Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

Abstract

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.

Fig. 1.

Cleft palate in Adamts9^+/–;bt/bt mice. (A) External appearance of newborn mice of the indicated genotypes. (B) Cleft palate in Adamts9^+/–;bt/bt mice (right, arrows). (C) mCT of the skull in three projections shows that all skull bones are present and similar in each genotype, other than abnormal formation of the palatine (asterisk) and basi-sphenoid, the anomalous development of which is presumed secondary to abnormal palatogenesis in Adamts9^+/–;bt/bt mice. The pterygoid processes of the sphenoid bone are indicated by arrows; their wider spacing in Adamts9^+/–;bt/bt mice is typical of unfused palates.

Fig. 2.

Adamts9 and Adamts20 participate independently and cooperatively in palatal shelf extension towards the midline. (A) The palatal fusion defect in Adamts9^+/–;bt/bt mice results from defective shelf elevation and extension to midline between E13.5 and E14.5. Coronal sections at various stages of palatal development are shown for the indicated genotypes. Palatal shelves are indicated by asterisks. There is reduced sculpting of the palatal shelves from bt/bt and Adamts9^+/–:bt/+ mice, as well as Adamts9^+/–;bt/bt mice. Scale bar: 100 μm. (B) Scanning electron microscopy of palates (inferior view) of various genotypes was obtained at the indicated gestational ages. The arrows indicate the location of the medial edge of the palatal shelf. Note the delayed closure at E14.5 in Adamts9^+/–;bt/+ and bt/bt mice, and failure to close by E15.5 in Adamts9^+/–;bt/bt mice.

Fig. 3.

Adamts9 and Adamts20 are expressed locally in palatal shelf, but in distinct cell types. (A,B) In situ hybridization (ISH) of Adamts9 (A) and Adamts20 (B) mRNA in the palatal shelf (asterisk) at E13.5 (coronal sections). ISH signal is red and nuclei blue (DAPI). Note that expression of both Adamts9 and Adamts20 is confined to mesenchyme, but in distinct distributions. (C,D) Combined β-gal histochemistry (blue) and endomucin immunohistochemistry (red) of Adamts9^+/– mice showing that Adamts9 expression is confined to microvascular endothelial cells. (C) An overview of staining in an E13.5 palatal shelf (D) Palatal capillaries (from C, boxed) at higher magnification. Note that endomucin-positive cytoplasm surrounds β-gal-stained nuclei. (E,F) Endomucin immunostaining shows comparable capillary density and distribution in Adamts9^+/–;bt/bt and bt/+ palatal shelf at E13.5. Scale bars: 50 μm.

Fig. 4.

Decreased cell proliferation and reduced cell density in Adamts9^+/–;bt/bt palatal shelves. (A) Anti-pH3 immunostaining (brown) in palatal shelves. Note the blue staining (β-gal) in capillaries of Adamts9^+/–;bt/bt palatal shelves. The genotype and gestational ages of the mice are indicated. The proliferation index was determined in the area between the dotted line and the medial edge of the shelf. Note the rounded shape of the Adamts9^+/–;bt/bt palatal shelves. (B) Proliferation index (pH3-stained nuclei/total nuclei counted) at E13.5 and E14.5 (n=5 for each genotype at each age). A statistically significant difference was observed at both ages (^*, P<0.05; #, P<0.005). (C) Cell density was quantified in the center of the palatal shelf by counting nuclei in sections from Adamts9^+/–;bt/bt and bt/+ palatal shelves (n=5 for each genotype) in the area indicated by the box. (D) A statistically significant reduction in cell density (^*P<0.05) at E13.5 and E14.5 was noted in Adamts9^+/–;bt/bt palatal shelves. Scale bars: 50 μm.

Fig. 5.

Versican is present normally in the palate but its processing is reduced in Adamts9^+/–;bt/bt palatal shelves. (A) Immunofluorescence microscopy of intact versican using a polyclonal antibody that recognizes the GAG-β domain in the V1 and V0 isoforms shows widespread versican localization throughout the palate mesenchyme. The boxed regions are shown at higher magnification to the right. There was no discernible difference in versican localization between the two genotypes. (B) Immunofluorescence microscopy of cleaved versican (using anti-DPEAAE) illustrates its broad distribution within the growing end of the bt/+ (control) palatal shelf. There is greatly reduced staining in the intermesenchymal ECM of the Adamts9^+/–;bt/bt palatal shelf, with residual staining present only around capillaries. The boxed regions are shown at higher magnification to the right. Scale bars: 50 μm.

