Abstract
Craniofacial defects are among the most common phenotypes caused by ciliopathies, yet the developmental and molecular etiology of these defects is poorly understood. We investigated multiple mouse models of human ciliopathies (including Tctn2, Cc2d2a and Tmem231 mutants) and discovered that each displays hypotelorism, a narrowing of the midface. As early in development as the end of gastrulation, Tctn2 mutants displayed reduced activation of the Hedgehog (HH) pathway in the prechordal plate, the head organizer. This prechordal plate defect preceded a reduction of HH pathway activation and Shh expression in the adjacent neurectoderm. Concomitant with the reduction of HH pathway activity, Tctn2 mutants exhibited increased cell death in the neurectoderm and facial ectoderm, culminating in a collapse of the facial midline. Enhancing HH signaling by decreasing the gene dosage of a negative regulator of the pathway, Ptch1, decreased cell death and rescued the midface defect in both Tctn2 and Cc2d2a mutants. These results reveal that ciliary HH signaling mediates communication between the prechordal plate and the neurectoderm to provide cellular survival cues essential for development of the facial midline.
Introduction
Primary cilia are microtubule-based organelles present on diverse vertebrate cell types and critical for development. Primary cilia function as specialized cellular signaling organelles that coordinate multiple signaling pathways, including the Hedgehog pathway (Zaghloul & Brugmann, 2011). Defects in the structure or signaling functions of cilia cause a group of human syndromes, collectively referred to as ciliopathies, which can manifest in diverse phenotypes including cystic kidneys, retinal degeneration, cognitive impairment, respiratory defects, left-right patterning defects, polydactyly, and skeletal defects (Baker & Beales, 2009; Schwartz, Hildebrandt, Benzing, & Katsanis, 2011; Tobin & Beales, 2009). In addition to these phenotypes, craniofacial defects including cleft lip/palate, high-arched palate, jaw disorders, midface dysplasia, craniosynostosis, tongue abnormalities, abnormal dentition and tooth number and exencephaly, are observed in approximately one-third of individuals with ciliopathies (Brugmann, Cordero, & Helms, 2010b; Zaghloul & Brugmann, 2011). The molecular and developmental etiology of these craniofacial abnormalities remains poorly understood.
HH signaling is intimately involved in forebrain and midface development (D. H. A. J. A. Helms, 1999; Rubenstein & Beachy, 1998). In humans, inherited mutations that compromise pathway activity impair forebrain development and cause hypotelorism (Fuccillo, Joyner, & Fishell, 2006; D. H. A. J. A. Helms, 1999; Hu & Marcucio, 2008; Marcucio, Cordero, Hu, & Helms, 2005; Muenke & Beachy, 2000; Young, Chong, Hu, Hallgrimsson, & Marcucio, 2010). For example, mutations in SHH lead to holoprosencephaly (Chiang et al., 1996; Cohen & Shiota, 2002). Meckel syndrome (MKS), a severe ciliopathy, is also characterized by holoprosencephaly and hypotelorism (Ben Chih et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011). MKS-associated genes encode proteins that form a complex that comprises part of the transition zone, a region of the ciliary base that regulates ciliogenesis and ciliary membrane protein composition in a tissue-specific manner (Ben Chih et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011; Roberson et al., 2015). Disruption of this transition zone complex results in impaired the ciliary localization of several membrane-associated signaling proteins including Smoothened (SMO), Adenylyl cyclase 3 (ADCY3), Polycystin 2 (PKD2) and ARL13B (Ben Chih et al., 2011; Garcia-Gonzalo et al., 2011; Roberson et al., 2015).
We investigated the molecular underpinnings of forebrain and midface defects in ciliopathies utilizing multiple mouse mutants affecting the transition zone. The mutants exhibited forebrain and midface defects by E9.5, which persisted throughout development. In these mutants, the prechordal plate, an organizer of anterior head development, displayed defects in HH pathway activation at E8.0. These early prechordal plate defects attenuated Shh expression in the adjacent ventral forebrain. Decreased HH signaling increased apoptosis in the ventral neurectoderm and facial ectoderm. Surprisingly, reducing Ptch1 gene dosage rescued the apoptosis and its corresponding midface defect. Thus, investigating the function of the transition zone has revealed a key role of prechordal plate-activated HH signaling in forebrain and midface cell survival. Moreover, our genetic results reveal that inhibition of PTCH1 can prevent ciliopathy-associated midface defects. Based on these mouse genetic models, we propose that the etiology of hypotelorism in human ciliopathies is a failure of the prechordal plate to induce SHH expression in the overlying ventral neuroectoderm.
