Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

Homozygous variant in C21orf2 in a case of Jeune syndrome with severe thoracic involvement: Extending the phenotypic spectrum.

We previously reported exome sequencing in a short-rib thoracic dystrophy (SRTD) cohort, in whom recessive mutations were identified in SRTD-associated genes in 10 of 11 cases. A heterozygous stop mutation in the known SRTD gene WDR60 was identified in the remaining case; no novel candidate gene/s were suggested by homozygous/compound heterozygous analysis. This case was thus considered unsolved. Re-analysis following an analysis pipeline update identified a homozygous mutation in C21orf2 (c.218G > C; p.Arg73Pro). This homozygous variant was previously removed at the quality control stage by the default GATK parameter "in-breeding co-efficient." C21orf2 was recently associated with both Jeune asphyxiating thoracic dystrophy (JATD) and axial spondylometaphyseal dysplasia (axial SMD); this particular mutation was reported in homozygous and compound heterozygous state in both conditions. Our case has phenotypic features of both JATD and axial SMD; and the extent of thoracic involvement appears more severe than in other C21orf2-positive cases. Identification of a homozygous C21orf2 mutation in this case emphasizes the value of exome sequencing for simultaneously screening known genes and identifying novel genes. Additionally, it highlights the importance of re-interrogating data both as novel gene associations are identified and as analysis pipelines are refined. Finally, the severity of thoracic restriction in this case adds to the phenotypic spectrum attributable to C21orf2 mutations.

Fryns Syndrome Associated with Recessive Mutations in PIGN in two Separate Families.

Fryns syndrome is an autosomal recessive condition characterized by congenital diaphragmatic hernia (CDH), dysmorphic facial features, distal digital hypoplasia, and other associated malformations, and is the most common syndromic form of CDH. No gene has been associated with this condition. Whole-exome sequence data from two siblings and three unrelated individuals with Fryns syndrome were filtered for rare, good quality, coding mutations fitting a recessive inheritance model. Compound heterozygous mutations in PIGN were identified in the siblings, with appropriate parental segregation: a novel STOP mutation (c.1966C>T: p.Glu656X) and a rare (minor allele frequency <0.001) donor splice site mutation (c.1674+1G>C) causing skipping of exon 18 and utilization of a cryptic acceptor site in exon 19. A further novel homozygous STOP mutation in PIGN (c.694A>T: p.Lys232X) was detected in one unrelated case. All three variants affected highly conserved bases. The two remaining cases were negative for PIGN mutations. Mutations in PIGN have been reported in cases with multiple congenital anomalies, including one case with syndromic CDH. Fryns syndrome can be caused by recessive mutations in PIGN. Whether PIGN affects other syndromic and non-syndromic forms of CDH warrants investigation.

Short-rib polydactyly and Jeune syndromes are caused by mutations in WDR60.

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.

Mutations in the gene encoding IFT dynein complex component WDR34 cause Jeune asphyxiating thoracic dystrophy.

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1(-/-) embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1(-/-) fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR-induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice.

The issues of whether and how some organs coordinate their size and shape with the blueprint of the embryo axis, while others appear to regulate their morphogenesis autonomously, remain poorly understood. Mutations in Ift144, encoding a component of the trafficking machinery of primary cilia assembly, result in a range of embryo patterning defects, affecting the limbs, skeleton and neural system. Here, we show that embryos of the mouse mutant Ift144(twt) develop gonads that are larger than wild-type. Investigation of the early patterning of the urogenital ridge revealed that the anterior-posterior domain of the gonad/mesonephros was extended at 10.5 dpc, with no change in the length of the metanephros. In XY embryos, this extension resulted in an increase in testis cord number. Moreover, we observed a concomitant extension of the trunk axis in both sexes, with no change in the length of the tail domain or somite number. Our findings support a model in which: (1) primary cilia regulate embryonic trunk elongation; (2) the length of the trunk axis determines the size of the urogenital ridges; and (3) the gonad domain is partitioned into a number of testis cords that depends on the available space, rather than being divided a predetermined number of times to generate a specific number of cords.

Patched1 is required in neural crest cells for the prevention of orofacial clefts.

