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

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 SOX9low GCP-like tumor cells constitute the bulk of both infant (Math1Cre:Ptch1lox/lox ) and adult (Ptch1LacZ/+ ) SHH-MB mouse models. Human medulloblastoma single-cell RNA data analyses reveal three distinct SOX9 populations present in SHH-MB and noticeably absent in other medulloblastoma subgroups: SOX9 + MATH1 + (GCP), SOX9 + GFAP + (astrocytes) and SOX9 + MATH1 + GFAP + (potential tumor-derived astrocytes). To functionally address whether SOX9 is required as a downstream effector of Hedgehog signaling in medulloblastoma tumor cells, we ablated Sox9 using a Math1Cre model system. Surprisingly, targeted ablation of Sox9 in GCPs (Math1Cre:Sox9lox/lox ) revealed no overt phenotype and loss of Sox9 in SHH-MB (Math1Cre:Ptch1lox/lox;Sox9lox/lox ) 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.

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

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.

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

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-1ß 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.

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

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 TReg 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.

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

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.

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

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.

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

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 NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox 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.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types.

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Patched Receptors Sense, Interpret, and Establish an Epidermal Hedgehog Signaling Gradient.

By using the sensitivity of single-molecule fluorescent in situ hybridization, we have precisely quantified the levels and defined the temporal and spatial distribution of Hedgehog signaling activity during embryonic skin development and discovered that there is a Hedgehog signaling gradient along the proximal-distal axis of developing hair follicles. To explore the contribution of Hedgehog receptors Ptch1 and Ptch2 in establishing the epidermal signaling gradient, we quantitated the level of pathway activity generated in Ptch1- and Ptch1;Ptch2-deficient skin and defined the contribution of each receptor to regulation of the levels of Hedgehog signaling identified in wild-type skin. Moreover, we show that both the cellular phenotype and level of pathway activity featured in Ptch1;Ptch2-deficient cells faithfully recapitulates the Peak level of endogenous Hedgehog signaling detected at the base of developing follicles, where the concentration of endogenous Shh is predicted to be highest. Taken together, these data show that both Ptch1 and Ptch2 play a crucial role in sensing the concentration of Hedgehog ligand and regulating the appropriate dose-dependent response.

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

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.

Sonic hedgehog and notch signaling can cooperate to regulate neurogenic divisions of neocortical progenitors.

BACKGROUND: Hedgehog (Hh) signaling is crucial for the generation and maintenance of both embryonic and adult stem cells, thereby regulating development and tissue homeostasis. In the developing neocortex, Sonic Hedgehog (Shh) regulates neural progenitor cell proliferation. During neurogenesis, radial glial cells of the ventricular zone (VZ) are the predominant neocortical progenitors that generate neurons through both symmetric and asymmetric divisions. Despite its importance, relatively little is known of the molecular pathways that control the switch from symmetric proliferative to differentiative/neurogenic divisions in neural progenitors. PRINCIPAL FINDINGS: Here, we report that conditional inactivation of Patched1, a negative regulator of the Shh pathway, in Nestin positive neural progenitors of the neocortex leads to lamination defects due to improper corticogenesis and an increase in the number of symmetric proliferative divisions of the radial glial cells. Hedgehog-activated VZ progenitor cells demonstrated a concomitant upregulation of Hes1 and Blbp, downstream targets of Notch signaling. The Notch signaling pathway plays a pivotal role in the maintenance of stem/progenitor cells and the regulation of glial versus neuronal identity. To study the effect of Notch signaling on Hh-activated neural progenitors, we inactivated both Patched1 and Rbpj, a transcriptional mediator of Notch signaling, in Nestin positive cells of the neocortex. CONCLUSIONS: Our data indicate that by mid neurogenesis (embryonic day 14.5), attenuation of Notch signaling reverses the effect of Patched1 deletion on neurogenesis by restoring the balance between symmetric proliferative and neurogenic divisions. Hence, our results demonstrate that correct corticogenesis is an outcome of the interplay between the Hh and Notch signaling pathways.

