Publications by authors named "Brandon J Wainwright"

52 Publications

SOX9 Defines Distinct Populations of Cells in SHH Medulloblastoma but Is Not Required for Math1-Driven Tumor Formation.

Mol Cancer Res 2021 Nov 30;19(11):1831-1839. Epub 2021 Jul 30.

The University of Queensland Diamantina Institute, University of Queensland, Woolloongabba, Queensland, Australia.

Medulloblastoma is the most common malignant pediatric brain tumor and there is an urgent need for molecularly targeted and subgroup-specific therapies. The stem cell factor SOX9, has been proposed as a potential therapeutic target for the treatment of Sonic Hedgehog medulloblastoma (SHH-MB) subgroup tumors, given its role as a downstream target of Hedgehog signaling and in functionally promoting SHH-MB metastasis and treatment resistance. However, the functional requirement for SOX9 in the genesis of medulloblastoma remains to be determined. Here we report a previously undocumented level of SOX9 expression exclusively in proliferating granule cell precursors (GCP) of the postnatal mouse cerebellum, which function as the medulloblastoma-initiating cells of SHH-MBs. Wild-type GCPs express comparatively lower levels of SOX9 than neural stem cells and mature astroglia and SOX9 GCP-like tumor cells constitute the bulk of both infant (Math1Cre: ) and adult ( ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: (GCP), (astrocytes) and (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated using a Math1Cre model system. Surprisingly, targeted ablation of in GCPs (Math1Cre: ) revealed no overt phenotype and loss of in SHH-MB (Math1Cre: ) does not affect tumor formation. IMPLICATIONS: Despite preclinical data indicating SOX9 plays a key role in SHH-MB biology, our data argue against SOX9 as a viable therapeutic target.
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http://dx.doi.org/10.1158/1541-7786.MCR-21-0117DOI Listing
November 2021

Tumor cells generate astrocyte-like cells that contribute to SHH-driven medulloblastoma relapse.

J Exp Med 2021 09 13;218(9). Epub 2021 Jul 13.

Cancer Biology Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, PA.

Astrocytes, a major glial cell type in the brain, play a critical role in supporting the progression of medulloblastoma (MB), the most common malignant pediatric brain tumor. Through lineage tracing analyses and single-cell RNA sequencing, we demonstrate that astrocytes are predominantly derived from the transdifferentiation of tumor cells in relapsed MB (but not in primary MB), although MB cells are generally believed to be neuronal-lineage committed. Such transdifferentiation of MB cells relies on Sox9, a transcription factor critical for gliogenesis. Our studies further reveal that bone morphogenetic proteins (BMPs) stimulate the transdifferentiation of MB cells by inducing the phosphorylation of Sox9. Pharmacological inhibition of BMP signaling represses MB cell transdifferentiation into astrocytes and suppresses tumor relapse. Our studies establish the distinct cellular sources of astrocytes in primary and relapsed MB and provide an avenue to prevent and treat MB relapse by targeting tumor cell transdifferentiation.
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http://dx.doi.org/10.1084/jem.20202350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282281PMC
September 2021

Systems pharmacogenomics identifies novel targets and clinically actionable therapeutics for medulloblastoma.

Genome Med 2021 06 21;13(1):103. Epub 2021 Jun 21.

The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD, 4102, Australia.

Background: Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects.

Methods: We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB.

Results: Our analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB.

Conclusions: Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.
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http://dx.doi.org/10.1186/s13073-021-00920-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215804PMC
June 2021

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype.

Nat Commun 2021 05 11;12(1):2678. Epub 2021 May 11.

Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.
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http://dx.doi.org/10.1038/s41467-021-22888-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113450PMC
May 2021

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1β release.

J Exp Med 2021 05;218(5)

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.
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http://dx.doi.org/10.1084/jem.20201452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933984PMC
May 2021

Genetic and functional interaction network analysis reveals global enrichment of regulatory T cell genes influencing basal cell carcinoma susceptibility.

Genome Med 2021 02 6;13(1):19. Epub 2021 Feb 6.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Background: Basal cell carcinoma (BCC) of the skin is the most common form of human cancer, with more than 90% of tumours presenting with clear genetic activation of the Hedgehog pathway. However, polygenic risk factors affecting mechanisms such as DNA repair and cell cycle checkpoints or which modulate the tumour microenvironment or host immune system play significant roles in determining whether genetic mutations culminate in BCC development. We set out to define background genetic factors that play a role in influencing BCC susceptibility via promoting or suppressing the effects of oncogenic drivers of BCC.

