Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

Effect of deletion of Ghrelin-O-Acyltransferase on the pulsatile release of growth hormone in mice

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat−/− mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat−/− mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat−/− mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat−/− mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat−/− mice.

Shrinking Violets

Description of the work Shrinking Violets is comprised of two half scale garments in laser cut silk organza, developed with a knotting device to allow for disassembly and reassembly. The first is a jacket in layered red organza including black storm flap details. The second is a vest in jade organza with circles of pink organza attached through a pattern of knots. Research Background This practice-led fashion design research sits within the field of Design for Sustainability (DfS) in fashion that seeks to mitigate the environmental and ethical impacts of fashion consumption and production. The research explores new systems of garment construction for DfS, and examines how these systems may involve ‘designing’ new user interactions with the garments. The garments’ construction system allows them to be disassembled and recycled or reassembled by users to form a new garment. Conventional garment design follows a set process of cutting and construction, with pattern pieces permanently machine-stitched together. Garments typically contain multiple fibre types; for example a jacket may be constructed from a shell of wool/polyester, an acetate lining, fusible interlinings, and plastic buttons. These complex inputs mean that textile recycling is highly labour intensive, first to separate the garment pieces and second to sort the multiple fibre types. This difficulty results in poor quality ‘shoddy’ comprised of many fibre types and unsuitable for new apparel, or in large quantities of recyclable textile waste sent to landfill (Hawley 2011). Design-led approaches that consider the garment’s end of life in the design process are a way of addressing this problem. In Gulich’s (2006) analysis, use of single materials is the most effective way to ensure ease of recycling, with multiple materials that can be detached next in effectiveness. Given the low rate of technological innovation in most apparel manufacturing (Ruiz 2011), a challenge for effective recycling is how to develop new manufacturing methods that allow for garments to be more easily disassembled at end-of-life. Research Contribution This project addresses the research question: How can design for disassembly be considered within the fashion design process? I have employed a practice-led methodology in which my design process leads the research, making use of methods of fashion design practice including garment and construction research, fabric and colour research, textile experimentation, drape, patternmaking, and illustration as well as more recent methods such as laser cutting. Interrogating the traditional approaches to garment construction is necessarily a technical process; however fashion design is as much about the aesthetic and desirability of a garment as it is about the garment’s pragmatics or utility. This requires a balance between the technical demands of designing for disassembly with the aesthetic demands of fashion. This led to the selection of luxurious, semi-transparent fabrics in bold floral colours that could be layered to create multiple visual effects, as well as the experimentation with laser cutting for new forms of finishing and fastening the fabrics together. Shrinking Violets makes two contributions to new knowledge in the area of design for sustainability within fashion. The first is in the technical development of apparel modularity through the system of laser cut holes and knots that also become a patterning device. The second contribution lies in the design of a system for users to engage with the garment through its ability to be easily reconstructed into a new form. Research Significance Shrinking Violets was exhibited at the State Library of Queensland’s Asia Pacific Design Library, 1-5 November 2015, as part of The International Association of Societies of Design Research’s (IASDR) biannual design conference. The work was chosen for display by a panel of experts, based on the criteria of design innovation and contribution to new knowledge in design. References Gulich, B. (2006). Designing textile products that are easy to recycle. In Y. Wang (Ed.), Recycling in Textiles (pp. 25-37). London: Woodhead. Hawley, J. M. (2011). Textile recycling options: exploring what could be. In A. Gwilt & T. Rissanen (Eds.), Shaping Sustainable Fashion: Changing the way we make and use clothes (pp. 143 - 155). London: Earthscan. Ruiz, B. (2014). Global Apparel Manufacturing. Retrieved 10 August 2014, from http://clients1.ibisworld.com/reports/gl/industry/default.aspx?entid=470