Patched1 inhibits epidermal progenitor cell expansion and basal cell carcinoma formation by limiting Igfbp2 activity.

Basal cell carcinoma (BCC) of the skin is the most common form of cancer, with the majority being caused by mutations in the Patched1 (Ptch1) gene, leading to activation of the Hedgehog (Hh) signaling pathway. Hh signaling is implicated in many tumor types; thus, defining the mechanisms by which Ptch1 regulates tissue proliferation is of paramount importance. Here, we show that the key role of Ptch1 in the skin is to limit the size of the epidermal stem/progenitor compartment and allow hair follicle differentiation. Specifically, loss of Ptch1 leads to the promotion of progenitor cell fate by increasing basal cell proliferation and limiting the progression of basal cells into differentiated hair follicle cell types. Our data indicate that BCCs likely result from hair follicle progenitor cells that, due to Hh signal activation, cannot progress through normal hair follicle differentiation. These data confirm the role of Ptch1 as a negative regulator of epidermal progenitor turnover and also show for the first time that Ptch1 plays a role in the differentiation of the hair follicle lineage. In addition, we show that insulin-like growth factor binding protein 2 (Igfbp2) is upregulated in both murine and human BCCs and that blocking Igfbp2 activity reduces the Hh-mediated expansion of epidermal progenitor cells. We propose that Igfbp2 mediates epidermal progenitor cell expansion and therefore represents an epidermal progenitor cell-specific target of Hh signaling that promotes BCC development.

Placental rescue reveals a sole requirement for c-Myc in embryonic erythroblast survival and hematopoietic stem cell function.

The c-Myc protein has been implicated in playing a pivotal role in regulating the expression of a large number of genes involved in many aspects of cellular function. Consistent with this view, embryos lacking the c-myc gene exhibit severe developmental defects and die before midgestation. Here, we show that Sox2Cre-mediated deletion of the conditional c-myc(flox) allele specifically in the epiblast (hence trophoectoderm and primitive endoderm structures are wild type) rescues the majority of developmental abnormalities previously characterized in c-myc knockout embryos, indicating that they are secondary defects and arise as a result of placental insufficiency. Epiblast-restricted c-Myc-null embryos appear morphologically normal and do not exhibit any obvious proliferation defects. Nonetheless, these embryos are severely anemic and die before E12. c-Myc-deficient embryos exhibit fetal liver hypoplasia, apoptosis of erythrocyte precursors and functionally defective definitive hematopoietic stem/progenitor cells. Specific deletion of c-myc(flox) in hemogenic or hepatocytic lineages validate the hematopoietic-specific requirement of c-Myc in the embryo proper and provide in vivo evidence to support a synergism between hematopoietic and liver development. Our results reveal for the first time that physiological levels of c-Myc are essential for cell survival and demonstrate that, in contrast to most other embryonic lineages, erythroblasts and hematopoietic stem/progenitor cells are particularly dependent on c-Myc function.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Patched1 functions as a gatekeeper by promoting cell cycle progression.

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G1-S phase and G2-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a "gatekeeper." In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b.

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.

Pathways to improving skin regeneration.

A significant proportion of the human population suffers from some form of skin disorder, whether it be from burn injury or inherited skin anomalies. The ideal treatment for skin disorders would be to regrow skin tissue from stem cells residing in the individual patient's skin. Locating these adult stem cells and elucidating the molecules involved in orchestrating the production of new skin cells are important steps in devising more-efficient methods of skin production and wound healing via the ex vivo expansion of patient keratinocytes in culture. This review focuses on the structure of the skin, the identification of skin stem cells, and the role of Notch, Wnt and Hedgehog signalling cascades in regulating the fate of epidermal stem cells.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis.

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Overexpression of Sonic Hedgehog suppresses embryonic hair follicle morphogenesis.

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development, we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype.