SP8 regulates signaling centers during craniofacial development

Abstract

Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.

Keywords: Anterior neural ridge; Craniofacial development; Cyclopamine; FGF17; FGF8; Neural crest; Olfactory pits; SP8; Signaling center.

Figure 1. Sp8 is required for proper nasal prominence morphogenesis

(A,B) E14.5 Sp8 mutant embryos (B) display severe failure of midline fusion and a loss of many facial structures compared to WT (A). (C–F) Wild type (C,E) and Sp8 mutant (D,F) skeletal preparations show loss of many NC derived bones in mutants, including the maxilla, palate, and frontal bone. Paraxial mesoderm-derived parietal and interparietal bones are also absent in Sp8 mutants. Dotted circle in panel F indicates a hole in the skull where the brain is exposed in the Sp8 mutant. (G–R) Facial prominences were digitally pseudo-colored red (maxillary prominence), blue (lateral nasal prominence), green (medial nasal prominence), and purple (frontonasal region) in E10.5, E11.5, and E12.5 WT (G,I,K,M,O,Q) and Sp8 mutants (H,J,L,N,P,R). Mutant lateral and medial nasal prominences are reduced in size and dysmorphic in shape. Exoccipital, EO; Frontal, FR; Interparietal, IP; Lateral Nasal Prominence, LNP; Mandible, MD; Maxilla, MX; Medial Nasal Prominence, MNP; Nasal, NS; Neuroepithelium, NE; Parietal, PR; Premaxilla, PMX; Supraoccipital, SO; Tongue, T

Figure 2. SP8 is made in the anterior neural ridge and olfactory pit signaling centers

(A,C) Immunofluorescence for SP8 on transverse sections of E8.5 embryos at 9 somites showed high SP8 levels in the hindbrain, anterior neural ridge (arrows), and epidermal ectoderm. (B,D) Sagittal sections at E9.5 revealed SP8 localization in the anterior neural ridge (arrow) and hindbrain. (E) Coronal section at E10.5 showed robust SP8 localization in the olfactory pit epithelium signaling center (arrows) and medial telencephalon (arrowhead). First Branchial Arch, 1BA; Foregut, FG; Forebrain, FB; Hindbrain, HB; Lateral Nasal Prominence, LNP; Medial Nasal Prominence, MNP; Midbrain, MB; Olfactory Pit, OP

Figure 3. Conditional Inactivation of Sp8 expression reveals important roles of Sp8 in Pax3-Cre and FoxG1-Cre expressing cells

Transgenic Cre lines selectively removed Sp8 expression in specific tissue compartments when combined with one floxed and one null Sp8 allele. Whole mount and skeletal preparations were performed. (A–L) Pax3-Cre conditional mutants displayed variable phenotypes. Embryos with severe malformations including excencephaly (black arrowhead), failure of facial prominence fusion along the midline (arrow), truncation of the anterior snout structures (white arrowhead), and an absence of many cranial bones closely resembled the Sp8 null mutant (n=4, A,D,G,J). The moderate phenotypes displayed cleft lip and palate, failure of facial prominence fusion along the midline (arrows), and a truncation of anterior structures (B,C, E,F, H,I, K,L). (O,R,U,X) The FoxG1-Cre conditional mutants displayed malformations of the telencephalon, cleft lip (arrow), and a truncation of anterior facial structures (arrowhead). (M,N, P,Q, S,T, V,W) Neither the Wnt1-Cre (M,P,S,V) nor the Mesp1-Cre (N,Q,T,W) conditional mutants displayed craniofacial phenotypes with the exception of one Mesp1-Cre mutant displaying a mild midline defect (not shown). Exoccipital, EO; Frontal, FR; Interparietal, IP; Mandible, MD; Maxilla, MX; Nasal, NS; Parietal, PR; Premaxilla, PMX; Supraoccipital, SO

Figure 4. Increased apoptosis and reduced proliferation in SP8^−/− facial mesenchyme

(A) Immunofluorescence for cleaved caspase 3 was used to label cells undergoing apoptosis. Wnt1-Cre activation of R26R-GFP labeled the neural crest. Apoptosis was quantified in various compartments of E8.5, E9.5, and E10.5 Sp8^−/− and WT embryos. Results showed a significant increase in apoptosis in the anterior neuroepithelium at E8.5. The neural crest underwent elevated apoptosis at E9.5 as did the medial nasal prominence, lateral nasal prominence, and olfactory pit at E10.5. (B) Immunofluorescence for phospho-histone H3 was used to label cells undergoing proliferation. The neural crest was labeled by Wnt1-Cre activation of R26R-GFP. Proliferation was quantified in various compartments of E8.5, E9.5, and E10.5 of Sp8^−/− and WT embryos. Results showed a significant decrease in the neural crest at E9.5 as well as in the lateral nasal prominence and olfactory pit at E10.5. Anterior Neuroepithelium, AN; Lateral Nasal Prominence, LNP; Medial Nasal Prominence, MNP; Neural Crest Cells, NCC; Olfactory Pit, OP p<0.05 (*), p<0.01 (**), p<0.001 (***)

Figure 5. Disrupted Fgf8, Fgf17 and Gli3 expression in Sp8^−/− embryos

(A–E) In-situ hybridization revealed a down-regulation of Fgf17, Fgf8, and FGF8 downstream targets Erm and Spry in E9.5 Sp8 mutant anterior neural ridge (arrows) (AD). Fgf17 expression was also lost along the midline (arrowhead) and in the olfactory pit (arrows) at E10.5 (D). (F) In-situ hybridization showed no change in expression of the E8.5 anterior neural ridge marker Dlx5 in E8.5 Sp8 mutants (arrows). (G) Q-PCR analysis showed a down-regulation of Fgf17 at E8.5, E9.5, and E10.5. Additionally at E9.5, Gli3, and Fgf8 expression levels were significantly decreased in mutants. (H–J) Western blot analysis (H) and relative levels (I) of GLI3 full length (transcriptional activator) and GLI3 repressor proteins showed a reduction in GLI3 full length and GLI3 repressor, although the change was not statistically significant. No significant change in the ratio of GLI3 full length to repressor levels was detected (J). p<0.05 (*), p<0.01 (**), p<0.001 (***)

Figure 6. Reduction of SHH signaling partially rescued Sp8 craniofacial malformations

(A–C) SHH levels were reduced in Sp8 mutants by deleting one allele of Shh. Sp8^−/−;Shh^−/+ mutants displayed partial craniofacial rescue of anterior, but not dorsal, structures at E18.5 (n=4, arrow in C). (D–G) SHH signaling was reduced through embryonic exposure to cyclopamine. Wild type embryos appeared normal after exposure to 150 mg/kg cyclopamine (F). Approximately one-third (n=13) of the cyclopamine treated mutants (n=41) displayed a partial rescue of facial midline (arrows), excencephaly, and hypertelorism (G). Sp8^−/− mutants exposed to vehicle alone showed the standard mutant phenotype (E).