Results
The ciliary MKS transition zone complex is essential for midline facial development
Individuals affected by developmental ciliopathies, such as Meckel, Orofaciodigital and Joubert syndromes, often display craniofacial phenotypes including holoprosencephaly and hypotelorism (Baker & Beales, 2009; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011). To explore the etiology of these craniofacial defects, we examined the craniofacial development in Tctn2 mouse mutants (Garcia-Gonzalo et al., 2011; Shaheen et al., 2011). TCTN2 is a component of the MKS transition zone complex critical for ciliary localization of several ciliary membrane proteins, including SMO, a key ciliary mediator of HH signaling (Ben Chih et al., 2011; Corbit et al., 2005; Garcia-Gonzalo et al., 2011) Mutations in human TCTN2 cause Meckel and Joubert syndromes (Huppke et al., 2015; Sang et al., 2011; Shaheen et al., 2011).
Tctn2+/- embryos were phenotypically indistinguishable from Tctn2+/+ embryos (Supplemental Figure 1). In contrast, embryonic day (E) 10.5 Tctn2-/- embryos exhibited an approximately 50% decrease in infranasal distance (the distance between the nasal pits, the nostril anlage) (Figure 1A). One day later in gestation (E11.5), Tctn2-/- embryos also exhibited midfacial narrowing, including hypoplasia of the frontonasal prominence and fusion of the two maxillary prominences at the midline (Figure 1A). Thus, TCTN2 is essential for development of the facial midline.
To assess whether this narrowing of the facial midline is specific to TCTN2, we examined the possible involvement of two other components of the MKS complex, TMEM231 and CC2D2A, in craniofacial development. Human CC2D2A mutations cause Meckel and Joubert syndromes, and TMEM231 mutations cause Meckel, Joubert and Orofaciodigital syndromes (Noor et al., 2008; Roberson et al., 2015; Shaheen, Ansari, Mardawi, Alshammari, & Alkuraya, 2013; Srour et al., 2012; Tallila, Jakkula, Peltonen, Salonen, & Kestilä, 2008). Similar to Tctn2 mutants, both E10.5 Cc2d2a and Tmem231 mutant embryos exhibited decreased infranasal distance (Figure 1B and Figure 1C, respectively). The similarity of the midline hypoplasia in all three transition zone mutants suggested a common etiology.
We also examined the involvement of a fourth member of the MKS complex, TMEM67, in craniofacial development. Human mutations in TMEM67 also cause Meckel and Joubert syndromes (Otto et al., 2009; Smith et al., 2006). Mutation of mouse Tmem67 causes phenotypes that are less severe than Tctn2, Tmem231 or Cc2d2a (Garcia-Gonzalo et al., 2011). The mild phenotype of Tmem67 mutants may be attributable to its dispensability for ciliary accumulation of HH pathway activator SMO (Garcia-Gonzalo et al., 2011). Unlike Tctn2, Tmem231 and Cc2d2a mutants, Tmem67 mutants did not exhibit altered infranasal distance (Figure 1D). Thus, some, but not all, MKS components are critical for early facial midline development.
Given the central role of the neural crest in craniofacial development as the main source of craniofacial mesenchyme (Santagati & Rijli, 2003), we tested whether transition zone disruption in this tissue is the origin of the midline defect seen in Tctn2 mutants. Interestingly, conditional deletion of Tctn2 in the neural crest using the Wnt1-Cre driver did not result in hypotelorism (Supplemental Figure 2). This result suggests that the neural crest is not the origin of the midline phenotype and led us to investigate a role for Tctn2 in the prechordal plate, an early organizing center critical for development of anterior head structures.
Tctn2 mutants exhibit defects in prechordal plate differentiation soon after gastrulation
As Tctn2, Cc2d2a and Tmem231 mutants all displayed facial midline defects at midgestation, we hypothesized that they shared a role in a patterning event early in craniofacial development. One organizing center critical for early forebrain and craniofacial development is the prechordal plate (Camus et al., 2000; Kiecker & Niehrs, 2001; Muenke & Beachy, 2000; Rubenstein & Beachy, 1998; Som, Streit, & Naidich, 2014). The prechordal plate is the anterior- most midline mesendoderm, immediately anterior to the notochord and in contact with the overlying ectoderm. Previous work demonstrated that surgical removal of the rat prechordal plate results in midface defects (Aoto et al., 2009) that seemed similar to those of the mouse Tctn2, Cc2d2a and Tmem231 mutants.
