Direct functional consequences of ZRS enhancer mutation combine with secondary long range SHH signalling effects to cause preaxial polydactyly.

Sonic hedgehog (SHH) plays a central role in patterning numerous embryonic tissues including, classically, the developing limb bud where it controls digit number and identity. This study utilises the polydactylous Silkie (Slk) chicken breed, which carries a mutation in the long range limb-specific regulatory element of SHH, the ZRS. Using allele specific SHH expression analysis combined with quantitative protein analysis, we measure allele specific changes in SHH mRNA and concentration of SHH protein over time. This confirms that the Slk ZRS enhancer mutation causes increased SHH expression in the posterior leg mesenchyme. Secondary consequences of this increased SHH signalling include increased FGF pathway signalling and growth as predicted by the SHH/GREM1/FGF feedback loop and the Growth/Morphogen models. Manipulation of Hedgehog, FGF signalling and growth demonstrate that anterior-ectopic expression of SHH and induction of preaxial polydactyly is induced secondary to increased SHH signalling and Hedgehog-dependent growth directed from the posterior limb. We predict that increased long range SHH signalling acts in combination with changes in activation of SHH transcription from the Slk ZRS allele. Through analysis of the temporal dynamics of anterior SHH induction we predict a gene regulatory network which may contribute to activation of anterior SHH expression from the Slk ZRS.

The chicken left right organizer has nonmotile cilia which are lost in a stage-dependent manner in the talpid(3) ciliopathy.

Motile cilia are an essential component of the mouse, zebrafish, and Xenopus laevis Left Right Organizers, generating nodal flow and allowing the reception and transduction of mechanosensory signals. Nonmotile primary cilia are also an important component of the Left Right Organizer's chemosensory mechanism. It has been proposed in the chicken that signaling in Hensen's node, the Left Right Organizer of the chicken, is independent of cilia, based on a lack of evidence of motile cilia or nodal flow. It is speculated that the talpid(3) chicken mutant, which has normal left-right patterning despite lacking cilia at many stages of development, is proof of this hypothesis. Here, we examine the evidence for cilia in Hensen's node and find that although cilia are present; they are likely to be immotile and incapable of generating nodal flow. Furthermore, we find that early planar cell polarity patterning and ciliogenesis is normal in early talpid(3) chicken embryos. We conclude that patterning and development of the early talpid(3) chicken is normal, but not necessarily independent of cilia. Although it appears that Hensen's node does not require motile cilia or the generation of motile flow, there may remain a requirement for cilia in the transduction of SHH signaling.

The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development.

Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly.

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.

Cell Cycle Heterogeneity Can Generate Robust Cell Type Proportioning.

Cell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.

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.