Ching-Fang Chang

The cellular and molecular etiology of the craniofacial defects in the avian ciliopathic mutant talpid2

talpid2 is an avian autosomal recessive mutant with a myriad of congenital malformations, including polydactyly and facial clefting. Although phenotypically similar to talpid3, talpid2 has a distinct facial phenotype and an unknown cellular, molecular and genetic basis. We set out to determine the etiology of the craniofacial phenotype of this mutant. We confirmed that primary cilia were disrupted in talpid2 mutants. Molecularly, we found disruptions in Hedgehog signaling. Post-translational processing of GLI2 and GLI3 was aberrant in the developing facial prominences. Although both GLI2 and GLI3 processing were disrupted in talpid2 mutants, only GLI3 activator levels were significantly altered in the nucleus. Through additional fine mapping and whole-genome sequencing, we determined that the talpid2 phenotype was linked to a 1.4 Mb region on GGA1q that contained the gene encoding the ciliary protein C2CD3. We cloned the avian ortholog of C2CD3 and found its expression was ubiquitous, but most robust in the developing limbs and facial prominences. Furthermore, we found that C2CD3 is localized proximal to the ciliary axoneme and is important for docking the mother centriole to the ciliary vesicle and cell membrane. Finally, we identified a 19 bp deletion in talpid2 C2CD3 that produces a premature stop codon, and thus a truncated protein, as the likely causal allele for the phenotype. Together, these data provide insight into the cellular, molecular and genetic etiology of the talpid2 phenotype. Our data suggest that, although the talpid2 and talpid3 mutations affect a common ciliogenesis pathway, they are caused by mutations in different ciliary proteins that result in differences in craniofacial phenotype.

Craniofacial Ciliopathies Reveal Specific Requirements for GLI Proteins during Development of the Facial Midline

Ciliopathies represent a broad class of disorders that affect multiple organ systems. The craniofacial complex is among those most severely affected when primary cilia are not functional. We previously reported that loss of primary cilia on cranial neural crest cells, via a conditional knockout of the intraflagellar transport protein KIF3a, resulted in midfacial widening due to a gain of Hedgehog (HH) activity. Here, we examine the molecular mechanism of how a loss of primary cilia can produce facial phenotypes associated with a gain of HH function. We show that loss of intraflagellar transport proteins (KIF3a or IFT88) caused aberrant GLI processing such that the amount of GLI3FL and GLI2FL was increased, thus skewing the ratio of GLIFL to GLIR in favor of the FL isoform. Genetic addition of GLI3R partially rescued the ciliopathic midfacial widening. Interestingly, despite several previous studies suggesting midfacial development relies heavily on GLI3R activity, the conditional loss of GLI3 alone did not reproduce the ciliopathic phenotype. Only the combined loss of both GLI2 and GLI3 was able to phenocopy the ciliopathic midfacial appearance. Our findings suggest that ciliopathic facial phenotypes are generated via loss of both GLI3R and GLI2R and that this pathology occurs via a de-repression mechanism. Furthermore, these studies suggest a novel role for GLI2R in craniofacial development.

Odd-skipped related-1 controls neural crest chondrogenesis during tongue development

Significance Development of the tongue is a major vertebrate adaptation to terrestrial life. Interestingly, although the tongues of birds and mammals initially develop similarly, the bird tongue is underpinned by an extensive internal skeleton, whereas the oral part of the mammalian tongue is boneless, which is critical not only for feeding but also for phonetic articulation and oral health. This paper shows that the mouse embryonic tongue, but not that in chick, expresses high levels of the odd-skipped related-1 (Osr1) transcription factor, which represses expression of SRY-box containing gene-9 (Sox9), the master regulator of cartilage formation. Mice lacking Osr1 function develop a cartilage in the anterior tongue reminiscent of that in chick. These data provide unique insights into the mechanisms of tongue development and evolution. The tongue is a critical element of the feeding system in tetrapod animals for their successful adaptation to terrestrial life. Whereas the oral part of the mammalian tongue contains soft tissues only, the avian tongue has an internal skeleton extending to the anterior tip. The mechanisms underlying the evolutionary divergence in tongue skeleton formation are completely unknown. We show here that the odd-skipped related-1 (Osr1) transcription factor is expressed throughout the neural crest-derived tongue mesenchyme in mouse, but not in chick, embryos during early tongue morphogenesis. Neural crest-specific inactivation of Osr1 resulted in formation of an ectopic cartilage in the mouse tongue, reminiscent in shape and developmental ontogeny of the anterior tongue cartilage in chick. SRY-box containing gene-9 (Sox9), the master regulator of chondrogenesis, is widely expressed in the nascent tongue mesenchyme at the onset of tongue morphogenesis but its expression is dramatically down-regulated concomitant with activation of Osr1 expression in the developing mouse tongue. In Osr1 mutant mouse embryos, expression of Sox9 persisted in the developing tongue mesenchyme where chondrogenesis is subsequently activated to form the ectopic cartilage. Furthermore, we show that Osr1 binds to the Sox9 gene promoter and that overexpression of Osr1 suppressed expression of endogenous Sox9 mRNAs and Sox9 promoter-driven reporter. These data indicate that Osr1 normally prevents chondrogenesis in the mammalian tongue through repression of Sox9 expression and suggest that changes in regulation of Osr1 expression in the neural crest-derived tongue mesenchyme underlie the evolutionary divergence of birds from other vertebrates in tongue morphogenesis.