Therefore, we analyzed the prechordal plate of Tctn2 mutants by examining the expression of axial mesendodermal markers T, Gsc and Shh. Gsc is expressed specifically in the prechordal mesoderm, while Shh and T are expressed in both the prechordal mesoderm and notochord (Dale et al., 1997; Herrmann, 1991; Schulte-Merker et al., 1994). In situ hybridization analysis revealed that in Tctn2 mutants, Shh and T expression in the prechordal plate and notochord were unaffected (Figure 2A). Therefore, TCTN2 is not essential for prechordal plate specification. In contrast, Tctn2 mutants exhibited abrogated Gsc expression in the prechordal plate (Figure 2B), indicating that TCTN2 is critical for prechordal plate differentiation.
The transition zone is critical for HH signaling, and one HH protein, SHH, is essential for Gsc expression in the prechordal plate (Aoto et al., 2009; Ben Chih et al., 2011; Garcia-Gonzalo et al., 2011). Therefore, we investigated whether TCTN2 is required for HH signaling in the prechordal plate by examining the expression of the transcriptional target Gli1. Tctn2 mutants exhibited reduced Gli1 expression throughout the axial mesendoderm, including the prechordal plate (Figure 2B). These results indicate that TCTN2 is dispensable for the formation of the prechordal plate, but is required for midline signaling by SHH to induce Gsc expression in this organizing center.
TCTN2 and other members of the MKS complex are required for proper cilia formation in some tissues but not in others (Garcia-Gonzalo et al., 2011). Therefore, we examined whether TCTN2 is required for ciliogenesis in the prechordal plate. The prechordal plate expresses FOXA2 (Jin, Harpal, Ang, & Rossant, 2001), (Figure 2C). Co-immunostaining embryos for FOXA2 and acetylated tubulin (TUBAc), a marker of cilia, revealed that Tctn2 mutants did not display decreased ciliogenesis in the E8.5 prechordal plate (Figure 2C, middle panel).
In cell types in which the MKS complex is dispensable for ciliogenesis, like neural progenitors, it is required for localization of ARL13B to cilia (Garcia-Gonzalo et al., 2011). Therefore, we examined ARL13B localization in E8.0 control embryos and Tctn2 mutants and discovered that ARL13B localization to prechordal plate cilia was attenuated without TCTN2 (Figure 2C, bottom panel). Thus, TCTN2 is not required for ciliogenesis in the prechordal plate, but does control ciliary composition.
Tctn2 mutants display decreased HH signaling in the ventral telencephalon
The axial mesendoderm helps pattern the overlying neurectoderm (Anderson & Stern, 2016; Rubenstein & Beachy, 1998). In the rostral embryo, the prechordal plate patterns the overlying ventral telencephalon via SHH (Chiang et al., 1996; Xavier et al., 2016). As extirpation of the prechordal plate results in decreased SHH activity in the basal telencephalon (Aoto et al., 2009; Aoto & Trainor, 2015), we investigated whether the prechordal plate defects observed in Tctn2 mutants results in mispatterning of the ventral telencephalon. Although Shh expression in the notochord was unaffected in Tctn2 mutants at E8.75, it was severely reduced in the ventral telencephalon (Figure 3A). This reduced expression of Shh in the ventral telencephalon persisted at E9.5 (Figure 3A).
To assess whether HH pathway activity was compromised by the absence of TCTN2, we assessed the expression of the HH transcriptional targets Gli1 and Ptch1. WM-ISH of Tctn2 mutants revealed dramatically reduced or absent expression of both Gli1 and Ptch1, especially in the basal forebrain (Figure 3B). Consistent with the WM-ISH data, qRT-PCR analysis of E8.5 (Figure 3C) and E9.5 (Figure 3D) Tctn2 mutant heads also revealed decreased expression of Shh, Ptch1 and Gli1, revealing that Tctn2 mutants exhibit both an early defect in prechordal plate differentiation and a defect in HH signaling in the adjacent neurectoderm.
