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

First published online 11 August 2004
doi: 10.1242/dev.01291

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development 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 Ruest, L.-B. Articles by Clouthier, D. E. Search for Related Content PubMed PubMed Citation Articles by Ruest, L.-B. Articles by Clouthier, D. E.

Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity

¹ Department of Molecular, Cellular and Craniofacial Biology and the Birth Defects Center, University of Louisville, Louisville, KY 40292, USA
² Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
³ UMR5166 CNRS/MNHN Evolution des Régulations Endocriniennes, 7 rue Cuvier, 75005 Paris, France

View larger version (66K): [in a new window]   Fig. 1. Analysis of mandible structure in Ednra–/– embryos. E18.5 wild-type (Ednra+/+; A,C,E,G,I) and Ednra–/– (B,D,F,H,J) embryos. (A,B) Unlike wild-type embryos (A), the lower jaw of an Ednra–/– embryo is shortened and is covered by vibrissae (asterisks in inset in B denote follicles; arrows denote actual vibrissae) (B). (C-J) Alizarin Red and Alcian Blue staining to visualize bone and cartilage structures, respectively. (C,D) In a lateral view, the mandible in Ednra–/– embryos appears shortened and flattened (D) compared with that of the wild-type embryo (C). This bone, the pseudo-maxilla (mx*) is aberrantly connected to the jugal bone (j) through a bone resembling a duplicated jugal (j*). Incisors (i) of Ednra–/– embryos are present (inset in D) but are set primarily in mesenchyme. (E,F) A ventral view shows bilateral foramina in the pseudo-maxilla of Ednra–/– embryos (F) as well as the presence of incisors (insets). (G,H) Removal of other structures emphasizes the mirror image appearance of the pseudo-maxilla and pseudo-jugal bones in Ednra–/– embryos (H). (I,J) A frontal view of the pseudo-maxilla shown in H shows pseudo-palatine bones (pl*) attached to the pseudo-maxilla in Ednra–/– embryos (J). (K,L) Two views of the alisphenoid bone in wild-type (Ednra+/+;bottom) and Ednra–/– (top) embryos. Both the ala temporalis (at) and lamina obturans (lo) regions of the alisphenoid appear to be duplicated in Ednra–/– embryos. at*, duplicated ala temporalis; bs, basisphenoid; lo*, duplicated lamina obturans; h, hyoid.

View larger version (67K): [in a new window]   Fig. 5. Fate of Hand2 daughter cells in Ednra–/–;R26R;Hand2-Cre embryos. E16.5 Ednra+/+;R26R;Hand2-Cre (A,C,E,G,I) and Ednra–/–;R26R;Hand2-Cre (B,D,F,H,J) embryos stained for ß-gal activity in whole-mount (A-F) or sections (G,H), or analyzed for Hand2 expression using sectional in situ hybridization (I,J). (A,B) In a lateral view, ß-gal-labeled cells are observed throughout the lower jaw of Ednra+/+;R26R;Hand2-Cre embryos (A). In Ednra–/–;R26R;Hand2-Cre embryos, staining is confined to ventral surface of the mandible (B). (C,D) In a ventral view, few labeled cells are present in the lower jaw in Ednra–/– embryos (D), although the labeling is very prominent in the cleft between the two mandibular halves (arrow in D). (E,F) After clearing in benzyl benzoate:benzyl alcohol, little structural detail is apparent in the lower jaw of wild-type embryos because of the extensive labeling (E). By contrast, labeled cells in Ednra–/– embryos are present in the epithelial seam between the two arch halves (arrow in F), hypoplastic tongue (t) and lower incisors (i). (G, H) Stained sagittal cryosections illustrate labeling in most lower jaw structures and surrounding soft tissue of wild-type embryos, including the mandible (md), Meckel's cartilage (mc) and dental pulp of the incisors (arrow in inset) (G). Labeling is not observed in the dental lamina (dl; * in inset). In Ednra–/– embryos, labeled cells are present in the dental pulp of the lower incisors (arrow in inset), but are more scattered compared with wild-type embryos. A few labeled cells are also present in the small residual bone and cartilage underneath the incisors and in the dental lamina (* in inset) (H). Rugae are obvious along the both the top and bottom of the oral cavity (arrows). (I,J) Hand2 expression is observed in the dental pulp of both wild-type and mutant embryos, with highest expression in the odontoblast layer (yellow arrow in insets). Expression is not observed in either embryo within the dental lamina (black arrow in insets). p, secondary palate tissue.