The ciliary baton: orchestrating neural crest cell development.

Primary cilia are cell surface, microtubule-based organelles that dynamically extend from cells to receive and process molecular and mechanical signaling cues. In the last decade, this organelle has gained increasing popularity due to its ability to act as a cellular antenna, receive molecular stimuli, and respond to the cells environment. A growing field of data suggests that various tissues utilize and interpret the loss of cilia in different ways. Thus, careful examination of the role of cilia on individual cell types and tissues is necessary. Neural crest cells (NCCs) are an excellent example of cells that survey their environment for developmental cues. In this review, we discuss how NCCs utilize primary cilia during their ontogenic development, paying special attention to the role primary cilia play in processing developmental signals required for NCC specification, migration, proliferation, and differentiation. We also discuss how the loss of functional cilia on cranial and trunk NCCs affects the development of various organ systems to which they contribute. A deeper understanding of ciliary function could contribute greatly to understanding the molecular mechanisms guiding NCC development and differentiation. Furthermore, superimposing the ciliary contribution on our current understanding of NCC development identifies new avenues for therapeutic intervention in neurocristopathies.

Using the avian mutant talpid2 as a disease model for understanding the oral-facial phenotypes of oral-facial-digital syndrome.

ABSTRACT Oral-facial-digital syndrome (OFD) is a ciliopathy that is characterized by oral-facial abnormalities, including cleft lip and/or palate, broad nasal root, dental anomalies, micrognathia and glossal defects. In addition, these individuals have several other characteristic abnormalities that are typical of a ciliopathy, including polysyndactyly, polycystic kidneys and hypoplasia of the cerebellum. Recently, a subset of OFD cases in humans has been linked to mutations in the centriolar protein C2 Ca2+-dependent domain-containing 3 (C2CD3). Our previous work identified mutations in C2CD3 as the causal genetic lesion for the avian talpid2 mutant. Based on this common genetic etiology, we re-examined the talpid2 mutant biochemically and phenotypically for characteristics of OFD. We found that, as in OFD-affected individuals, protein-protein interactions between C2CD3 and oral-facial-digital syndrome 1 protein (OFD1) are reduced in talpid2 cells. Furthermore, we found that all common phenotypes were conserved between OFD-affected individuals and avian talpid2 mutants. In light of these findings, we utilized the talpid2 model to examine the cellular basis for the oral-facial phenotypes present in OFD. Specifically, we examined the development and differentiation of cranial neural crest cells (CNCCs) when C2CD3-dependent ciliogenesis was impaired. Our studies suggest that although disruptions of C2CD3-dependent ciliogenesis do not affect CNCC specification or proliferation, CNCC migration and differentiation are disrupted. Loss of C2CD3-dependent ciliogenesis affects the dispersion and directional persistence of migratory CNCCs. Furthermore, loss of C2CD3-dependent ciliogenesis results in dysmorphic and enlarged CNCC-derived facial cartilages. Thus, these findings suggest that aberrant CNCC migration and differentiation could contribute to the pathology of oral-facial defects in OFD. Summary: This study utilizes a naturally occurring avian mutant known as talpid2 to determine the cellular basis for the oral-facial phenotypes present in oral-facial-digital syndrome.

A mutation in FRIZZLED2 impairs Wnt signaling and causes autosomal dominant omodysplasia

Autosomal dominant omodysplasia is a rare skeletal dysplasia characterized by short humeri, radial head dislocation, short first metacarpals, facial dysmorphism and genitourinary anomalies. We performed next-generation whole-exome sequencing and comparative analysis of a proband with omodysplasia, her unaffected parents and her affected daughter. We identified a de novo mutation in FRIZZLED2 (FZD2) in the proband and her daughter that was not found in unaffected family members. The FZD2 mutation (c.1644G>A) changes a tryptophan residue at amino acid 548 to a premature stop (p.Trp548*). This altered protein is still produced in vitro, but we show reduced ability of this mutant form of FZD2 to interact with its downstream target DISHEVELLED. Furthermore, expressing the mutant form of FZD2 in vitro is not able to facilitate the cellular response to canonical Wnt signaling like wild-type FZD2. We therefore conclude that the FRIZZLED2 mutation is a de novo, novel cause for autosomal dominant omodysplasia.

