Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion - PubMed (original) (raw)

Comparative Study

Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion

Marek Dudas et al. Dev Biol. 2006.

Abstract

Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.

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Figures

Fig. 1

Epithelial deletion of Alk5 causes impaired fusion of palatal shelves and posterior palatal cleft. Alk5/K14-Cre pups (G–J) are born with a defect in the posterior part of the soft palate (arrow in panel G). In contrast, _Tgf-β_3 null newborns have a complete bilateral palatal cleft (M–P; arrows in panel M), whereas control newborns (A–D) have palates fully fused. The upper row shows stereoscopic images of formalin-fixed newborn heads after removal of the mandible (magnification 5×), the next three rows show histological sections through three different antero-posterior positions (a, b, c), as indicated in panel M (hematoxylin–eosin, magnification 20×). Alk5/ K14-Cre palatal shelves dissected on E14 fail to fuse in organ culture (L), whereas wild-type shelves (F) fuse normally (hematoxylineosin, magnification 40×). The fourth row (E, K, Q) demonstrates decreased proportion of cells with filopodia (asterisks) by E14 at the edges of pre-fusion palatal shelves in the Alk5/K14-Cre mutants (K) and _Tgf-β_3 null embryos (Q), in comparison with controls (E) (Scanning electron microscopy, magnification 5 000×). (R) Comparison of proportion of cells with filopodia in pre-fusion palatal shelves. Cells were counted in three areas (anterior, medium, posterior; 20×40μm) of the two palatal shelves for each genotype, and averaged within each group. f—the site of fusion of palatal shelves; ns—nasal septum; *****—cells with filopodia.

Fig. 2

TGF-β-induced apoptosis in the midline epithelial seam is required for palatal fusion. Comparison of apoptotic cell death in control (A), _Tgf-_β_3_−/ − (B) and Alk5/ K14-Cre (C) palatal midline regions using TUNEL assay (green fluorescent signal) in embryonic heads fixed on E14.5. Intense clusters of apoptotic cells can be seen in controls (A), whereas positively staining cells cannot be seen in _Tgf-β_3 and Alk5/K14-Cre mutants. Arrow in panel A points to TUNEL-positive nuclei inside the disappearing midline seam. (D–F) Implantation of TGF-β3 beads (E) into the palatal midline region of E14 _Tgf-_β_3_−/ − explants leads to increased apoptosis of adjacent epithelial cells up to a level seen in control wild-type explants treated with BSA (bovine serum albumin) beads (F). DAPI counterstain, magnification 40×.

Fig. 3

Fate mapping of midline epithelial cells in Alk5/K14-Cre mutants. Transverse palatal sections of E17 embryos expressing K14-Cre in the Rosa26 reporter background were stained for β-galactosidase activity (blue), which permanently marks epithelial cells and their descendants (eosin counterstain, magnification 20×). (A) Control palates have cells derived from the epithelium exclusively on the oral and nasal side of the palate, whereas the fully confluent mesenchyme (pink) along the entire anterior–posterior axis is devoid of positively staining cells. (B–F) Alk5/K14-Cre mutants show either no adhesion (cleft) or adhesion with a lack of fusion detected as persistent midline epithelial seam in the anterior palate (blue staining and arrows). Moreover, the nasal septum has failed to fuse with palatal shelves (B). The posterior part of the palate shows a stretched epithelial bridge (E), followed by palatal cleft in the most posterior region of the soft palate (F).

Fig. 4

External facial phenotype of Alk5/Wnt1-Cre mutants. Lateral view of a newborn control (A) and a mutant (B) showing the malformed face and head. Mutant embryos are noticeably pale in comparison with controls. (C) Detailed view of the oral cavity of the newborn mutant. Arrow points to a cleft snout and upper lip; undersized palatal shelves are marked with P. Coronal sections through the palatal region of control (D) and Alk5/Wnt1-Cre mutant embryos (E) at E18 (hematoxylin and eosin, ×4). S—nasal septum; P—palatal shelves; T—tongue.