Defects such as cleft lip with or without cleft palate (CL/P) are among the most common craniofacial birth defects in humans. In many cases, the underlying molecular and cellular mechanisms that result in these debilitating anomalies remain largely unknown. Perturbed hedgehog (HH) signalling plays a major role in craniofacial development, and mutations in a number of pathway constituents underlie craniofacial disease. In particular, mutations in the gene encoding the major HH receptor and negative regulator, patched1 (PTCH1), are associated with both sporadic and familial forms of clefting, yet relatively little is known about how PTCH1 functions during craniofacial morphogenesis. To address this, we analysed the consequences of conditional loss of Ptch1 in mouse neural crest cell-derived facial mesenchyme. Using scanning electron microscopy (SEM) and live imaging of explanted facial primordia, we captured defective nasal pit invagination and CL in mouse embryos conditionally lacking Ptch1. Our analysis demonstrates interactions between HH and FGF signalling in the development of the upper lip, and reveals cell-autonomous and non-autonomous roles mediated by Ptch1. In particular, we show that deletion of Ptch1 in the facial mesenchyme alters cell morphology, specifically in the invaginating nasal pit epithelium. These findings highlight a critical link between the neural crest cells and olfactory epithelium in directing the morphogenesis of the mammalian lip and nose primordia. Importantly, these interactions are critically dependent on Ptch1 function for the prevention of orofacial clefts.

A genome-wide association study identifies five loci influencing facial morphology in Europeans.

Inter-individual variation in facial shape is one of the most noticeable phenotypes in humans, and it is clearly under genetic regulation; however, almost nothing is known about the genetic basis of normal human facial morphology. We therefore conducted a genome-wide association study for facial shape phenotypes in multiple discovery and replication cohorts, considering almost ten thousand individuals of European descent from several countries. Phenotyping of facial shape features was based on landmark data obtained from three-dimensional head magnetic resonance images (MRIs) and two-dimensional portrait images. We identified five independent genetic loci associated with different facial phenotypes, suggesting the involvement of five candidate genes--PRDM16, PAX3, TP63, C5orf50, and COL17A1--in the determination of the human face. Three of them have been implicated previously in vertebrate craniofacial development and disease, and the remaining two genes potentially represent novel players in the molecular networks governing facial development. Our finding at PAX3 influencing the position of the nasion replicates a recent GWAS of facial features. In addition to the reported GWA findings, we established links between common DNA variants previously associated with NSCL/P at 2p21, 8q24, 13q31, and 17q22 and normal facial-shape variations based on a candidate gene approach. Overall our study implies that DNA variants in genes essential for craniofacial development contribute with relatively small effect size to the spectrum of normal variation in human facial morphology. This observation has important consequences for future studies aiming to identify more genes involved in the human facial morphology, as well as for potential applications of DNA prediction of facial shape such as in future forensic applications.

Mutations in mouse Ift144 model the craniofacial, limb and rib defects in skeletal ciliopathies.

Mutations in components of the intraflagellar transport (IFT) machinery required for assembly and function of the primary cilium cause a subset of human ciliopathies characterized primarily by skeletal dysplasia. Recently, mutations in the IFT-A gene IFT144 have been described in patients with Sensenbrenner and Jeune syndromes, which are associated with short ribs and limbs, polydactyly and craniofacial defects. Here, we describe an N-ethyl-N-nitrosourea-derived mouse mutant with a hypomorphic missense mutation in the Ift144 gene. The mutant twinkle-toes (Ift144(twt)) phenocopies a number of the skeletal and craniofacial anomalies seen in patients with human skeletal ciliopathies. Like other IFT-A mouse mutants, Ift144 mutant embryos display a generalized ligand-independent expansion of hedgehog (Hh) signalling, in spite of defective ciliogenesis and an attenuation of the ability of mutant cells to respond to upstream stimulation of the pathway. This enhanced Hh signalling is consistent with cleft palate and polydactyly phenotypes in the Ift144(twt) mutant, although extensive rib branching, fusion and truncation phenotypes correlate with defects in early somite patterning and may reflect contributions from multiple signalling pathways. Analysis of embryos harbouring a second allele of Ift144 which represents a functional null, revealed a dose-dependent effect on limb outgrowth consistent with the short-limb phenotypes characteristic of these ciliopathies. This allelic series of mouse mutants provides a unique opportunity to uncover the underlying mechanistic basis of this intriguing subset of ciliopathies.

Transmembrane protein 2 (Tmem2) is required to regionally restrict atrioventricular canal boundary and endocardial cushion development.