The Ciliary Baton

Primary cilia are cell surface, microtubule-based organelles that dynamically extend from cells to receive and process molecular and mechanical signaling cues. In the last decade, this organelle has gained increasing popularity due to its ability to act as a cellular antenna, receive molecular stimuli, and respond to the cells environment. A growing field of data suggests that various tissues utilize and interpret the loss of cilia in different ways. Thus, careful examination of the role of cilia on individual cell types and tissues is necessary. Neural crest cells (NCCs) are an excellent example of cells that survey their environment for developmental cues. In this review, we discuss how NCCs utilize primary cilia during their ontogenic development, paying special attention to the role primary cilia play in processing developmental signals required for NCC specification, migration, proliferation, and differentiation. We also discuss how the loss of functional cilia on cranial and trunk NCCs affects the development of various organ systems to which they contribute. A deeper understanding of ciliary function could contribute greatly to understanding the molecular mechanisms guiding NCC development and differentiation. Furthermore, superimposing the ciliary contribution on our current understanding of NCC development identifies new avenues for therapeutic intervention in neurocristopathies.

Utilizing the chicken as an animal model for human craniofacial ciliopathies

The chicken has been a particularly useful model for the study of craniofacial development and disease for over a century due to their relatively large size, accessibility, and amenability for classical bead implantation and transplant experiments. Several naturally occurring mutant lines with craniofacial anomalies also exist and have been heavily utilized by developmental biologist for several decades. Two of the most well known lines, talpid(2) (ta(2)) and talpid(3) (ta(3)), represent the first spontaneous mutants to have the causative genes identified. Despite having distinct genetic causes, both mutants have recently been identified as ciliopathic. Excitingly, both of these mutants have been classified as models for human craniofacial ciliopathies: Oral-facial-digital syndrome (ta(2)) and Joubert syndrome (ta(3)). Herein, we review and compare these two models of craniofacial disease and highlight what they have revealed about the molecular and cellular etiology of ciliopathies. Furthermore, we outline how applying classical avian experiments and new technological advances (transgenics and genome editing) with naturally occurring avian mutants can add a tremendous amount to what we currently know about craniofacial ciliopathies.

Unique spatiotemporal requirements for intraflagellar transport genes during forebrain development.

Primary cilia are organelles extended from virtually all cells and are required for the proper regulation of a number of canonical developmental pathways. The role in cortical development of proteins important for ciliary form and function is a relatively understudied area. Here we have taken a genetic approach to define the role in forebrain development of three intraflagellar transport proteins known to be important for primary cilia function. We have genetically ablated Kif3a, Ift88, and Ttc21b in a series of specific spatiotemporal domains. The resulting phenotypes allow us to draw several conclusions. First, we conclude that the Ttc21b cortical phenotype is not due to the activity of Ttc21b within the brain itself. Secondly, some of the most striking phenotypes are from ablations in the neural crest cells and the adjacent surface ectoderm indicating that cilia transduce critical tissue—tissue interactions in the developing embryonic head. Finally, we note striking differences in phenotypes from ablations only one embryonic day apart, indicating very discrete spatiotemporal requirements for these three genes in cortical development.

A tissue-specific role for intraflagellar transport genes during craniofacial development

Primary cilia are nearly ubiquitous, cellular projections that function to transduce molecular signals during development. Loss of functional primary cilia has a particularly profound effect on the developing craniofacial complex, causing several anomalies including craniosynostosis, micrognathia, midfacial dysplasia, cleft lip/palate and oral/dental defects. Development of the craniofacial complex is an intricate process that requires interactions between several different tissues including neural crest cells, neuroectoderm and surface ectoderm. To understand the tissue-specific requirements for primary cilia during craniofacial development we conditionally deleted three separate intraflagellar transport genes, Kif3a, Ift88 and Ttc21b with three distinct drivers, Wnt1-Cre, Crect and AP2-Cre which drive recombination in neural crest, surface ectoderm alone, and neural crest, surface ectoderm and neuroectoderm, respectively. We found that tissue-specific conditional loss of ciliary genes with different functions produces profoundly different facial phenotypes. Furthermore, analysis of basic cellular behaviors in these mutants suggests that loss of primary cilia in a distinct tissue has unique effects on development of adjacent tissues. Together, these data suggest specific spatiotemporal roles for intraflagellar transport genes and the primary cilium during craniofacial development.