Fig. 5

Bone and cartilage defects resulting from deletion of Alk5 in neural crest cells. Bone (red) and cartilage (blue) staining in E18 embryos; controls are on the left side, mutants on the right. Stereoscopic magnification ×5; magnification ×7 used for panels G and H, ×15 for panels I and J. (A–B) Lateral views demonstrate a large defect in calvaria, which is almost completely missing in Alk5/Wnt1-Cre mutants. Only small portions of the parietal and frontal bones remain and form a thin ring around the cranial base (long arrowheads). Shortened mandible (arrow) and multiple anomalies in the region of the absent temporomandibular joint (circled) are other prominent skeletal features of Alk5/Wnt1-Cre embryos, together with almost completely missing laryngeal cartilages and hyoid bone (short arrowhead). (C–D) Superior view shows a lack of all three components of the zygomatic arch (arrowheads), cleft in the region of nasal cartilages underlying the cleft snout (arrows), and missing calvaria as mentioned above (long arrowheads). (E–F) Inferior view after removal of the mandible with middle ear ossicles reveals a large defect in the bony palate (circled) due to rudimentary palatine, maxillary, and premaxillary palatal components. Arrowheads point to locations of missing squamosal and alisphenoid bones; asterisk marks missing presphenoid ossification. (G–H) Lateral view of the mandibular complexes with Meckel's cartilage and middle ear ossicles. Mutants show remarkable differences when compared to control littermates in the persistence and curvature of Meckel's cartilage (asterisk), defects in formation of the secondary cartilages, and impaired middle ear bones (missing incus and stapes, incomplete tympanic ring). (I–J) Semilateral view of the laryngeal region. In mutants, rudiments of the hyoid bone and thyroid cartilage appear as multiple small elements scattered in an anatomically correct location (double arrowheads). The cricoarytenoid complex is positioned abnormally low, at the level of the upper thoracic aperture, resulting in a gap in the laryngeal skeleton (arrow). The long arrowhead points to a midline cartilage of unknown origin. alsph—alisphenoid bone; alsph.l.obtur—lamina obturans; alsph.pter—pterygoid process of alisphenoid bone; ang.sc—secondary cartilage of the angular process; ary—arytenoid cartilage; bsph—basisphenoid bone; cochl—cochlear part of the temporal bone; cond.sc—secondary cartilage of the condylar process; cric—cricoid cartilage; f—frontal bone; hyo—hyoid bone; hyo.grhorn—greater horn of the hyoid bone; hyo.lhorn—lesser horn of the hyoid bone; i—incus; ip—intraparietal bone; f.car—foramen caroticum; f.ov—foramen ovale; gon—gonium; m—malleus; Meck.ant—anterior process of Meckel's cartilage; mx—maxilla; mx.alv—alveolar process of maxilla; mx.palat—palatal process of maxilla; mx.zyg—zygomatic process of maxilla; nas —nasal cartilages; no—used for missing structures; occ.b—basis of the occipital bone; p—parietal bone; palat—palatal bone; palat.palat—palatal process of palatal bone; pmx—premaxilla; pmx.palat—palatal process of premaxilla; s—stapes; sq—squamous bone; sq.rtp—retrotympanic process of the squamal bone; styl—styloid process; thyr—thyroid cartilage; tymp.r—tympanic ring; zyg—zygomatic (jugal) bone.

Fig. 6

Normal neural crest cell migration but pronounced hypomorphism of mandibular, maxillary, and nasal processes in Alk5/Wnt1-Cre mutants. Embryos carrying the Rosa26 Cre reporter were stained for β-galactosidase activity. (A–D) Migration of neural crest cells (blue) in controls at E8.5 (A–B) is indistinguishable from mutants (C–D). (E–F) Control at E11. (G–H) Alk5/Wnt1-Cre mutant at E11. White mx in G marks an undersized maxillary process, arrow in panel H points to an enlarged gap between nasal pits, corresponding to the future clefting region (compare with panel L). Long arrowhead—forebrain vesicles protruding frontally (aggravated in panel K); short arrowhead—hypoplastic maxillary process. (I–J) Control littermate at E15. (K–L) Alk5/Wnt1-Cre mutant at E15. Long arrowhead points to frontally protruding forebrain region, double arrowhead points to a small mandible, and arrow indicates the facial cleft. II—2nd pharyngeal arch; fnm—frontonasal mass; mnd—mandibular process of the 1st pharyngeal arch; mx—maxillary process of the 1st pharyngeal arch.

Fig. 7

Increased apoptosis in pharyngeal arches of Alk5/Wnt1-Cre mutants. Frontal sections show a pronounced increase in number of TUNEL-positive apoptotic cells (green signal) in the maxillary (mx) and mandibular (mnd) processes of Alk5/Wnt1-Cre mutants on E11, but not on E10, when compared to controls. Cell proliferation (anti-phosphohistone H3 immunostaining, red signal) is comparable between mutants and controls (blue counterstaining with DAPI; magnification 20×).

