Cleft palate formation in fetal Br mice with midfacial retrusion: tenascin, fibronectin, laminin, and type IV collagen immunolocalization.
OBJECTIVE
This study tested the hypothesis that altered craniofacial morphology does not affect the expression of extracellular matrix (ECM) molecules such as fibronectin (FN), laminin (LN), type IV collagen, and tenascin-C (TN) but is associated with failure of palatal shelf elevation and fusion concomitant with cleft palate formation.


METHODS
To test this hypothesis, a comparative immunohistological analysis of FN, LN, type IV collagen, and TN was undertaken on brachyrrhine (Br/Br) mice and normal (+/+) fetuses during secondary palate formation. Normal and Br/Br fetuses were collected at gestational days E13 and E14 (representing prefusion stages) and E15 and E18 (representing postfusion stages). Cryostat palatal sections (8 microm) were postfixed in methanol, washed, and stained with primary antibody. All sections were washed and coated with secondary antibody (swine-anti-rabbit IgG) and mounted with citifluor.


RESULTS
Immunohistological analysis showed that LN and type IV collagen were located near the presumptive medial epithelial seam (MES) or edge (MEE) in +/+ or Br/Br fetuses, respectively. Fibronectin showed a homogeneous distribution at all stages in both groups of mice. In contrast, TN became localized below the presumptive MES or MEE in both groups of mice at E14. In +/+ animals at E15, TN dissipated and became confined to the oral basement membrane by E18. At E15 and E18 in cleft Br/Br mutants, TN stained beneath the MEE.


CONCLUSIONS
Although the distributions of ECM molecules are similar during normal and cleft palatogenesis, differences in TN expression are associated with cleft palate formation.
Singh
G D
GD
Department of Dental Surgery and Periodontology, Dundee Dental Hospital and School, University of Dundee, Scotland, UK. g.d.singh@dundee.ac.uk
Johnston
J
J
Ma
W
W
Lozanoff
S
S
D000818:Animals; D000906:Antibodies; D001485:Basement Membrane / Q000196:embryology; D002972:Cleft Palate / Q000196:embryology* / Q000378:metabolism; D003094:Collagen / Q000032:analysis* / Q000235:genetics; D004396:Coloring Agents; D004848:Epithelium / Q000196:embryology; D005109:Extracellular Matrix / Q000235:genetics; D005145:Face / Q000002:abnormalities* / Q000196:embryology; D005353:Fibronectins / Q000032:analysis* / Q000235:genetics; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D018507:Gene Expression Regulation, Developmental; D005865:Gestational Age; D007150:Immunohistochemistry; D007797:Laminin / Q000032:analysis* / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D009061:Mouth Mucosa / Q000196:embryology; D010159:Palate / Q000196:embryology; D019063:Tenascin / Q000032:analysis* / Q000235:genetics
D000906:Antibodies; D004396:Coloring Agents; D005353:Fibronectins; D005456:Fluorescent Dyes; D007797:Laminin; D019063:Tenascin; D003094:Collagen; D016650:Fluorescein-5-isothiocyanate