The atrioventricular canal (AVC) physically separates the atrial and ventricular chambers of the heart and plays a crucial role in the development of the valves and septa. Defects in AVC development result in aberrant heart morphogenesis and are a significant cause of congenital heart malformations. We have used a forward genetic screen in zebrafish to identify novel regulators of cardiac morphogenesis. We isolated a mutant, named wickham (wkm), that was indistinguishable from siblings at the linear heart tube stage but exhibited a specific loss of cardiac looping at later developmental stages. Positional cloning revealed that the wkm locus encodes transmembrane protein 2 (Tmem2), a single-pass transmembrane protein of previously unknown function. Expression analysis demonstrated myocardial and endocardial expression of tmem2 in zebrafish and conserved expression in the endocardium of mouse embryos. Detailed phenotypic analysis of the wkm mutant identified an expansion of expression of known myocardial and endocardial AVC markers, including bmp4 and has2. By contrast, a reduction in the expression of spp1, a marker of the maturing valvular primordia, was observed, suggesting that an expansion of immature AVC is detrimental to later valve maturation. Finally, we show that immature AVC expansion in wkm mutants is rescued by depleting Bmp4, indicating that Tmem2 restricts bmp4 expression to delimit the AVC primordium during cardiac development.

Twist2 contributes to termination of limb bud outgrowth and patterning through direct regulation of Grem1.

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues including the limb. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of organs. Expression of Twist2 within the developing limbs begins after formation of the limb bud and persists within the peripheral mesenchyme until digital rays condense. We have used RCAS-mediated overexpression in chick to investigate the function of Twist2 in limb development. Viral misexpression following injection into the lateral plate mesoderm results in a spectrum of hypoplastic limb phenotypes. These include generalized shortening of the entire limb, fusion of the autopod skeletal elements, loss of individual digits or distal truncation resulting in complete loss of the autopod. These phenotypes appear to result from a premature termination of limb outgrowth and manifest as defective growth in both the proximal-distal and anterior-posterior axes. In situ hybridisation analysis demonstrates that many components of the Shh/Grem1/Fgf regulatory loop that controls early limb growth and patterning are downregulated by Twist2 overexpression. Grem1 has a complementary expression pattern to Twist2 within the limb primordia and co-expression of both Grem1 and Twist2 results in a rescue of the Twist2 overexpression phenotype. We demonstrate that Twist proteins directly repress Grem1 expression via a regulatory element downstream of the open reading frame. These data indicate that Twist2 regulates early limb morphogenesis through a role in terminating the Shh/Grem1/Fgf autoregulatory loop.

Patched 1 is a crucial determinant of asymmetry and digit number in the vertebrate limb.

The vertebrate hedgehog receptor patched 1 (Ptc1) is crucial for negative regulation of the sonic hedgehog (Shh) pathway during anterior-posterior patterning of the limb. We have conditionally inactivated Ptc1 in the mesenchyme of the mouse limb using Prx1-Cre. This results in constitutive activation of hedgehog (Hh) signalling during the early stages of limb budding. Our data suggest that variations in the timing and efficiency of Cre-mediated excision result in differential forelimb and hindlimb phenotypes. Hindlimbs display polydactyly (gain of digits) and a molecular profile similar to the Gli3 mutant extra-toes. Strikingly, forelimbs are predominantly oligodactylous (displaying a loss of digits), with a symmetrical, mirror-image molecular profile that is consistent with re-specification of the anterior forelimb to a posterior identity. Our data suggest that this is related to very early inactivation of Ptc1 in the forelimb perturbing the gene regulatory networks responsible for both the pre-patterning and the subsequent patterning stages of limb development. These results establish the importance of the downstream consequences of Hh pathway repression, and identify Ptc1 as a key player in limb patterning even prior to the onset of Shh expression.

Inactivation of Patched1 in the mouse limb has novel inhibitory effects on the chondrogenic program.

