A dosage-dependent role for Spry2 in growth and patterning during palate development

Abstract

The formation of the palate involves the coordinated outgrowth, elevation and midline fusion of bilateral shelves leading to the separation of the oral and nasal cavities. Reciprocal signaling between adjacent fields of epithelial and mesenchymal cells directs palatal shelf growth and morphogenesis. Loss of function mutations in genes encoding FGF ligands and receptors have demonstrated a critical role for FGF signaling in mediating these epithelial-mesenchymal interactions. The Sprouty family of genes encode modulators of FGF signaling. We have established that mice carrying a deletion that removes the FGF signaling antagonist Spry2 have cleft palate. We show that excessive cell proliferation in the Spry2-deficient palate is accompanied by the abnormal progression of shape changes and movements required for medially directed shelf outgrowth and midline contact. Expression of the FGF responsive transcription factors Etv5, Msx1, and Barx1, as well as the morphogen Shh, is restricted to specific regions of the developing palate. We detected elevated and ectopic expression of these transcription factors and disorganized Shh expression in the Spry2-deficient palate. Mice carrying a targeted disruption of Spry2 fail to complement the craniofacial phenotype characterized in Spry2 deletion mice. Furthermore, a Spry2-BAC transgene rescues the palate defect. However, the BAC transgenic mouse lines express reduced levels of Spry2. The resulting hypomorphic phenotype demonstrates that palate development is Spry2 dosage sensitive. Our results demonstrate the importance of proper FGF signaling thresholds in regulation of epithelial-mesenchymal interactions and cellular responses necessary for coordinated morphogenesis of the face and palate.

Fig. 1

Altered morphogenic progression of the 36Pub mutant palate. H&E stained serial frontal sections through the entire anterior-posterior length of E14.5 and E15.5 palates were grouped into four regions each representing one quarter of the total palatal length and the morphology of the palate within each region was compared between wildtype (E14.5 A-D; E15.5 I-L) and mutant (E14.5 E-H; E15.5 M-P). Images show the right side palatal shelf of the wildtype beside the contralateral mutant shelf from the same region for comparative purposes. By E14.5 the anterior palatal shelves (A) of the wildtype have elevated above the tongue and in the middle region (B) the shelves are in contact although still separated by the medial epithelial seam. The posterior palate (C) and region of the soft palate (D) of the E14.5 wildtype exhibit a medially projecting prominence indicative of directed growth towards the midline (black arrows). E14.5 36Pub mutant palates have not elevated in the anterior half (E, F) and the posterior palate (G) and region of the soft palate (H) have failed to acquire the shape changes associated with the transition from vertically- to medially-directed outgrowth. The E15.5 wildtype palate is closed along its entire A-P length (I-L) and fusion of the shelves by breakdown of the medial epithelial seam is nearly complete (asterisks). By E15.5 the 36Pub mutant palate has still not elevated dorsal to the tongue (M,N) although the posterior half of the palate does exhibit limited growth towards the midline (arrows in O&P). Arrowheads point to sites of mesodermal condensation and matrix deposition associated with incipient intramembranous ossification that is particularly impacted in the middle and posterior of the mutant palate. Abbreviations: m, Meckel's cartilage; mes, medial epithelial seam; ns, nasal septum; t, tongue.

Fig. 2

Spry2 expression during orofacial development. (A, B) Spry2 is expressed in structures that will outgrow and fuse to form the primary palate and jaw. (A) At E9.5 Spry2 is expressed in the telencephalon and olfactory placode as well as in both the maxillary and mandibular processes of the first branchial arch (arrow). By E11.5 (B) Spry2 expression is seen in the area of fusion between the frontonasal process and the anterior maxillary process of the first branchial arch (white arrow). (C, D) Wholemount view of the oronasal cavity (rostrocaudal from top to bottom) showing dynamic expression of Spry2 during palate development between E12.5 and E14.5. (C) At E12.5, Spry2 is strongly expressed in the epithelium and mesenchyme of the anterior half of the oral cavity that is derived from the mid-maxilla of the first branchial arch. (D) Prior to contact of the shelves at E14.5 Spry2 expression is seen in a broad domain in the mid-posterior palatal epithelium (arrow) and the along the medial aspect of the approaching shelves with strongest expression in the posterior half of the palate (white arrowhead). (E-H) Wholemount view of oronasal cavity (rostrocaudal from top to bottom) and (I-T) frontal section views of gene expression at E13.5 showing Spry2 spatially referenced to the expression of components of the FGF signaling pathway (Fgf10: E, I, J, K; Fgfr2: F, L, M, N; Spry2: G, O, P, Q; Etv5: H, R, S, T). (E) Fgf10 is expressed in the mesenchyme of the anterior two-thirds of the palate and becomes progressively localized to the oral side of the palatal shelf in the posterior region of its domain (I, J, & K). (F) Fgfr2b and Fgfr2c expression is detected with a single probe recognizing both isoforms. Fgfr2b is broadly expressed throughout the palatal epithelium (L, M, & N) whereas mesenchyme specific Fgfr2c is localized to discrete domains of osteogenic mesenchyme in the anterior maxilla and underlying the nasal epithelium (black arrowheads). (G,P) Spry2 is expressed throughout the palatal epithelium with strongest levels on the oral side of the mid-palate. Spry2 in the mesenchyme is strongly expressed in the anterior (O) and at slightly lower levels in the posterior (Q) with expression in the mid-palate tightly restricted to the mesenchyme subjacent to the nasal epithelium (P). (H) Etv5 in the epithelium is restricted to the anterior two-thirds of the palate in the oral surface directly overlaying the Fgf10 domain. Etv5 in the mesenchyme is broadly expressed in the anterior palatal shelves (R), although more medially restricted than that of Spry2 (compare O and R), and becomes progressively restricted to the mesenchyme subjacent to the nasal epithelium (S, T).