View larger version (81K): [in a new window]   Fig. 2. Gene expression changes in Ednra–/– embryos. Whole-mount in situ hybridization analysis of gene expression in E10.5 wild-type (Ednra+/+) and Ednra–/– embryos using digoxigenin-labeled cRNA riboprobes. Embryos are shown in ventral view, with the heart and outflow tract removed to aid in visualization. (A,B) Wnt5a expression spreads into the distal half of the mandibular arch in Ednra–/– embryos (black arrow in B). (C,D) Dlx1 expression spreads into the distal mandibular arch in Ednra–/– embryos (C; compare broken yellow lines in C and D). (E,F) Msx1 expression appears to spread proximally in Ednra–/– embryos (F; compare broken black lines in E and F). (G,H) Twist expression expands distally in Ednra–/– embryos (H; compare broken yellow lines in G and H). (I,J) Dlx2 expression also spreads more distally in Ednra–/– embryos (J; compare broken yellow line in I and J). This expansion is accompanied by loss of epithelial expression (compare arrows in insets). (K,L) Bmp4 expression is also downregulated on the rostral epithelium of Ednra–/– embryos (L; compare arrows in insets in K and L). (M,N) Dlx5 expression is almost completely lost in the mandibular arch of Ednra–/– embryos (N). (O,P) Dlx6 expression is also absent in the mandibular arch of Ednra–/– embryos (P). (Q,R) Hand2 expression, which is observed in the distal two-thirds of wild-type embryos (Q), is almost completely absent in Ednra–/– embryos, though some expression remains in the distal arch (R; arrow in inset). 1, mandibular region of first pharyngeal arch; 2, second pharyngeal arch.

View larger version (105K): [in a new window]   Fig. 3. Hand2 expression in Ednra–/– and Dlx5/6–/– embryos. (A-H) Ednra+/+;R26R;Hand2-Cre and Ednra–/–;R26R;Hand2-Cre embryos stained in whole mount for ß-gal activity and shown in both lateral (A,B) and ventral views (C,D). For ventral views, the heart and outflow tract have been removed. Embryos (not necessarily those shown in A-D) were then sectioned along either sagittal (E,F) or frontal (G,H) planes and counterstained with nuclear Fast Red. (A,C) In E10.5 Ednra+/+;R26R;Hand2-Cre embryos, ß-gal-labeled cells are observed throughout pharyngeal arches 1 and 2. (B,D) In Ednra–/–;R26R;Hand2-Cre embryos, labeled cells are observed only along the distocaudal aspect of mandibular arch. (E,G) In sagittal sections through the arches of Ednra–/–;R26R;Hand2-Cre embryos, labeled cells are confined to the neural crest-derived mesenchyme. (F,H) In sagittal sections through the arches of Ednra–/–;R26R;Hand2-Cre embryos, scattered labeled cells are observed in the distal mesenchyme, with more intense labeling observed in cells in the surrounding arch epithelium. (I-L) Analysis of Hand2 expression in Dlx5/Dlx6–/– embryos. Normal Hand2 expression (I,K) is absent in the pharyngeal arches of Dlx5/Dlx6–/– embryos (J,L), although residual expression is still observed in the distal mandibular arch (yellow arrow in inset). A small expression domain is present in the second arch (black arrows in L).

View larger version (105K): [in a new window]   Fig. 4. Gata3 in mandibular arch development. E10.5 wild-type (Gata3+/+, A,C,E,G; Ednra+/+, I,K), Gata3–/– (B,D,F,H) and Ednra–/– (J,L) embryos following whole-mount in situ hybridization analysis using DIG-labeled riboprobes against Hand2 (A-D), Dlx5 (E-H) and Gata3 (I-L). Embryos are shown in both lateral (A,B,E,F,I,J) and ventral (C,D,G,H,K,L) views. (A-D) Unlike wild-type embryos (A,C), Hand2 expression in Gata3–/– embryos is observed only in the rostral mandibular arch (B,D); expression is also decreased in the second arch. There is an overall decrease in arch size in Gata3–/– embryos. (E-H) Dlx5 expression is observed only along the rostral half of the mandibular arch of Gata3–/– embryos (F,H). (I-L) Mandibular arch expression of Gata3, observed in the distal arch mesenchyme of wild-type embryos (I,K), appears unchanged in Ednra–/– embryos (J,L). 1, mandibular region of the first pharyngeal arch; 2, second pharyngeal arch; h, heart; lb, limb bud; np, nasal prominence.

View larger version (25K): [in a new window]   Fig. 6. Gene expression domains in E10.5 Ednra+/+ (top) and Ednra–/– (bottom) embryos. The sketches illustrate a ventral view of the mandibular arch, with the orientation of each arch half depicted above. Color-coded keys define both mesenchymal and epithelial gene expression domains. (A) In wild-type (Ednra+/+) embryos, the overlapping expression domains of Dlx5 and Dlx6 are shown on the left-hand side, while the right side depicts Msx1, Dlx2, Twist and Gata3 expression domains. Epithelial expression of Fgf8, Dlx2 and Edn1 are also shown on both sides. In Ednra–/– embryos, expression of both Dlx5 and Dlx6 is disrupted in the arch mesenchyme, as is Dlx2 expression in the rostral arch epithelium. By contrast, mesenchymal expression domains of Msx1, Dlx2 and Twist expand either proximally or distally, creating a large region of overlap. The Gata3 expression domain appears unchanged. (B) Hand2 expression is shown in two domains: one that requires Ednra signaling and one that does not require Ednra signaling.