Fig. 8

Increased apoptosis and proliferation rate in the palatal mesenchyme of Alk5/Wnt1-Cre mutants and comparison with Alk5/K14-Cre mutants and _Tgf-β_3 knockouts. (A–B) Proliferating cells labeled with anti-phospho-histone H3 antibody (red fluorescent signal) at E14. (C–D) Apoptotic cells labeled using a TUNEL assay at E14 (green fluorescent signal). Magnification 20×, DAPI counterstain. (E) Histogram demonstrating statistically significant increase in the absolute number of proliferating cells in the mesenchyme of Alk5/Wnt1-Cre mutants in comparison with wild-type controls, Alk5/K14-Cre mutants, and _Tgf-β_3 knockouts (Wilcoxon rank score test, P <0.05; error bars =standard deviations; _N_=number of samples; sec=total number of sections analyzed).

Fig. 9

Attenuated expression of Msx1 and changes in expression patterns of Fgf8 and Tgif in Alk5/Wnt1-Cre mutants. Endogenous gene expression was visualized by in situ hybridization with biotinylated riboprobes (blue signal, magnification 5× for E10 and 4× for E11 samples). Whereas Msx2 did not show any change, Msx1 shows a remarkable decrease in expression in the maxillary process and frontonasal mass in mutants at E10 (B, arrowheads) and in the upper nasal pit region at E11 (D, arrowhead). Fgf8 expression in Alk5/Wnt1-Cre mutants spreads more anteriorly along the lower edge of the maxillary process at E11 (J, arrowhead), when compared with the control (I). Tgif expression shows a slight increase in the mandibular process and the 2nd pharyngeal arch in mutants at E11, whereas its expression in mutants of the same age is attenuated or absent in the upper portion of the maxillary process of the 1st pharyngeal arch, frontonasal process, temporoparietal region, and the nuchal area (K–L, arrowheads) The nasal pits and most of the calvaria show a comparable staining pattern and intensity in both mutants and controls.

Fig. 10

Endogenous co-expression of Alk5, type-II receptors, and Gdf11 in the facial primordia demonstrated by in situ hybridization. (A–C) Expression of Gdf11 in the tail and craniofacial primordia. Long arrowhead points to a strong signal in the mandibular arch at E10.5. (D–H) Whole-mount in situ hybridization with riboprobes for Alk5 (D), Acvr2A (E), Acvr2B (F), Bmpr2 (G), and Tgfbr2 (H). Arrowheads point to a positive signal in the nasal, maxillary and mandibular processes, second pharyngeal arch, and forebrain. ov—otic vesicle.

Fig. 11

Activation of ALK5 by two different type II receptors in vitro. Regulated heterodimerization was used as a model system to test whether ALK5 may also be activated by ACVRIIB in addition to canonical TGFβRII. (A) Expression constructs for type II receptors contained N-terminal myristoylation signal (Myr), two copies of FK506 binding domains (FKBP), and regions coding for kinase domains of ACVRIIB (construct IIA in panel A) and TGFβRII (construct IIT in panel A), respectively. The ALK5 cytoplasmic domain was fused to FKBP-rapamycin binding domain (FRB*, see Materials and methods; construct I in panel A). (B) Heterodimerizing agent (AP21967, Ariad Pharmaceuticals; brown in the scheme) can be used to induce regulated heterodimerization of chimeric type I and type II receptor fusion proteins expressed in cell culture. (C) Western blot analysis shows that the kinase domain of ACVRIIB was able to activate the ALK5 fusion protein in _Tgfbr2_-deficient DR26 cells co-transfected with the Smad2 expression vector, albeit less efficiently than the corresponding TGFβRII domain, as demonstrated by phosphorylation of the effector protein Smad2. Left panel-exposure 5min; right panel—exposure 20min.

References

1. Akhurst RJ, Fitzpatrick DR, Gatherer D, Lehnert SA, Millan FA. Transforming growth factor betas in mammalian embryogenesis. Prog Growth Factor Res. 1990;2:153–168. -PubMed
1. Albertson RC, Yelick PC. Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton. Dev Biol. 2005;283:310–321. -PubMed
1. Andl T, Ahn K, Kairo A, Chu EY, Wine-Lee L, Reddy ST, Croft NJ, Cebra-Thomas JA, Metzger D, Chambon P, Lyons KM, Mishina Y, Seykora JT, Crenshaw EB, III, Millar SE. Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development. Development. 2004;131:2257–2268. -PubMed
1. Barlow AJ, Bogardi JP, Ladher R, Francis-West PH. Expression of chick Barx-1 and its differential regulation by FGF-8 and BMP signaling in the maxillary primordia. Dev Dyn. 1999;214:291–302. -PubMed
1. Bei M, Maas R. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development. 1998;125:4325–4333. -PubMed

Epithelial and ectomesenchymal role of the type I TGF-beta receptor ALK5 during facial morphogenesis and palatal fusion - PubMed (original) (raw)