Fig. 3

Spatial expression pattern from Spry2-BAC transgene is similar to endogenous Spry2 expression but at reduced levels. (A-F) The Spry2-BAC drives expression in a pattern comparable to the endogenous locus. Spry2 transcripts are detected in the developing branchial arches and facial primordia of wildtype embryos at E10.5 (A) and in a frontal view of E13.5 wildtype palatal shelves (D). The Spry2-BAC hemizygous, 36Pub mutant shows comparable expression in the developing E10.5 embryo (B) as well as in the palatal shelves at E13.5 (E), note reduced expression levels (arrowheads). Spry2 transcripts are absent in the non-transgenic 36Pub mutant E10.5 embryo (C) and E13.5 palatal shelves (F). (G) Quantitative RT-PCR analysis of Spry2 expression in Spry2-BAC transgenic line 2 and 69 using E14.5 palatal tissue shows Spry2 is expressed at ∼12.5% and ∼25% of endogenous levels, respectively.

Fig. 4

Hypomorphic rescue by Spry2-BAC transgene of craniofacial defects observed in 36Pub mutant mice. (A-C) Spry2-BAC transgene rescues defects of the primary palate. E15.5 wildtype (A) and E15.5 Spry2-BAC hemizygous, 36Pub mutant mice (B) show normal development of the primary palate and lip whereas E15.5 36Pub mutant mice (C) exhibit facial clefting. (D-F) Rescue of cleft palate at E18.5 is shown by complete closure in wildtype (D) and Spry2-BAC hemizygous, 36Pub mutant mice (E) compared with cleft palate in the 36Pub mutant (F).

Fig. 5

Altered gene expression along the anterior-posterior axis of the 36Pub mutant palate. (A-D) Etv5 expression. (A) Etv5 expression in the E13.5 wildtype palate is restricted to segmented domains in the anterior two-thirds of the epithelium and in the mesenchyme to the medial aspect of the vertical shelf. Expression of Etv5 in E13.5 36Pub (B) mutant is upregulated and expanded in both the epithelial and mesenchymal domains (arrows). (C) Wildtype expression of Etv5 in the mesenchyme at E14.5 becomes restricted to the anterior two-thirds of the palatal shelf. In E14.5 36Pub mutants (D) Etv5 remains upregulated and expanded in the epithelium and ectopic mesenchymal expression extends into the posterior of the shelves (arrows in D). (E-H) Barx1 expression. (E) E13.5 wildtype Barx1 expression is restricted to segmented domains in the anterior epithelium and to the mid-posterior half of the palatal mesenchyme. In the E13.5 36Pub mutant (F) Barx1 expression is elevated in the anterior epithelium and the posterior mesenchymal domain is expanded. Note arrows marking elevated medial domain of Barx1 in the posterior of mutants compared to wildtype, also compare bars marking length of Barx1 negative domains in the anterior mesenchyme. (G) Barx1 in the E14.5 wildtype is expressed in anterior epithelium and posterior mesenchyme and elevated and expanded expression domains persist in the E14.5 36Pub mutant (arrows in H). (I-L) Msx1 expression. (I) Msx1 is expressed at low levels in the mesenchyme in the anterior third of the E13.5 wildtype palate. (J) The 36Pub mutant palate shows elevated Msx1 expression anteriorly (arrows) and ectopic expression in the mid-posterior palate (arrowheads). (K) Weak Msx1 expression in the E14.5 wildtype anterior palate compared with (L) continued elevated expression in the E14.5 36Pub mutant palate (arrows). (M-P) Tbx22 expression. (M) Tbx22 is strongly expressed in the mesenchyme of the mid-posterior of the E13.5 wildtype palate. (N) We detected no difference in Tbx22 expression in the E13.5 36Pub mutant. (O) At E14.5 as the posterior palate initiates medially directed growth the wildtype Tbx22 expression domain extends to the posterior end of the palate. (P) Failure to initiate posterior expansion of Tbx22 expression at E14.5 (white arrowheads) is associated with altered morphological progression of the posterior palate in 36Pub mutants. Dashed lines outline posterior palatal shelf to highlight altered morphology of E14.5 36Pub mutants.