Garden of Shrinking Violets

Description of the work Garden of Shrinking Violets is a collection of six half scale garments and three illustrations, continuing the practice-led research project into design for disassembly, developed in the work Shrinking Violets (2015). All garments are constructed in laser cut modules that enable the items to be reassembled in new combinations. The project extended the materials used to include ahimsa (peace) silk, silk organza and silk twill. The pattern pieces have internal laser cut grids of 5mm circles, allowing the textiles to be layered, threaded and knotted to achieve rich embellished surfaces that play with the transparencies and colour overlays of the sheer and opaque silks. Research Background Conceptually grounded in design for sustainability, the aim of the work is to develop approaches to garment construction that could allow users to engage with the garments by adding, removing and reconfiguring elements. This approach to design considers the use and end-of-life phases of the transient fashion garment through considering how the garments can be later disassembled. Research Contribution This construction process is unique in being not only a patterning device but also integral to the garment’s construction. This work sits at the intersection of technical design and craft: the laser cutting and technical approach to developing new forms of garment construction is coupled with the artisanal approach of hand-knotting, a reference to traditional quilting techniques, as a method to layer and pattern the textiles. The technique developed in Shrinking Violets was extended to experiment with different grid structures, knotting devices, and decorative fringing. The result is a proposed construction system in which the laser cut grid and knotting form a decorative patterning device, but are also integral to the garments’ construction. Research Significance Garden of Shrinking Violets was exhibited at artisan gallery’s Ivory Street window, Brisbane, January 18 – February 28 2016. The work was selected by artisan gallery exhibition curators. As part of artisan gallery’s public programming, the author participated in a panel discussion: ‘Constructive conversations: deconstruction and reconstruction in contemporary craft and design’ with jeweller Elizabeth Shaw and visual arts lecturer Courtney Pedersen, 20 February 2016. Photography used in illustrations by Jonathan Rae

Implantation Induced Hardening of Nanocrystalline Titanium Thin Films

Formation of nanocrystalline TiN at low temperatures was demonstrated by combining Pulsed Laser Deposition (PLD) and ion implantation techniques. The Ti films of nominal thickness similar to 250 nm were deposited at a substrate temperature of 200 degrees C by ablating a high pure titanium target in UHV conditions using a nanosecond pulsed Nd:YAG laser operating at 1064 nm. These films were implanted with 100 keV N+ ions with fluence ranging from 1.0 x 10(16) ions/cm(2) to 1.0 x 10(17) ions/cm(2). The structural, compositional and morphological evolutions were tracked using Transmission Electron Microscopy (TEM), Secondary Ion Mass Spectrometry (SIMS) and Atomic Force Microscopy (AFM), respectively. TEM analysis revealed that the as-deposited titanium film is an fcc phase. With increasing ion fluence, its structure becomes amorphous phase before precipitation of nanocrystalline fcc TiN phase. Compositional depth profiles obtained from SIMS have shown the extent of nitrogen concentration gradient in the implantation zone. Both as-deposited and ion implanted films showed much higher hardness as compared to the bulk titanium. AFM studies revealed a gradual increase in surface roughness leading to surface patterning with increase in ion fluence.

