) and anterior neural plate from X. laevis (Xl, inner pigmented cells). The section was stained with PAS. No cement gland cells were observed in E. coqui tissue. (D) Section of E. coqui (Ec) ectoderm explant stained with PAS.
	FIG. 7. Assay of induced X. Iaevis N-CAM gene expression in recombinants by RT - PCR. Lane 1 (XI ec + Ec np): RNA was extracted from recombinants made of ventral surface ectoderm of stage 10 X. Iaevis embryos and anterior neural plate of stage 13 E. coqui embryos. N-CAM transcripts were detected in the RNA sample, indicating that neural tissue was induced in X. Iaevis ectoderm. Lane 2 (XI ec): RNA was extracted from ventral surface ectoderm of stage 10 X. Iaevis embryos. Ectodermal explants were cultured for the same period of time as the recombinants before RNA extraction. N-CAM transcripts were barely amplified. Lane 3 (XI st.28 emb.): RNA was extracted from stage 28 X. Iaevis embryos. N-CAM transcripts were detected. Lane 4 (Ec np): RNA was extracted from E. coqui anterior neural plate explants which were cultured for the same period of time as the recombinants. No NCAM molecule was amplified, indicating that theN-CAM primers specifically amplify X. Iaevis N-CAM. EF-1a was used to demonstrate that roughly comparable amounts of RNA were used in each reaction.