TCTN2 protects neurectoderm and facial ectoderm from apoptosis
In the developing craniofacial complex, SHH induces cell proliferation (D. H. A. J. A. Helms, 1999; Hu et al., 2015). Therefore, we assessed if the reduction in facial midline width in Tctn2 mutants was due to decreased cell proliferation. More specifically, we measured cell proliferation by quantitating phospho-histone H3 in the components of the craniofacial complex – the forebrain, hindbrain, facial ectoderm and mesenchyme (Figure 4A,B). Tctn2 mutants showed no differences in amount or spatial distribution of cell proliferation.
In other developmental contexts, HH signaling promotes cell survival (Ahlgren & Bronner-Fraser, 1999; Aoto et al., 2009; Aoto & Trainor, 2015; Litingtung & Chiang, 2000). Therefore, we assessed apoptosis in the craniofacial complex of Tctn2 mutants. Quantification of TUNEL staining revealed that apoptosis in the mesenchyme was unaffected, but increased in the neurectoderm and facial ectoderm of Tctn2 mutants compared to controls, and most dramatically in the ventral telencephalon (Figure 4C,D). To further test whether apoptosis is increased in the absence of TCTN2, we stained for activators of the intrinsic apoptotic pathway, cleaved Caspase-3 and Caspase-9 (activated CASP3 and CASP9). Both activated CASP3 and CASP9 were increased in the ventral telencephalon and facial ectoderm of Tctn2 mutants at E9.5 (Figure 4E). These data indicate that TCTN2 is required to protect against cell death, but does not affect proliferation, in the neurectoderm and non-neural ectoderm. As SHH also protects neurectoderm from apoptosis (Thibert et al., 2003), we propose that TCTN2 mediates cell survival by promoting HH signaling and that the increase in cell death underlies the midfacial hypoplasia in transition zone mutants.
Reducing Ptch1 gene dosage rescues the facial midline defect in transition zone mutants
To assess whether decreased HH signaling is not just correlated with midfacial hypoplasia in transition zone mutants, but is causative, we investigated whether modulating the HH pathway could rescue the midface defects. We employed a strategy targeting Ptch1, a negative regulator of the HH pathway, by generating Tctn2-/- Ptch1-/+ embryos and comparing them to Tctn2-/- Ptch1+/+ embryos. Surprisingly, removing a single allele of Ptch1 in Tctn2 mutants restored midface width at E11.5 (Figure 5A,B).
To assess whether removing a single allele of Ptch1 restores midface width in other transition zone mutants, we generated Cc2d2a-/- Ptch1-/+ and Cc2d2a-/- Ptch1+/+ embryos. As with Tctn2, removing a single allele of Ptch1 restored midface width in Cc2d2a mutants (Figure 5C,D). Thus, reducing Ptch1 gene dosage rescues midface expansion in both models of ciliopathy-associated hypoplasia.
As we had hypothesized that increased apoptosis underlay the midface hypoplasia of Tctn2-/- Ptch1+/+ embryos, we assessed apoptosis in Tctn2-/- Ptch1+/- embryos via TUNEL staining. Tctn2-/- Ptch1+/- embryos exhibited less apoptosis than Tctn2-/- Ptch1+/+ embryos, with a restriction of apoptosis in the ventral telencephalon (Figure 5E-F). These results bolster the hypothesis that increased midline apoptosis accounts for the midline hypoplasia of transition zone mutants.
As genes encoding transition zone MKS components are epistatic to Ptch1 (Reiter, 2006), we pondered how reducing Ptch1 gene dosage restored facial midline development to transition zone MKS component mutants. The best studied role for PTCH1 is in repression of the HH signal transduction pathway. Therefore, we examined HH signal transduction pathway activity in Tctn2-/- Ptch1+/+ and Tctn2-/- Ptch1+/- embryos. WM-ISH revealed that expression of neither Shh nor Gli1 was increased in the ventral telencephalons of Tctn2-/- Ptch1+/- embryos in comparison to Tctn2-/- Ptch1+/+ embryos (Figure 5G). Thus, the restoration of midface growth by reduction of Ptch1 gene dosage is not due to a restoration of HH signal transduction.