Radiation effects in supported nanoparticles

Nanotechnology applications are entering the market in increasing numbers, nanoparticles being among the main classes of materials used. Particles can be used, e.g., for catalysing chemical reactions, such as is done in car exhaust catalysts today. They can also modify the optical and electronic properties of materials or be used as building blocks for thin film coatings on a variety of surfaces. To develop materials for specific applications, an intricate control of the particle properties, structure, size and shape is required. All these depend on a multitude of factors from methods of synthesis and deposition to post-processing. This thesis addresses the control of nanoparticle structure by low-energy cluster beam deposition and post-synthesis ion irradiation. Cluster deposition in high vacuum offers a method for obtaining precisely controlled cluster-assembled materials with minimal contamination. Due to the clusters small size, however, the cluster-surface interaction may drastically change the cluster properties on deposition. In this thesis, the deposition process of metal and alloy clusters on metallic surfaces is modelled using molecular dynamics simulations, and the mechanisms influencing cluster structure are identified. Two mechanisms, mechanical melting upon deposition and thermally activated dislocation motion, are shown to determine whether a deposited cluster will align epitaxially with its support. The semiconductor industry has used ion irradiation as a tool to modify material properties for decades. Irradiation can be used for doping, patterning surfaces, and inducing chemical ordering in alloys, just to give a few examples. The irradiation response of nanoparticles has, however, remained an almost uncharted territory. Although irradiation effects in nanoparticles embedded inside solid matrices have been studied, almost no work has been done on supported particles. In this thesis, the response of supported nanoparticles is studied systematically for heavy and light ion irradiation. The processes leading to damage production are identified and models are developed for both types of irradiation. In recent experiments, helium irradiation has been shown to induce a phase transformation from multiply twinned to single-crystalline nanoparticles in bimetallic alloys, but the nature of the transition has remained unknown. The alloys for which the effect has been observed are CuAu and FePt. It is shown in this thesis that transient amorphization leads to the observed transition and that while CuAu and FePt do not amorphize upon irradiation in bulk or as thin films, they readily do so as nanoparticles. This is the first time such an effect is demonstrated with supported particles, not embedded in a matrix where mixing is always an issue. An understanding of the above physical processes is essential, if nanoparticles are to be used in applications in an optimal way. This thesis clarifies the mechanisms which control particle morphology, and paves way for the synthesis of nanostructured materials tailored for specific applications.

Proteomic identification of putative microRNA394 target genes in Arabidopsis thaliana identifies MLP family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem (SAM) organisation. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Major Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development.

Traditional patterns and textures as values for meaningful automatization in music

This contribution focuses on the accelerated loss of traditional sound patterning in music, parallel to the exponential loss of linguistic and cultural variety in a world increasingly 'globalized' by market policies and economic liberalization, in which scientific or technical justification plays a crucial role. As a suggestion to an alternative trend, composers and music theorists are invited to explore the world of design and patterning by grammar rules from non-dominant cultures, and to make an effort to understand their contextual usage and its transformation, in order to appreciate their symbolism and aesthetic depth. Practical examples are provided.

Laser processing of high-Tc superconducting thin films

High-Tc superconducting thin films can be deposited and processed by pulsed and CW lasers, and a respectable materials technology for the Y-Ba-Cu-O superconductor is rapidly emerging. The pulsed laser deposition technique is simple because it produces films with compositions nearly identical to those of the target pellets. A larger variety of substrates can be used, compared to other deposition technologies, because of the relatively low temperature requirements. The laser deposition mechanism has been investigated. As-deposited superconducting films, epitaxial films with smooth surfaces, and multilayer structures with abrupt interfaces have been produced. The electrical transport properties can be changed locally using a focused argon-ion laser by modifying the oxygen stoichiometry. This laser writing can be erased by room-temperature exposure to an oxygen plasma. Other laser patterning methods such as material removal, melt-quench, and direct pattern transfer are being developed.

