A novel ciliopathic skull defect arising from excess neural crest.

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

An essential role for LPA signalling in telencephalon development.

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Hedgehog activity controls opening of the primary mouth.

To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes.

PAR-1 promotes primary neurogenesis and asymmetric cell divisions via control of spindle orientation.

In both invertebrate and vertebrate embryonic central nervous systems, deep cells differentiate while superficial (ventricular) epithelial cells remain in a proliferative, stem cell state. The conserved polarity protein PAR-1, which is basolaterally localised in epithelia, promotes and is required for differentiating deep layer cell types, including ciliated cells and neurons. It has recently been shown that atypical protein kinase C (aPKC), which is apically enriched, inhibits neurogenesis and acts as a nuclear determinant, raising the question of how PAR-1 antagonises aPKC activity to promote neurogenesis. Here we show that PAR-1 stimulates the generation of deep cell progeny from the superficial epithelium of the neural plate and that these deep cells have a corresponding (i.e. deep cell) neuronal phenotype. We further show that gain- and loss-of-function of PAR-1 increase and decrease, respectively, the proportion of epithelial mitotic spindles with a vertical orientation, thereby respectively increasing and decreasing the number of cleavages that generate deep daughter cells. PAR-1 is therefore a crucial regulator of the balance between symmetric (two superficial daughters) and asymmetric (one superficial and one deep daughter) cell divisions. Vertebrate PAR-1 thus antagonises the anti-neurogenic influence of apical aPKC by physically partitioning cells away from it in vivo.

Cilia-mediated Hedgehog signaling controls form and function in the mammalian larynx.

Acoustic communication is fundamental to social interactions among animals, including humans. In fact, deficits in voice impair the quality of life for a large and diverse population of patients. Understanding the molecular genetic mechanisms of development and function in the vocal apparatus is thus an important challenge with relevance both to the basic biology of animal communication and to biomedicine. However, surprisingly little is known about the developmental biology of the mammalian larynx. Here, we used genetic fate mapping to chart the embryological origins of the tissues in the mouse larynx, and we describe the developmental etiology of laryngeal defects in mice with disruptions in cilia-mediated Hedgehog signaling. In addition, we show that mild laryngeal defects correlate with changes in the acoustic structure of vocalizations. Together, these data provide key new insights into the molecular genetics of form and function in the mammalian vocal apparatus.

The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.

Fuz mutant mice reveal shared mechanisms between ciliopathies and FGF-related syndromes.

Ciliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.

Electroporation of craniofacial mesenchyme.

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse (1-10). However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme. When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis (11-13). In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation. Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers (14). Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (˜100 million years closer) (15). Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.