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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00849

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Genetic dissection of Pitx2 in craniofacial development uncovers new functions in branchial arch morphogenesis, late aspects of tooth morphogenesis and cell migration

Alkek Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 Holcombe Blvd, Houston, TX 77030, USA

View larger version (49K): [in a new window]   Fig. 1. Pitx2 alleles and Pitx2 isoform expression in developing teeth. (A) Summary of Pitx2 isoforms. Numbered boxes represent exons and black boxes the homeodomain. (B) Pitx2 abccreneo targeting strategy. At the top is a wild-type allele, the targeting vector is in the middle and at the bottom is the targeted allele. Numbered boxes represent exons and lines the intervening introns. Black shaded areas are homeobox. p1 and p2 are two alternate promoters located upstream of exon 1 and exon 4. On the right are Southern blots with 5' and 3' flanking probes. (C) Five Pitx2 alleles: Pitx2a and Pitx2b isoform-specific deletions, abhypoc and ab alleles (Liu et al., 2001). The abcnull allele is a homeobox deletion and a Pitx2-null allele (Lu et al., 1999) and c allele is and isoform-specific deletion of Pitx2c. (D) lacZ staining of ab+/– embryo showing expression in oral ectoderm (arrow). (E,F) Coronal (E) and parasagittal (F) sections through molar cap stage tooth showing lacZ expression in epithelial components and enamel knot (arrows). (G) Whole-mount in situ with Pitx2c probe showing expression in oral ectoderm (arrow). (H,I) Coronal (H) and parasagittal (I) section in situ with Pitx2c probe showing expression in epithelium of cap stage tooth. (J) RTPCR of Pitx2 isoforms in oral and dental epithelium. Specific amplified bands for each isoform (arrow). + indicates inclusion of reverse transcriptase;–is control without reverse transcriptase. md, mandibular process; mx maxillary process.

View larger version (118K): [in a new window]   Fig. 2. Histological analysis of tooth morphology in Pitx2 isoform deletions. (A-F) Coronal sections through molar teeth of 14.5 and 16.5 dpc embryos stained with Hematoxylin and Eosin. Genotypes and stage are labeled. (G-I) Parasagittal sections stained with Hematoxylin and Eosin through molar teeth (18.5 dpc).

View larger version (93K): [in a new window]   Fig. 3. Fgf8 signaling pathways require low doses of Pitx2. (A,B) In situ analysis of 9.5 dpc wild-type (A) and Pitx2 abcnull embryo (B) with Fgf8 probe. (C,D) Parasagittal cryosections of 9.5 dpc Fgf8 whole-mount of wild-type (C) and Pitx2 abcnull mutant (D) embryos. (E-N) In situ of wild-type (E,G,I,K,M), Pitx2 abcnull homozygous mutant embryos (F,H,J,L,N). (O,P) Cartilage staining of 13.5 dpc wild-type (O), Pitx2 abcnull homozygous mutant (P) embryos. (Q-S) In situ of 10.5 dpc wild-type (Q), Pitx2 abcnull homozygous mutant (R) and abcnull; abhypoc (S) embryos with Fgf8. Arrows indicate areas of oral ectoderm expression. (T-V) In situ of 10.5 dpc wild-type (T), Pitx2 abcnull homozygous mutant (U) and abcnull; abhypoc (V) embryos with a Barx1 probe. Arrows denote expression in proximal mandibular mesenchyme that is absent in Pitx2 abcnull mutant. Arrowhead indicates expression in caudal mandibular arch mesenchyme that is probably induced by Fgf8 signaling from the caudal mandibular ectoderm. (W-Y) In situ of 12.0 dpc wild-type (W), Pitx2 abcnull homozygous mutant (X) and abcnull; abhypoc (Y) embryos with Pax9. Arrows (mandibular incisor) and arrowheads (mandibular molar) indicate areas of expression in dental mesenchyme that are reduced in abcnull homozygous mutant. md, mandibular process; mx maxillary process.

View larger version (58K): [in a new window]   Fig. 4. Repression of Bmp4-signaling pathways requires high doses of Pitx2. (A-G) Whole-mount in situ analysis of 10.5 dpc Pitx2 abcnull homozygous mutant (A), Pitx2 wild-type (B,E), abcnull; abhypoc (C,F) abcnull; ab (D,G) embryos with a Bmp4 probe. (H-K) Whole-mount in situ analysis of 10.5 dpc Pitx2 wild-type (H), abcnull homozygous mutant (I), abcnull; abhypoc (J) and abcnull; ab (K) embryos with Msx2. (L-O) Whole-mount in situ of 10.5 dpc wild-type (L), Pitx2 abcnull homozygous mutant (M), abcnull; abhypoc (N) and abcnull; ab (O) embryos with Msx1 showing normal (arrows) and expanded (arrowheads) areas of expression. *Areas in wild type with expanded expression in mutant. md, mandibular process; mx maxillary process.

View larger version (84K): [in a new window]   Fig. 5. Tooth phenotypes of Pitx2 mutant allelic combinations. (A-D) Low-power view of coronal Hematoxylin and Eosin stained sections through molar teeth of 16.5 dpc wild type (A), Pitx2 abcnull; abhypoc (B), abcnull; ab (C), ab; ab (D). (E-H) High-power view of coronal Hematoxylin and Eosin stained sections through molar teeth of 16.5 dpc wild type (E), Pitx2 abcnull; abhypoc (F), abcnull; ab (G), ab; ab (H). (I-K) Parasagittal cryosections through 16.5 dpc embryos stained for lacZ. Wild-type (I) and abcnull; ab (J) show cap stage teeth, whereas abcnull; abhypoc (K) reveals a defect prior to cap formation (arrows). dc, dental cord; dp, dental papilla; e, epithelium: ek, enamel knot; mc, Meckel's cartilage; t, tongue;

View larger version (72K): [in a new window]   Fig. 6. Fate mapping of Pitx2 daughters in craniofacial development. (A,B) 11.5 dpc abccre+/– (A) and abccre ;abcnull (B) embryos hybridized to cre. Cre expression is restricted to oral ectoderm and is not found in the facial ectoderm (asterisk). (C-E) Sections of 11.5 dpc embryos hybridized to cre. Coronal (C,D) and parasagittal sections (E) show restricted expression of cre in oral ectoderm (arrows). Boxed area in C corresponds to higher-power image in D. (F,G) abccre+/– (F), abccre ;abcnull (G) rosa26 reporter+/– 11.5 dpc embryos. (H,I) Ventral view of abccre+/– rosa26 reporter compound heterozygous 11.5 dpc embryos stained for lacZ (H) showing lacZ-positive Pitx2 daughter cells (arrowhead). (J,K) Lateral view of abccre+/– (J) and abccre ;abcnull (K) rosa26 reporter+/– 11.5 dpc embryos stained for lacZ. Arrowhead (J) indicates lacZ-positive Pitx2 daughter cells and arrow (K) denotes cells that move ectopically. (L-O) Oral view of floor of mouth (L,M) or roof of mouth (N,O) from abccre+/– (L,N) and abccre ;abcnull (M,O) rosa26 reporter +/– 11.5 dpc embryos showing migrating Pitx2 daughter cells (arrows). (P-S) Parasagittal sections of 10.5 dpc (P,Q) and 12.5 dpc (R,S) abccre+/– (P,R) and abccre ;abcnull (Q,S) rosa26 reporter+/– embryos. Arrowheads indicate lacZ-positive Pitx2 daughter cells contributing to oral cavity roof and asterisk indicates region that has diminished contribution in Pitx2 mutant. Arrows indicate Pitx2 daughters contributing to mandibular oral and dental epithelium. e, eye; g, gut; fn, frontonasal process; lb, limb bud; md, mandible; mx, maxilla; nc, nasal cartilage; nt, neural tube; oe, oral ectoderm; rp, Rathke's pouch; to, tongue.