Molecular background of three lethal fetal syndromes

This study identified the molecular defects underlying three lethal fetal syndromes. Lethal Congenital Contracture Syndrome 1 (LCCS1, MIM 253310) and Lethal Arthrogryposis with Anterior Horn Cell Disease (LAAHD, MIM 611890) are fetal motor neuron diseases. They affect the nerve cells that control voluntary muscle movement, and eventually result in severe atrophy of spinal cord motor neurons and fetal immobility. Both LCCS1 and LAAHD are caused by mutations in the GLE1 gene, which encodes for a multifunctional protein involved in posttranscriptional mRNA processing. LCCS2 and LCCS3, two syndromes that are clinically similar to LCCS1, are caused by defective proteins involved in the synthesis of inositol hexakisphosphate (IP6), an essential cofactor of GLE1. This suggests a common mechanism behind these fetal motor neuron diseases, and along with accumulating evidence from genetic studies of more late-onset motor neuron diseases such as Spinal muscular atrophy (SMA) and Amyotrophic lateral sclerosis (ALS), implicates mRNA processing as a common mechanism in motor neuron disease pathogenesis. We also studied gle1-/- zebrafish in order to investigate whether they would be a good model for studying the pathogenesis of LCCS1 and LAAHD. Mutant zebrafish exhibit cell death in their central nervous system at two days post fertilization, and the distribution of mRNA within the cells of mutant zebrafish differs from controls, encouraging further studies. The third lethal fetal syndrome is described in this study for the first time. Cocoon syndrome (MIM 613630) was discovered in a Finnish family with two affected individuals. Its hallmarks are the encasement of the limbs under the skin, and severe craniofacial abnormalities, including the lack of skull bones. We showed that Cocoon syndrome is caused by a mutation in the gene encoding the conserved helix-loop-helix ubiquitous kinase CHUK, also known as IκB kinase α (IKKα). The mutation results in the complete lack of CHUK protein expression. CHUK is a subunit of the IκB kinase enzyme that inhibits NF-κB transcription factors, but in addition, it has an essential, independent role in controlling keratinocyte differentiation, as well as informing morphogenetic events such as limb and skeletal patterning. CHUK also acts as a tumor suppressor, and is frequently inactivated in cancer. This study has brought significant new information about the molecular background of these three lethal fetal syndromes, as well as provided knowledge about the prerequisites of normal human development.

Functional Surfaces for Microfluidics in Proteomic Analysis

Microchips for use in biomolecular analysis show a lot of promise for medical diagnostics and biomedical basic research. Among the potential advantages are more sensitive and faster analyses as well as reduced cost and sample consumption. Due to scaling laws, the surface are to volume ratios of microfluidic chips is very high. Because of this, tailoring the surface properties and surface functionalization are very important technical issues for microchip development. This thesis studies two different types of functional surfaces, surfaces for open surface capillary microfluidics and surfaces for surface assisted laser desorption ionization mass spectrometry, and combinations thereof. Open surface capillary microfluidics can be used to transport and control liquid samples on easily accessible open surfaces simply based on surface forces, without any connections to pumps or electrical power sources. Capillary filling of open partially wetting grooves is shown to be possible with certain geometries, aspect ratios and contact angles, and a theoretical model is developed to identify complete channel filling domains, as well as partial filling domains. On the other hand, partially wetting surfaces with triangular microstructures can be used for achieving directional wetting, where the water droplets do not spread isotropically, but instead only spread to a predetermined sector. Furthermore, by patterning completely wetting and superhydrophobic areas on the same surface, complex droplet shapes are achieved, as the water stretches to make contact with the wetting surface, but does not enter into the superhydrophobic domains. Surfaces for surface assisted laser desorption ionization mass spectrometry are developed by applying various active thin film coatings on multiple substrates, in order to separate surface and bulk effects. Clear differences are observed between both surface and substrate layers. The best performance surfaces consisted of amorphous silicon coating and an inorganic-organic hybrid substrate, with nanopillars and nanopores. These surfaces are used for matrix-free ionization of drugs, peptides and proteins, and for some analytes, the detection limits were in the high attomoles. Microfluidics and laser desorption ionization surfaces are combined on a functionalized drying platforms, where the surface is used to control the shape of the deposited analyte droplet, and the shape of the initial analyte droplet affects the dried droplet solute deposition pattern. The deposited droplets can then directly detected by mass spectrometry. Utilizing this approach, results of analyte concentration, splitting and separation are demonstrated.

The determination of spatial pattern in Dictyostelium discoideum

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other, and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.