In addition to its role in regulating HH signal transduction, PTCH1 exhibits pro-apoptotic activity in vitro (Thibert et al., 2003). As reducing Ptch1 gene dosage reduces apoptosis without increasing HH signal transduction in Tctn2 mutants, we conclude that it is the PTCH1-mediated death of the midline neurectorderm and facial ectoderm, and not alterations in HH signal transduction within those cells, that is the etiology of midface defects in transition zone mutants. Taken together, these results suggest a working model for how ciliary HH signaling regulates midface development.
In wild-type embryos, HH signaling within the prechordal plate is critical for Gsc expression and the induction of Shh in the adjacent neurectoderm and inhibition of apoptosis (Figure 6A). In transition zone mutants, defects in prechordal plate signaling cause reduced SHH in the neurectoderm, resulting in increased PTCH1-mediated cell death and midline collapse (Figure 6B). In transition zone mutants lacking a single allele of Ptch1, reduced SHH in the neurectoderm persists, but the attenuated PTCH1 is no longer sufficient to induce extensive cell death, allowing for normal midline facial development (Figure 6C).
Discussion
Using a combination of genetic, developmental and biochemical techniques, we have identified a common etiology by which disruption of MKS transition zone proteins (TCTN2, CC2D2A, and TMEM231) result in midline hypoplasia and hypotelorism. We traced the origin of the molecular defect contributing to the midline phenotype to the prechordal plate, defects in which resulted in reduced HH pathway activation and cell survival in the adjacent neurectoderm and facial midline collapse. We uncovered Ptch1 gene dosage as a key mediator of cell survival in the facial midline of transition zone mutants, as loss of a single allele of Ptch1 rescued cell survival and midline development in Tctn2 mutants. Together, these results reveal a new paradigm whereby primary cilia mediate signal crosstalk from the prechordal plate to the adjacent neurectoderm to promote cell survival, without which the facial midline collapses and hypotelorism results.
Different ciliopathies are associated with either narrowing or expansion of the facial midline (hypotelorism or hypertelorism) (Schock et al., 2015; Zaghloul & Brugmann, 2011). Severe ciliopathies associated with perinatal lethality, such as Meckel syndrome, can present with hypotelorism or hypertelorism while other ciliopathies such as Joubert syndrome typically present with hypertelorism (Brugmann, Cordero, & Helms, 2010b; MacRae, Howard, Albert, & Hsia, 1972; Schock & Brugmann, 2017). How disruption in primary cilia can give rise to these opposing phenotypes has been an active area of interest. Hypertelorism is attributable to roles for cilia in promoting Gli3 repressor formation in neural crest cells (Brugmann, Allen, James, Mekonnen, et al., 2010a; C. F. Chang et al., 2014; C.-F. Chang, Chang, Millington, & Brugmann, 2016; Liu, Chen, Johnson, & Helms, 2014). Our work implicates a distinct etiology of hypoterlorism: rather than involving neural crest, midline hypoplasia can be caused by defects in the ciliary transition zone in the prechordal plate.
The earliest alteration we detected in Tctn2-/- signaling centers that regulate craniofacial development was in the prechordal plate at the end of gastrulation. TCTN2 was dispensable for the expression of Shh and T in the prechordal plate, indicating that induction and specification of the prechordal plate were unaffected. In contrast, TCTN2 was essential for prechordal plate expression of Gli1 and Gsc. In tissues such as the limb bud, TCTN2 is dispensable for ciliogenesis but critical for ciliary HH signaling and the induction of HH target genes such as Gli1 (Ben Chih et al., 2011; Dowdle et al., 2011; Garcia-Gonzalo et al., 2011). We found that, similarly in the prechordal plate, TCTN2 is dispensable for ciliogenesis but critical for induction of Gli1.