The bones of the vertebrate limb form by the process of endochondral ossification, whereby limb mesenchyme condenses to form an intermediate cartilage scaffold that is then replaced by bone. Although Indian hedgehog (IHH) is known to control hypertophic differentiation of chondrocytes during this process, the role of hedgehog signaling in the earlier stages of chondrogenesis is less clear. We have conditionally inactivated the hedgehog receptor Ptc1 in undifferentiated limb mesenchyme of the mouse limb using Prx1-Cre, thus inducing constitutively active ligand-independent hedgehog signaling. In addition to major patterning defects, we observed a marked disruption to the cartilage elements in the limbs of Prx1-Cre:Ptc1(c/c) embryos. Using an in vitro micromass culture system we show that this defect lies downstream of mesenchymal cell condensation and likely upstream of chondrocyte differentiation. Despite early increases in levels of chondrogenic genes, soon after mesenchymal condensation the stromal layer of Prx1-Cre:Ptc1(c/c)-derived micromass cultures is characterized by a loss of cell integrity, which is associated with increased cell death and a striking decrease in Alcian blue staining cartilage nodules. Furthermore, inhibition of the hedgehog pathway activation using cyclopamine was sufficient to essentially overcome this chondrogenic defect in both micromass and ex vivo explant assays of Prx1-Cre:Ptc1(c/c) limbs. These data demonstrate for the first time the inhibitory effect of cell autonomously activated hedgehog signaling on chondrogenesis, and stress the importance of PTC1 in maintaining strict control of signaling levels during this phase of skeletal development.

Inheritance of a novel mutated allele of the OCA2 gene associated with high incidence of oculocutaneous albinism in a Polynesian community.

Oculocutaneous albinism type 2 (OCA2) is a human autosomal-recessive hypopigmentation disorder associated with pathological mutations of the OCA2 gene. In this study, we investigated a form of OCA in a Polynesian population with an observed phenotype characterized by fair skin, some brown nevi present in the sun-exposed areas and green or blue eyes. Hair presented with a unique red coloration since birth, with tones ranging across individuals from Yellow-Red to Brown-Red, or Auburn. We genetically screened for mutations in the OCA2 and MC1R genes as their products have previously been shown to be associated with red hair/fair skin and OCA2. The SLC45A2 gene was also screened to identify any possible relation to skin color variation. We have identified a novel missense substitution in the OCA2 gene (Gly775Asp) responsible for OCA2 in individuals of Polynesian heritage from Tuvalu. The estimated incidence of this form of OCA2 in the primary study community is believed to occur at one of the highest recorded rates of albinism at approximately 1 per 669 individuals. In addition, we have analyzed four unrelated individuals with albinism who have Polynesian heritage from three other separate communities and found they carry the same OCA2 mutation. We also analyzed an out-group comprising three unrelated individuals with albinism of Melanesian ancestries from two separate communities, one Australian Aboriginal and three Australian Caucasians, and did not detect this mutation. We hypothesize that this mutation may be Polynesian specific and that it originated from a common founder.

The metalloendopeptidase gene Pitrm1 is regulated by hedgehog signaling in the developing mouse limb and is expressed in muscle progenitors.

Pitrm1 is a zinc metalloendopeptidase that has been implicated in Alzheimer's disease and mitochondrial peptide degradation, but to date no major role in embryonic development has been documented. In a screen for genes regulated by hedgehog signaling in the mouse limb, we showed that expression of Pitrm1 is upregulated in response to loss of the Gli3 transcription factor. Here we confirm spatial changes in Pitrm1 expression in the Gli3 mutant mouse limb and examine Pitrm1 expression in Shh null and Ptch1 conditional deletion mouse mutants. In wild-type mice, Pitrm1 is expressed in a number of developing tissues known to be patterned by Sonic hedgehog, including the limbs, face, cortex, hippocampus, cerebellum, tectum, sub-mandibular gland, lung, genital tubercle, hair follicles, and the enamel knot of the teeth. Additionally, Pitrm1 is expressed in Pax3-expressing myoblast progenitors in the limb, the dermomyotome, and developing muscles of the face and torso.

Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.

Murine embryonic expression of the gene for the UV-responsive protein p15(PAF).

In a screen for genes expressed in the embryonic mouse facial primordia, we identified the gene sequence annotated as KIAA0101, which has previously been shown to encode a novel proliferating cell nuclear antigen (PCNA)-interacting protein named p15(PAF). We have since demonstrated that this protein also interacts in a complex with the tumour suppressor product p33ING1b, and that overexpression results in a decrease in UV-induced cell death. Although available data suggest widespread or ubiquitous expression in the adult, here we report highly restricted expression of the p15(PAF) gene in a spatio-temporal manner during mouse embryogenesis. Major sites of expression include the facial prominences, limbs, somites, brain, spinal cord and hair follicles. Based on the nature of its interacting partners, p15(PAF) is proposed to play a role in tumorigenesis. Our data also suggest a role in embryonic development, consistent with findings that a wide range of tumours result from aberrant activity of key developmental pathways.