VIP blockade leads to microcephaly in mice via disruption of Mcph1-Chk1 signaling

Autosomal recessive primary microcephaly (MCPH) is a genetic disorder that causes a reduction of cortical outgrowth without severe interference with cortical patterning. It is associated with mutations in a number of genes encoding protein involved in mitotic spindle formation and centrosomal activities or cell cycle control. We have shown previously that blocking vasoactive intestinal peptide (VIP) during gestation in mice by using a VIP antagonist (VA) results in microcephaly. Here, we have shown that the cortical abnormalities caused by prenatal VA administration mimic the phenotype described in MCPH patients and that VIP blockade during neurogenesis specifically disrupts Mcph1 signaling. VA administration reduced neuroepithelial progenitor proliferation by increasing cell cycle length and promoting cell cycle exit and premature neuronal differentiation. Quantitative RT-PCR and Western blot showed that VA downregulated Mcph1. Inhibition of Mcph1 expression led to downregulation of Chk1 and reduction of Chk1 kinase activity. The inhibition of Mcph1 and Chk1 affected the expression of a specific subset of cell cycle-controlling genes and turned off neural stem cell proliferation in neurospheres. Furthermore, in vitro silencing of either Mcph1 or Chk1 in neurospheres mimicked VA-induced inhibition of cell proliferation. These results demonstrate that VIP blockade induces microcephaly through Mcph1 signaling and suggest that VIP/Mcph1/Chk1 signaling is key for normal cortical development.

Dynamic contact angle beating from drops impacting onto solid surfaces exhibiting anisotropic wetting

This paper reports an experimental investigation of low Weber number water drops impacting onto solid surfaces exhibiting anisotropic wetting. The wetting anisotropy is created by patterning the solid surfaces with unidirectional parallel grooves. Temporal measurements of impacting drop parameters such as drop base contact diameter, apparent contact angle of drop, and drop height at the center are obtained from high-speed video recordings of drop impacts. The study shows that the impact of low Weber number water drops on the grooved surface exhibits beating phenomenon in the temporal variations of the dynamic contact angle anisotropy and drop height at the center of the impacting drop. It is observed that the beating phenomenon of impacting drop parameters is caused by the frequency difference between the dynamic contact angle oscillations of impacting drop liquid oriented perpendicular and parallel to the direction of grooves on the grooved surface. The primary trigger for the phenomenon is the existence of non-axisymmetric drop flow on the grooved surface featuring pinned and free motions of drop liquid in the directions perpendicular and parallel to the grooves, respectively. The beat frequency is almost independent of the impact drop Weber number. Further experimental measurements with solid surfaces of different groove textures show that the grooved surface with larger wetting anisotropy may be expected to show a dominant beating phenomenon. The phenomenon is gradually damped out with time and is fully unrecognizable at higher drop impact Weber numbers. (C) 2011 Elsevier B.V. All rights reserved.

Expression of Sonic Hedgehog During Cell Proliferation in the Human Cerebellum

The regulation of cell proliferation in the external granular layer (EGL) of the developing cerebellum is important for its normal patterning. An important signal that regulates EGL cell proliferation is Sonic hedgehog (Shh). Shh is secreted by the Purkinje cells (PC) and has a mitogenic effect on the granule cell precursors of the EGL. Deregulation of Shh signaling has been associated with abnormal development, and been implicated in medulloblastomas, which are tumors that arise from the cerebellum. Given the importance of the Shh pathway in cerebellum development and disease, there has been no systematic study of its expression pattern during human cerebellum development. In this study, we describe the expression pattern of Shh, its receptor patched, smoothened, and its effectors that belong to the Gli family of transcription factors, during normal human cerebellum development from 10 weeks of gestational age, and in medulloblastomas that represents a case of abnormal cell proliferation in the cerebellum. This expression pattern is compared to equivalent stages in the normal development of cerebellum in mouse, as well as in tumors. Important differences between human and mouse that reflect differences in the normal developmental program between the 2 species are observed. First, in humans there appears to be a stage of Shh signaling within the EGL, when the PC are not yet the source of Shh. Second, unlike in the postnatal mouse cerebellum, expression of Shh in the PC in the postnatal human cerebellum is downregulated. Finally, medulloblastomas in the human but not in patched heterozygote mouse express Shh. These results highlight cross-species differences in the regulation of the Shh signaling pathway.