Fig. 6.

High incidence of cleft palate in bt/bt;Vcan^hdf/+ mice. (A) The incidence of cleft palate in crosses between bt/+;Vcan^hdf/+ and bt/bt mice. (B) Complete cleft of the secondary palate in bt/bt;Vcan^hdf/+ mice. In the bt/bt;Vcan^hdf/+ palate, the edges of the palatal shelves are indicated by arrows. The appearance of the palate is similar to that of the Adamts9^+/–;bt/bt shelves in Fig. 1B. (C) Histology of palatal closure (coronal sections stained with Hematoxylin and Eosin) in the indicated genotypes. Note the retarded growth of palatal shelves of bt/bt;Vcan^hdf/+ mice, but their relatively normal shape compared with Adamts9^+/–;bt/bt shelves (see Fig. 2A), and the failure to establish contact (asterisk at E16.5). Scale bar: 100 μm.

Fig. 7.

Decreased cell proliferation but normal cell density in bt/bt;Vcan^hdf/+ palatal shelves. (A) Anti-pH3 immunostaining in palatal shelves. The genotype and gestational ages of the mice are indicated. The proliferation index was determined in the area between the dotted line and the medial edge of the shelf. (B) Quantification of proliferation (pH3-stained nuclei/total nuclei counted) at E13.5 and E14.5. ^*, P<0.05 (n=4). (C) Cell density was quantified in the center of the palatal shelf by counting all nuclei in sections from bt/bt;Vcan^hdf/+ and bt/+ palatal shelves in the area indicated by the box. (D) There is no significant alteration in cell density at E13.5 or E14.5 in bt/bt;Vcan^hdf/+ palatal shelves (n=4 for each genotype). Scale bars: 50 μm.

Fig. 8.

Versican GAG-β and anti-DPEAAE immunofluorescence reflects both reduced Vcan gene dosage and reduced versican processing in bt/bt;Vcan^hdf/+ palatal shelves. (A) Immunofluorescence microscopy of intact versican using a polyclonal antibody that recognizes the GAG-β domain in the V1 and V0 isoforms, showing reduced staining in bt/bt;Vcan^hdf/+ palatal shelves, consistent with Vcan haploinsufficiency. (B) Immunofluorescence microscopy of cleaved versican (using anti-DPEAAE) illustrates greatly reduced staining in the bt/bt;Vcan^hdf/+ palatal shelves. In bt/+ palatal shelves, note the bright signal around palatal capillaries and its reduction in the bt/bt;Vcan^hdf/+ palatal shelf. Scale bars: 50 μm.

Fig. 9.

Models of versican and ADAMTS function in the mesenchyme. The models summarize the experimental observations in wild-type, Adamts9^+/–;bt/bt (at E13.5 and E14.5) and bt/bt;Vcan^hdf/+ (at E14.5) palatal shelves, and the proposed underlying mechanisms. In wild-type and Adamts9^+/–;bt/bt mice, a deep shade of blue indicates normal versican levels in the ECM, whereas bt/bt;Vcan^hdf/+ mice have half the normal amount (light-blue). The wild-type model indicates that cleaved versican is normally present in both the pericapillary and intermesenchymal ECM. In both Adamts9^+/–;bt/bt and bt/bt;Vcan^hdf/+ palates there is a paucity of intermesenchymal cleaved versican because ADAMTS20 is absent. In Adamts9^+/–;bt/bt palates there is also a paucity of cleaved versican around capillaries because of Adamts9 haploinsufficiency. In bt/bt;Vcan^hdf/+ palates there is less cleaved versican around capillaries owing to Vcan haploinsufficiency, although Adamts9 dosage is not reduced. We propose that the amount of cleaved versican affects mesenchymal cell proliferation. In addition, the model depicts reduced cell density (i.e. the cell-to-matrix ratio) in the Adamts9^+/–;bt/bt palate, which results from both reduced clearance of versican (i.e. increased ECM) and decreased cell proliferation. Note that these models do not include the epithelium, as the observed mechanism seems to be wholly operational in mesenchyme.