Fig. 6

Shh expression in the rugae highlights regional growth of the palate and Shh expression is altered by loss of Spry2. (A) Shh expression on the oral surface of the palatal shelves in wildtype embryos from E11.5 through E14.5. Expression is restricted to stripes corresponding to developing rugae (numbered 1-7) and a posterior domain (pd) corresponding to the presumptive soft palate. The number of Shh expressing rugae increases as palate development progresses. Using expression in the molar tooth bud (asterisks) and the first distinct stripe of Shh (1) as landmarks illustrates that the relative position of these two domains remains constant whereas the number of rugae increases anterior to this position. Because new rugae are formed just anterior to stripe #1, numbering the rugae in order of their formation places each subsequently formed rugae (#3-7) between the anterior most stripe #2 and the posterior stripe #1. This results in the apparent posterior regression of the rugae forming region and presumptive soft palate relative to the rostral extension of the anterior palate (compare relative A-P positions of red asterisks). (B&C) Well defined domains of Shh expression in the rugae of E14.5 wildtype (B), Shh expression in the E14.5 36Pub mutant palate is both diffuse and fragmented into isolated clumps of expressing cells (arrowheads in C). (D&E) Wildtype expression of Ptc1 is restricted to the mesenchyme immediately adjacent to the overlying Shh domain in the rugae and broadly expressed in a posterior domain (pd) of the mesenchyme of the presumptive soft palate (D). In the E14.5 36Pub mutant palate (E) Ptc1 expression is disorganized and ectopically expressed in expanded domains of the palate. Note that Ptc1 expression in stripe #1 appears less impacted than in more anterior domains (arrowheads in E). (F&G) Rugae morphology at E18.5. The wildtype palate typically has an orderly array of 7 to 9 rugae, the 3 anterior most of which fuse across the midline (F). Rugae morphology is disorganized and fragmented in the cleft palates of both 36Pub homozygous (not shown) and 36Pub/ Spry^2Δ_ORF compound mutants (arrows in G) at E18.5.

Fig. 7

Altered proliferation in the 36Pub mutant palate. Frontal sections in the mid-posterior region of E14.5 wildtype (A) and E14.5 36Pub mutant (B) palates show increased proliferation in the mutant shelves. Mitotic cells (green) were detected using a BrdU incorporation assay. Nuclei are counterstained with DAPI (blue). Note increased labeling of mitotic cells throughout both the overlying epithelium and underlying mesenchyme particularly along the medial aspect of the palatal shelf and mesenchyme underlying the MEE (white arrows). (C) A regional proliferation map of the mitotic index along the A-P axis of the palate highlights increased cell proliferation in the mutant palate (asterisks p<0.05, see Supplementary Table 1).

Fig. 8

Altered morphology of skeletal elements in the Spry2-deficient palate. (A-C) E18.5 alcian blue and alizarin red stained skeletal preparations identify a hypomorphic phenotype of reduced skeletal differentiation in Spry2-BAC hemizygous, 36Pub mutant mice. Compared to E18.5 wildtype (A) the palatal processes of the maxilla and palatine bones of the hard palate (outlined in dashed black line in A and B) are reduced in size and show altered morphology in the closed palates of E18.5 Spry2-BAC hemizygous, 36Pub mutant mice (B). In the nontransgenic E18.5 36Pub mutant (C) the palatal process of the maxilla is highly hypoplastic (outlined in dashed white line) while the more posterior process of the palatine bone is completely absent (arrows). Abbreviations: ppmx, palatal process of the maxilla; pppl, palatal process of the palatine bone; vm, vomeronasal.

Fig. 9

Model of epithelial-mesenchymal signaling regulating cell proliferation during palatogenesis. In the anterior palate, mesenchymal proliferation is controlled by a Bmp4/Msx1 feedback loop required for Shh and Bmp2 expression. Proliferation and Shh expression in the epithelium is also dependent on Fgf10 signaling through Fgfr2b. Fgf9 is a candidate for an epithelial source of FGF regulating mesenchymal proliferation and gene expression. The palatal mesenchyme is patterned into anterior Msx1 and posterior Barx1 domains. The expression of these genes is responsive to thresholds of FGF signaling. Elevated FGF activity in the absence of Spry2 antagonism leads to increased proliferation, elevated and expanded Etv5 and Barx1 expression, ectopic expression of Msx1 (dashed arrow), and altered Shh expression. Therefore FGF signaling thresholds must be properly modulated to coordinate shelf outgrowth and loss of the FGF antagonist Spry2 results in perturbations of gene expression, cell proliferation and timing of palatal development.