Methods: We performed genome-wide association studies (GWAS) on 17,416 cases and 375,455 controls. We subsequently performed statistical analysis by integrating data from population-based genetic studies of multi-omics data, including blood- and skin-specific expression quantitative trait loci and methylation quantitative trait loci, thereby defining a list of functionally relevant candidate BCC susceptibility genes from our GWAS loci. We also constructed a local GWAS functional interaction network (consisting of GWAS nearest genes) and another functional interaction network, consisting specifically of candidate BCC susceptibility genes.

Results: A total of 71 GWAS loci and 46 functional candidate BCC susceptibility genes were identified. Increased risk of BCC was associated with the decreased expression of 26 susceptibility genes and increased expression of 20 susceptibility genes. Pathway analysis of the functional candidate gene regulatory network revealed strong enrichment for cell cycle, cell death, and immune regulation processes, with a global enrichment of genes and proteins linked to T cell biology.

Conclusions: Our genome-wide association analyses and functional interaction network analysis reveal an enrichment of risk variants that function in an immunosuppressive regulatory network, likely hindering cancer immune surveillance and effective antitumour immunity.
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http://dx.doi.org/10.1186/s13073-021-00827-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866769PMC
February 2021

Patient-derived orthotopic xenograft models of medulloblastoma lack a functional blood-brain barrier.

Neuro Oncol 2021 05;23(5):732-742

The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland, Woolloongabba, Queensland, Australia.

Background: Novel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB.

Methods: Dynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models.

Results: Contrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models.

Conclusions: These results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.
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http://dx.doi.org/10.1093/neuonc/noaa266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099473PMC
May 2021

Germline Elongator mutations in Sonic Hedgehog medulloblastoma.

Nature 2020 04 1;580(7803):396-401. Epub 2020 Apr 1.

Department of Neurosciences, University of California San Diego and Rady Children's Hospital, San Diego, CA, USA.

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MB) ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MB. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U) position. Tumours from patients with ELP1-associated MB were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.
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http://dx.doi.org/10.1038/s41586-020-2164-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430762PMC
April 2020

Common Regulatory Targets of NFIA, NFIX and NFIB during Postnatal Cerebellar Development.

Cerebellum 2020 Feb;19(1):89-101

The School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Australia.

Transcriptional regulation plays a central role in controlling neural stem and progenitor cell proliferation and differentiation during neurogenesis. For instance, transcription factors from the nuclear factor I (NFI) family have been shown to co-ordinate neural stem and progenitor cell differentiation within multiple regions of the embryonic nervous system, including the neocortex, hippocampus, spinal cord and cerebellum. Knockout of individual Nfi genes culminates in similar phenotypes, suggestive of common target genes for these transcription factors. However, whether or not the NFI family regulates common suites of genes remains poorly defined. Here, we use granule neuron precursors (GNPs) of the postnatal murine cerebellum as a model system to analyse regulatory targets of three members of the NFI family: NFIA, NFIB and NFIX. By integrating transcriptomic profiling (RNA-seq) of Nfia- and Nfix-deficient GNPs with epigenomic profiling (ChIP-seq against NFIA, NFIB and NFIX, and DNase I hypersensitivity assays), we reveal that these transcription factors share a large set of potential transcriptional targets, suggestive of complementary roles for these NFI family members in promoting neural development.
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http://dx.doi.org/10.1007/s12311-019-01089-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815246PMC
February 2020

Granule neuron precursor cell proliferation is regulated by NFIX and intersectin 1 during postnatal cerebellar development.

Brain Struct Funct 2019 Mar 3;224(2):811-827. Epub 2018 Dec 3.

The School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Australia.

Cerebellar granule neurons are the most numerous neuronal subtype in the central nervous system. Within the developing cerebellum, these neurons are derived from a population of progenitor cells found within the external granule layer of the cerebellar anlage, namely the cerebellar granule neuron precursors (GNPs). The timely proliferation and differentiation of these precursor cells, which, in rodents occurs predominantly in the postnatal period, is tightly controlled to ensure the normal morphogenesis of the cerebellum. Despite this, our understanding of the factors mediating how GNP differentiation is controlled remains limited. Here, we reveal that the transcription factor nuclear factor I X (NFIX) plays an important role in this process. Mice lacking Nfix exhibit reduced numbers of GNPs during early postnatal development, but elevated numbers of these cells at postnatal day 15. Moreover, Nfix GNPs exhibit increased proliferation when cultured in vitro, suggestive of a role for NFIX in promoting GNP differentiation. At a mechanistic level, profiling analyses using both ChIP-seq and RNA-seq identified the actin-associated factor intersectin 1 as a downstream target of NFIX during cerebellar development. In support of this, mice lacking intersectin 1 also displayed delayed GNP differentiation. Collectively, these findings highlight a key role for NFIX and intersectin 1 in the regulation of cerebellar development.
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http://dx.doi.org/10.1007/s00429-018-1801-3DOI Listing
March 2019

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior.

Nat Commun 2018 08 10;9(1):3195. Epub 2018 Aug 10.

Institute for Molecular Bioscience, the University of Queensland, Brisbane, QLD, 4072, Australia.

Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.
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http://dx.doi.org/10.1038/s41467-018-05765-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086839PMC
August 2018

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

Oncotarget 2017 Oct 19;8(48):84006-84018. Epub 2017 Sep 19.

Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, Universidad de Chile, Santiago, Chile.

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of receptor, and ligands with respect to , the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how expression decreases throughout BCC progression in the K14-Cre:Ptch1 mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.
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http://dx.doi.org/10.18632/oncotarget.21061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663572PMC
October 2017

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

J Am Soc Nephrol 2018 02 6;29(2):532-544. Epub 2017 Nov 6.

Program in Developmental and Stem Cell Biology,

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing , a Hedgehog signaling target. Constitutive expression of GLI3 repressor in deficient mice rescued ectopic expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in -deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.
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http://dx.doi.org/10.1681/ASN.2017050482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791051PMC
February 2018

Patched1 patterns Fibroblast growth factor 10 and Forkhead box F1 expression during pulmonary branch formation.

Mech Dev 2017 10 20;147:37-48. Epub 2017 Sep 20.

Institute for Molecular Bioscience, The University of Queensland, Australia. Electronic address:

Hedgehog (Hh) signalling, Fibroblast growth factor 10 (Fgf10) and Forkhead box F1 (Foxf1) are each individually important for directing pulmonary branch formation but their interactions are not well understood. Here we demonstrate that Hh signalling is vital in regulating Foxf1 and Fgf10 expression during branching. The Hedgehog receptor Patched1 (Ptch1) was conditionally inactivated in the lung mesenchyme by Dermo1-Cre in vivo or using a recombinant Cre recombinase protein (HNCre) in lung cultures resulting in cell autonomous activation of Hh signalling. Homozygous mesenchymal Ptch1 deleted embryos (Dermo1Cre+/-;Ptch1) showed secondary branching and lobe formation defects. Fgf10 expression is spatially reduced in the distal tip of Dermo1Cre+/-;Ptch1 lungs and addition of Fgf10 recombinant protein to these lungs in culture has shown partial restoration of branching, indicating Ptch1 function patterns Fgf10 to direct lung branching. Foxf1 expression is upregulated in Dermo1Cre+/-;Ptch1 lungs, suggesting Foxf1 may mediate Hh signalling effects in the lung mesenchyme. In vitro HNCre-mediated Ptch1 deleted lung explants support the in vivo observations, with evidence of mesenchyme hyperproliferation and this is consistent with the previously reported role of Hh signalling in maintaining mesenchymal cell survival. Consequently it is concluded that during early pseudoglandular stage of lung development Ptch1 patterns Fgf10 and regulates Foxf1 expression in the lung mesenchyme to direct branch formation and this is essential for proper lobe formation and lung function.
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http://dx.doi.org/10.1016/j.mod.2017.09.001DOI Listing
October 2017

Inhibition of CDK4/6 by Palbociclib Significantly Extends Survival in Medulloblastoma Patient-Derived Xenograft Mouse Models.

Clin Cancer Res 2017 Oct 21;23(19):5802-5813. Epub 2017 Jun 21.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Bioinformatics analysis followed by studies in patient-derived xenograft (PDX) models were used to identify and validate CDK 4/6 inhibition as an effective therapeutic strategy for medulloblastoma, particularly group 3 -amplified tumors that have the worst clinical prognosis. A protein interaction network derived from a mutagenesis model of medulloblastoma was used to identify potential novel therapeutic targets. The top hit from this analysis was validated using PDX models of medulloblastoma implanted subcutaneously in the flank and orthotopically in the cerebellum of mice. Informatics analysis identified the CDK4/6/CYCLIN D/RB pathway as a novel "druggable" pathway for multiple subgroups of medulloblastoma. Palbociclib, a highly specific inhibitor of CDK4/6, was found to inhibit RB phosphorylation and cause G arrest in PDX models of medulloblastoma. The drug caused rapid regression of Sonic hedgehog (SHH) and -amplified group 3 medulloblastoma subcutaneous tumors and provided a highly significant survival advantage to mice bearing -amplified intracranial tumors. Inhibition of CDK4/6 is potentially a highly effective strategy for the treatment of SHH and -amplified group 3 medulloblastoma. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6939669PMC
October 2017

Cell-type-specific expression of NFIX in the developing and adult cerebellum.

Brain Struct Funct 2017 Jul 23;222(5):2251-2270. Epub 2016 Nov 23.

The School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Australia.

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.
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http://dx.doi.org/10.1007/s00429-016-1340-8DOI Listing
July 2017

MicroRNA Biogenesis and Hedgehog-Patched Signaling Cooperate to Regulate an Important Developmental Transition in Granule Cell Development.

Genetics 2016 Mar 15;202(3):1105-18. Epub 2016 Jan 15.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia.

The Dicer1, Dcr-1 homolog (Drosophila) gene encodes a type III ribonuclease required for the canonical maturation and functioning of microRNAs (miRNAs). Subsets of miRNAs are known to regulate normal cerebellar granule cell development, in addition to the growth and progression of medulloblastoma, a neoplasm that often originates from granule cell precursors. Multiple independent studies have also demonstrated that deregulation of Sonic Hedgehog (Shh)-Patched (Ptch) signaling, through miRNAs, is causative of granule cell pathologies. In the present study, we investigated the genetic interplay between miRNA biogenesis and Shh-Ptch signaling in granule cells of the cerebellum by way of the Cre/lox recombination system in genetically engineered models of Mus musculus (mouse). We demonstrate that, although the miRNA biogenesis and Shh-Ptch-signaling pathways, respectively, regulate the opposing growth processes of cerebellar hypoplasia and hyperplasia leading to medulloblastoma, their concurrent deregulation was nonadditive and did not bring the growth phenotypes toward an expected equilibrium. Instead, mice developed either hypoplasia or medulloblastoma, but of a greater severity. Furthermore, some genotypes were bistable, whereby subsets of mice developed hypoplasia or medulloblastoma. This implies that miRNAs and Shh-Ptch signaling regulate an important developmental transition in granule cells of the cerebellum. We also conclusively show that the Dicer1 gene encodes a haploinsufficient tumor suppressor gene for Ptch1-induced medulloblastoma, with the monoallielic loss of Dicer1 more severe than biallelic loss. These findings exemplify how genetic interplay between pathways may produce nonadditive effects with a substantial and unpredictable impact on biology. Furthermore, these findings suggest that the functional dosage of Dicer1 may nonadditively influence a wide range of Shh-Ptch-dependent pathologies.
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http://dx.doi.org/10.1534/genetics.115.184176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788112PMC
March 2016

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

Cerebellum 2015 Dec;14(6):688-98

Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St Lucia, Queensland, 4072, Australia.

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.
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http://dx.doi.org/10.1007/s12311-015-0672-xDOI Listing
December 2015

Nuclear factor one transcription factors: Divergent functions in developmental versus adult stem cell populations.

Dev Dyn 2015 Mar 11;244(3):227-38. Epub 2014 Sep 11.

The School of Biomedical Sciences, The University of Queensland, Brisbane, Australia.

Nuclear factor one (NFI) transcription factors are a group of site-specific DNA-binding proteins that are emerging as critical regulators of stem cell biology. During development NFIs promote the production of differentiated progeny at the expense of stem cell fate, with Nfi null mice exhibiting defects such as severely delayed brain and lung maturation, skeletomuscular defects and renal abnormalities, phenotypes that are often consistent with patients with congenital Nfi mutations. Intriguingly, recent research suggests that in adult tissues NFI factors play a qualitatively different role than during development, with NFIs serving to promote the survival and maintenance of slow-cycling adult stem cell populations rather than their differentiation. Here we review the role of NFI factors in development, largely focusing on their role as promoters of stem cell differentiation, and attempt to reconcile this with the emerging role of NFIs in adult stem cell niches.
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http://dx.doi.org/10.1002/dvdy.24182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563865PMC
March 2015

Patched 1 and patched 2 redundancy has a key role in regulating epidermal differentiation.

J Invest Dermatol 2014 Jul 3;134(7):1981-1990. Epub 2014 Feb 3.

Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. Electronic address:

The Patched 1 (Ptch1) receptor has a pivotal role in inhibiting the activity of the Hedgehog (Hh) pathway and is therefore critical in preventing the onset of many human developmental disorders and tumor formation. However, the functional role of the mammalian Ptch2 paralogue remains elusive, particularly the extent to which it contributes to regulating the spatial and temporal activity of Hh signaling. Here we demonstrate in three independent mouse models of epidermal development that in vivo ablation of both Ptch receptors results in a more severe phenotype than loss of Ptch1 alone. Our studies indicate that concomitant loss of Ptch1 and Ptch2 activity inhibits epidermal lineage specification and differentiation. These results reveal that repression of Hh signaling through a dynamic Ptch regulatory network is a crucial event in lineage fate determination in the skin. In general, our findings implicate Ptch receptor redundancy as a key issue in elucidating the cellular origin of Hh-induced tumors.
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http://dx.doi.org/10.1038/jid.2014.63DOI Listing
July 2014

Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.

Proc Natl Acad Sci U S A 2013 Nov 28;110(46):E4325-34. Epub 2013 Oct 28.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.

The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups.
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http://dx.doi.org/10.1073/pnas.1318639110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832011PMC
November 2013

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

Hum Mol Genet 2013 Dec 29;22(24):5026-35. Epub 2013 Jul 29.

Institute for Molecular Bioscience and.

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.
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http://dx.doi.org/10.1093/hmg/ddt353DOI Listing
December 2013

Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

PLoS One 2013 11;8(6):e65818. Epub 2013 Jun 11.

Laboratory of Stem Cells and Development, FONDAP Center for Genome Regulation, Faculty of Science, University of Chile, Santiago, Chile.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065818PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679138PMC
January 2014

Neogenin1 is a Sonic Hedgehog target in medulloblastoma and is necessary for cell cycle progression.

Int J Cancer 2014 Jan 24;134(1):21-31. Epub 2013 Jul 24.

Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile; FONDAP Center for Genome Regulation, Santiago, Chile.

The canonical Sonic Hedgehog (Shh)/Gli pathway plays multiples roles during central nervous system (CNS) development. To elucidate the molecular repertoire of Shh mediators, we have recently described novel transcriptional targets in response to Shh pathway modulation. Among them, we were able to identify Neogenin1 (Neo1), a death dependence receptor, as a new direct Shh downstream regulator in neural precursor proliferation. As appropriate Shh signaling is required for cerebellar growth and alterations cause Shh-driven medulloblastoma (MB), here we have addressed the role of the Shh/Neogenin1 interaction in the context of cerebellar development and cancer. We demonstrate that the Shh pathway regulates Neogenin1 expression in mouse models that recapitulate the Shh MB subtype. We show that the canonical Shh pathway directly regulates the Neo1 gene acting through an upstream sequence in its promoter both in vitro and in vivo in granule neuron precursor cells. We also identified and characterized a functional Gli-binding site in the first intron of the human NEO1 gene. Gene expression profiling of more than 300 MB shows that NEO1 is indeed upregulated in SHH tumors compared to the other MB subgroups. Finally, we provide evidence that NEO1 is necessary for cell cycle progression in a human MB cell line, because a loss of function of NEO1 arrests cells in the G2/M phase. Taken together, these results highlight Neogenin1 as a novel downstream effector of the Shh pathway in MB and a possible therapeutic target.
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http://dx.doi.org/10.1002/ijc.28330DOI Listing
January 2014

Murine basal cell carcinoma leads to tumor-mediated alterations in endocrine Igf1 signaling.

Endocr Relat Cancer 2013 Jun 20;20(3):273-81. Epub 2013 May 20.

Institute for Molecular Bioscience, University of Queensland, 360 Carmody Road, St Lucia, Brisbane, Queensland 4072, Australia.

The intrinsic properties underlying cancer development are extensively studied while the effect of a cancer on the host is often overlooked. Activation of the Hedgehog (Hh) signaling pathway underlies a number of types of common human cancers, yet little is known concerning endocrine signaling in such tumors. Here, we investigated endocrine signaling in a murine model of basal cell carcinoma (BCC) of the skin, the most common cancer. BCCs were generated by the activation of Hh signaling resulting from the specific deletion of the Ptch1 gene in the developing epidermis. Subsequently, a severe growth deficiency was observed in the murine BCC model, and we identified a deficiency of circulating IGF1 (Igf1). We demonstrate that Hh pathway activation in murine BCC induces IGF binding proteins, thereby regulating Igf1 sequestration into the skin and skewing Igf endocrine signaling. Significantly, these results show that Hh-induced tumors can have endocrine effects on normal tissues that in turn can greatly impact the host. This study not only identifies that Igf is important in Hh-associated skin tumors but also exemplifies the need to consider endocrine signaling when interpreting complex in vivo tumor models.
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http://dx.doi.org/10.1530/ERC-12-0307DOI Listing
June 2013

Kif7 regulates Gli2 through Sufu-dependent and -independent functions during skin development and tumorigenesis.

Development 2012 Nov 3;139(22):4152-61. Epub 2012 Oct 3.

Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

Abnormal activation of Hedgehog (Hh) signaling leads to basal cell carcinoma (BCC) of the skin, the most common human cancer. Gli2, the major transcriptional activator of Hh signaling, is essential for hair follicle development and its overexpression in epidermis induces BCC formation and maintains tumor growth. Despite its importance in skin development and tumorigenesis, little is known about the molecular regulation of Gli2. Sufu and Kif7 are two evolutionarily conserved regulators of Gli transcription factors. Here, we show that Sufu and Kif7 regulate Gli2 through distinct mechanisms in keratinocytes. Sufu restricts the activity of Gli2 through cytoplasmic sequestration. Kif7 possesses Sufu-dependent and -independent regulatory functions in Hh signaling: while it promotes Hh pathway activity through the dissociation of Sufu-Gli2 complex, it also contributes to the repression of Hh target genes in the absence of Sufu. Deletion of both Sufu and Kif7 in embryonic skin leads to complete loss of follicular fate. Importantly, although inactivation of Sufu or Kif7 alone in adult epidermis cannot promote BCC formation, their simultaneous deletion induces BCC. These studies establish Sufu and Kif7 as crucial components in the regulation of Gli2 localization and activity, and illustrate their overlapping functions in skin development and tumor suppression.
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http://dx.doi.org/10.1242/dev.081190DOI Listing
November 2012

Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model.

Proc Natl Acad Sci U S A 2012 May 1;109(20):7859-64. Epub 2012 May 1.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.
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http://dx.doi.org/10.1073/pnas.1114718109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356655PMC
May 2012

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

Hum Mol Genet 2012 Apr 6;21(8):1808-23. Epub 2012 Jan 6.

Epigenetics Laboratory, Queensland Institute for Medical Research, Herston, Queensland 4006, Australia.

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.
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http://dx.doi.org/10.1093/hmg/ddr613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3313797PMC
April 2012

Hedgehog/Notch-induced premature gliogenesis represents a new disease mechanism for Hirschsprung disease in mice and humans.

J Clin Invest 2011 Sep 15;121(9):3467-78. Epub 2011 Aug 15.

Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong, China.

Hirschsprung (HSCR) disease is a complex genetic disorder attributed to a failure of the enteric neural crest cells (ENCCs) to form ganglia in the hindgut. Hedgehog and Notch are implicated in mediating proliferation and differentiation of ENCCs. Nevertheless, how these signaling molecules may interact to mediate gut colonization by ENCCs and contribute to a primary etiology for HSCR are not known. Here, we report our pathway-based epistasis analysis of data generated by a genome-wide association study on HSCR disease, which indicates that specific genotype constellations of Patched (PTCH1) (which encodes a receptor for Hedgehog) and delta-like 3 (DLL3) (which encodes a receptor for Notch) SNPs confer higher risk to HSCR. Importantly, deletion of Ptch1 in mouse ENCCs induced robust Dll1 expression and activation of the Notch pathway, leading to premature gliogenesis and reduction of ENCC progenitors in mutant bowels. Dll1 integrated Hedgehog and Notch pathways to coordinate neuronal and glial cell differentiation during enteric nervous system development. In addition, Hedgehog-mediated gliogenesis was found to be highly conserved, such that Hedgehog was consistently able to promote gliogenesis of human neural crest-related precursors. Collectively, we defined PTCH1 and DLL3 as HSCR susceptibility genes and suggest that Hedgehog/Notch-induced premature gliogenesis may represent a new disease mechanism for HSCR.
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http://dx.doi.org/10.1172/JCI43737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163945PMC
September 2011
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