In many developmental events, such as limb patterning, SHH signals to neighboring cells to induce a pattern (Panman & Zeller, 2003; Zhulyn et al., 2014). In other developmental events, such as notochord to neural tube signaling, SHH signals produced by the notochord induce the expression of Shh in the overlaying neural tube (Fuccillo et al., 2006). SHH produced by the prechordal plate may fall into the latter category, as the absence of Shh expression in Tctn2-/- mutants presages reduced Shh expression and reduced expression of HH pathway transcriptional targets Gli1 and Ptch1 in the region of the basal forebrain sometimes referred to as the rostral diencephalon ventral midline. Thus, in the posterior midline, the notochord induces Shh expression in overlying neuroectoderm, and in the anterior midline, the prechordal plate induces Shh expression in the overlying neuroectoderm. In the failure of the prechordal plate to induce Shh expression in the overlying neurectoderm, Tctn2 mutants recapitulate previous observations of Lrp2 mutants which display attenuated responses to SHH (Christ et al., 2012). One possible mechanism by which SHH may activate expression of Shh in the basal forebrain is via the induction of the transcription factor SIX3. SIX3 is regulated by HH signaling and required for the induction of Shh in the developing forebrain (Geng et al., 2008; Jeong et al., 2008). However, our observation that Six3 expression is unaltered in in the forebrains of Tctn2 mutants diminishes support for this hypothesis.
In caudal neural tube and limb patterning, HH signals induce patterning. In hair follicles and cerebellar granule cells, HH signaling promote proliferation. In addition to roles in patterning and proliferation, HH signals can bind to PTCH1 to inhibit apoptosis (Aoto & Trainor, 2015; Borycki et al., 1999; Thibert et al., 2003). Our data are consistent with a role of PTCH1 in promoting apoptosis in the neuroectoderm and facial ectoderm which is inhibited by prechordal plate-produced SHH. In the absence of TCTN2, the prechordal plate does not produce SHH, releasing PTCH1 to promote apoptosis in the midline and resulting in midface hypoplasia. This model is consistent with previous data demonstrating that surgical ablation of the prechordal plate reduces the forebrain (Aoto et al., 2009). Increased cleaved Caspase-3 and Caspase-9 staining in the basal forebrain and facial ectoderm of E9.5 Tctn2 mutants provides further support for apoptosis contributing to the midline defect.
We sought to identify where TZ function is critical to coordinate normal midline facial development by taking a tissue-specific approach to delete Tctn2 in the various tissues that comprise the craniofacial complex. Deletion of Tctn2 in the prechordal plate (via Isl1-cre) or the neurectoderm (via Sox1-cre) did not recapitulate the midline hypoplasia seen in Tctn2 global null mutants (Supplemental figure 3). Similarly, deletion of Tctn2 in the facial ectoderm (via Crect-cre) or in the forebrain and facial ectoderm (via Foxg1-cre) also failed to result in midline collapse (Supplemental figure 4). This result could be due to residual TCTN2 function at these early timepoints as indicated by persistent ARL13B localization at the cilium, low turnover of TZ components, and an inability to delete Tctn2 at an early developmental timepoint where the TZ coordinates midline facial development. New genetic tools that will allow an earlier deletion of Tctn2 in craniofacial complex tissues will be critical to uncover the tissue-specific requirements of Tctn2 in midline development.
Surprisingly, removing one allele of Ptch1 fully rescues the midface defect in both Tctn2 and Cc2d2a transition zone cilia mutants. Even more surprisingly, this phenotypic rescue is not associated with restoration of either Shh expression or HH pathway activation in the basal forebrain. We propose that reducing Ptch1 levels attenuates the PTCH1-mediated pro-apoptotic program normally attenuated by SHH in the basal forebrain.
In summary, we have identified the primary cilia transition zone as a critical regulator of facial midline development. The transition zone component TCTN2 was critical for SHH signaling in the prechordal plate and uncovered a signaling paradigm whereby the transition zone promotes cell survival by mediating crosstalk between the prechordal plate and neurectoderm to promote HH pathway activation. These results provide new insights into how primary cilia mediate cell survival to promote facial development.
Materials and Methods
Mouse Strains
All mouse protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco. Tctn2+/- (Tctn2tm1.1Reit), Cc2d2a+/- (Cc2d2aGt(AA0274)Wtsi), Tmem231+/- (Tmem231Gt(OST335874)Lex), and Tmem67+/- (Tmem67tm1Dgen) mouse alleles have been previously described. Wnt1-cre (Tg(Wnt1-cre)11Rth) and Islet1-cre (Isl1tm1(cre)Sev) mice were obtained from Brian Black, Sox1-cre (Sox1tm1(cre)Take) mice were obtained from Jeff Bush, Foxg1-cre (Foxg1tm1(cre)Skm) mice were obtained from Stavros Lomvardas, and Crect-cre (Tg(Tcfap2a-cre)1Will) mice were obtained from Trevor Williams. The Ptch1tm1Mps allele was used in this study as a null allele. All mice were maintained on a C57BL/6J background. For timed matings, noon on the day a copulation plug was detected was considered to be 0.5 days postcoitus.
Immunofluorescence
The antibodies used in this study were rabbit α-Arl13b (1:1000, Proteintech 17711-1-AP), rabbit α-cleaved-caspase 3 (Asp175) (1:400, Cell Signaling #9664), rabbit α-cleaved-caspase 9 (Asp353) (1:100, Cell Signaling #9509), rabbit α-Phospho-Histone H3 (Ser28) (1:400, Cell Signaling #9713), chicken anti-GFP (1:1000, Aves labs GFP-1020), goat gamma-tubulin (1:200, Santa Cruz sc7396), rat E-Cadherin (1:1000, Invitrogen 13-1900), and rabbit FoxA2 (1:400 abcam ab108422). The In Situ Cell Death Detection Kit, Fluorescein (Roche) was used for TUNEL cell death assay. For immunofluorescence antibody staining of frozen tissue sections, embryos were fixed overnight in 4% PFA/PBS, washed in PBS and cryopreserved via overnight incubation in 30% sucrose/PBS. Embryos were embedded in OCT and frozen at -80C. Frozen OCT blocks were cut into 10μM sections. For immunostaining, frozen sections were washed 3×5’ in PBST (0.1%Tween-20/PBS) followed by blocking for 2 hours in blocking solution (5% donkey serum in PBS+ 0.3% Triton X-100 + 0.2% Na-azide). Slides were incubated overnight in primary antibody diluted in blocking solution at 4 degrees. The following day, slides were washed 3×10’ in PBST, stained with appropriate AlexaFluor 488, 568, or 647 conjugated secondary antibodies (Life Technologies) at 1:1000 and Hoecsht or Dapi nuclear stain in blocking buffer for 1 hour, rinsed 3×10’ with PBST and mounted using Fluoromount-G (Southern Biotech). All steps performed at room temperature unless otherwise noted. *Note: For gamma- tubulin antibody staining, antigen retrieval by incubating with 1%SDS/PBST for 5 mins prior to blocking and primary antibody incubation in required for good staining. Stained samples were imaged on a Leica SP-5 confocal microscope. Images were processed using FIJI (ImageJ).
In Situ Hybridization
Whole mount in situ hybridization was performed as previously described (Harrelson, Kaestner, & Evans, 2012). DIG-labeled riboprobes were made using plasmids from the following sources: Shh (Echelard et al., 1993), Gsc (Blum et al., 1992), Ptch1 (Goodrich, 1999), Foxa2 (Brennan et al., 2001), Gli1 (EST W65013).
RT-qPCR
For gene expression studies, RNA was extracted from E8.5/E9.5 embryo heads using an RNAeasy Micro Kit (QIAGEN), and cDNA synthesis was performed using the iScript cDNA synthesis kit (BioRad). RT-qPCR was performed using EXPRESS Sybr GreenER 2X master mix with ROX (Invitrogen) and primers homologous to Shh, Ptch1, Gli1, or Six3 on an ABI 7900HT real-time PCR machine. Expression levels were normalized to the geometric mean of three control genes (Actb, Hprt, and Ubc), average normalized Ct values for control and experimental groups determined and relative expression levels determined by Δ ΔCt. The RT-qPCR of each RNA sample was performed in quadruplicate and each experiment was replicated.
Embryo processing for midface imaging
Embryos were harvested in ice-cold PBS, staged by counting somite number, and fixed o/n at 4 degrees in 4%PFA/PBS. Embryo heads were removed and stained in 0.01% ethidium bromide in PBS at room temperature for 15 minutes. Embryos were positioned using glass beads in PBS and imaged on a Leica MZ16 F fluorescence stereomicroscope.
Image Quantification
For 2D midface width quantification the infranasal distance was measure using FIJI software by drawing a line between the center of each nasal pit. For quantification of cell death and proliferation, 6 sections per embryo were quantified. Staining with epithelial marker E-cadherin was used for quantification of facial ectoderm while nuclear morphology was used to separate mesenchyme, hindbrain and forebrain tissue compartments. For quantification, threshold was first set for each image followed by binary watershed separation to obtain accurate nuclei counts. The percentage of TUNEL+ or pHH3+ nuclei were compared between Tctn2 mutant and control samples.