Feather arrays are patterned by interacting signalling and cell density waves.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation.

Experimental evidence that preaxial polydactyly and forearm radial deficiencies may share a common developmental origin.

Preaxial polydactyly is a congenital hand anomaly predominantly of sporadic occurrence, which is frequently associated with abnormalities of the Sonic hedgehog signalling pathway. In experimentally induced preaxial polydactyly, radial aplasia is also frequently observed. To determine if there is a correlation between preaxial polydactyly and radial aplasia, we induced ectopic Sonic hedgehog signalling during chicken limb development with application of a smoothened-agonist (SAG) or retinoic acid. Application of SAG caused malformations in 71% limbs including preaxial polydactyly (62%) and forearm abnormalities (43%). Retinoic acid application induced malformations in 56% of limb including preaxial polydactyly (45%) and forearm abnormalities (50%). Radial dysplasia and ulnar dimelia were observed in both experimental conditions. We demonstrate that ectopic Sonic hedgehog signalling may cause both preaxial polydactyly and predictable forearm anomalies and that these conditions could potentially be classified as one embryological group. We propose a unifying model based on known models of ectopic Sonic hedgehog signalling.

Canine Brachycephaly Is Associated with a Retrotransposon-Mediated Missplicing of SMOC2.

In morphological terms, "form" is used to describe an object's shape and size. In dogs, facial form is stunningly diverse. Facial retrusion, the proximodistal shortening of the snout and widening of the hard palate is common to brachycephalic dogs and is a welfare concern, as the incidence of respiratory distress and ocular trauma observed in this class of dogs is highly correlated with their skull form. Progress to identify the molecular underpinnings of facial retrusion is limited to association of a missense mutation in BMP3 among small brachycephalic dogs. Here, we used morphometrics of skull isosurfaces derived from 374 pedigree and mixed-breed dogs to dissect the genetics of skull form. Through deconvolution of facial forms, we identified quantitative trait loci that are responsible for canine facial shapes and sizes. Our novel insights include recognition that the FGF4 retrogene insertion, previously associated with appendicular chondrodysplasia, also reduces neurocranium size. Focusing on facial shape, we resolved a quantitative trait locus on canine chromosome 1 to a 188-kb critical interval that encompasses SMOC2. An intronic, transposable element within SMOC2 promotes the utilization of cryptic splice sites, causing its incorporation into transcripts, and drastically reduces SMOC2 gene expression in brachycephalic dogs. SMOC2 disruption affects the facial skeleton in a dose-dependent manner. The size effects of the associated SMOC2 haplotype are profound, accounting for 36% of facial length variation in the dogs we tested. Our data bring new focus to SMOC2 by highlighting its clinical implications in both human and veterinary medicine.

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.

TALPID3 controls centrosome and cell polarity and the human ortholog KIAA0586 is mutated in Joubert syndrome (JBTS23).

Joubert syndrome (JBTS) is a severe recessive neurodevelopmental ciliopathy which can affect several organ systems. Mutations in known JBTS genes account for approximately half of the cases. By homozygosity mapping and whole-exome sequencing, we identified a novel locus, JBTS23, with a homozygous splice site mutation in KIAA0586 (alias TALPID3), a known lethal ciliopathy locus in model organisms. Truncating KIAA0586 mutations were identified in two additional patients with JBTS. One mutation, c.428delG (p.Arg143Lysfs*4), is unexpectedly common in the general population and may be a major contributor to JBTS. We demonstrate KIAA0586 protein localization at the basal body in human and mouse photoreceptors, as is common for JBTS proteins, and also in pericentriolar locations. We show that loss of TALPID3 (KIAA0586) function in animal models causes abnormal tissue polarity, centrosome length and orientation, and centriolar satellites. We propose that JBTS and other ciliopathies may in part result from cell polarity defects.

Loss of cilia causes embryonic lung hypoplasia, liver fibrosis, and cholestasis in the talpid3 ciliopathy mutant.

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid(3) chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid(3) phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid(3) chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid(3) chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development.

The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.