RESUMEN
Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin, encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.
Asunto(s)
Proteínas de Unión al ADN , Epitelio , Regulación del Desarrollo de la Expresión Génica , Mesodermo , Morfogénesis , Animales , Humanos , Ratones , Dermis/citología , Dermis/embriología , Dermis/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Ectodermo/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/embriología , Factor de Transcripción GATA3 , Mesodermo/metabolismo , Mutación , Organoides , Transactivadores , Factores de Transcripción/metabolismoRESUMEN
During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh(-/-) skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh-Noggin signaling loop to drive hair follicle morphogenesis.
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Folículo Piloso/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Piel/citología , Piel/metabolismo , Animales , Femenino , Técnicas de Silenciamiento del Gen , Cabello/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Receptores Acoplados a Proteínas G/genética , Receptor SmoothenedRESUMEN
Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.
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Epidermólisis Ampollosa de la Unión/genética , Terapia Genética/métodos , Recombinación Homóloga/genética , Laminina/genética , Animales , Colágeno Tipo VII/genética , Dependovirus/genética , Epidermólisis Ampollosa de la Unión/patología , Epidermólisis Ampollosa de la Unión/terapia , Xenoinjertos , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , MutaciónRESUMEN
We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.
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Tratamiento Basado en Trasplante de Células y Tejidos , Colágeno Tipo VII , Epidermólisis Ampollosa Distrófica , Células Madre Pluripotentes Inducidas , Humanos , Epidermólisis Ampollosa Distrófica/terapia , Epidermólisis Ampollosa Distrófica/genética , Animales , Células Madre Pluripotentes Inducidas/trasplante , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Ratones , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Fibroblastos/metabolismo , Diferenciación Celular , Queratinocitos/metabolismo , Queratinocitos/trasplante , Piel/metabolismo , Trasplante Autólogo , Masculino , Mutación , Femenino , Trasplante de Piel/métodos , Edición Génica/métodos , Sistemas CRISPR-CasRESUMEN
Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.
RESUMEN
Human pluripotent stem cell-derived tissue engineering offers great promise in designer cell-based personalized therapeutics. To harness such potential, a broader approach requires a deeper understanding of tissue-level interactions. We previously developed a manufacturing system for the ectoderm-derived skin epithelium for cell replacement therapy. However, it remains challenging to manufacture the endoderm-derived esophageal epithelium, despite both possessing similar stratified structure. Here we employ single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for human esophageal development. We illuminate the cellular diversity, dynamics and signal communications for the developing esophageal epithelium and stroma. Using the machine-learning based Manatee, we prioritize the combinations of candidate human developmental signals for in vitro derivation of esophageal basal cells. Functional validation of the Manatee predictions leads to a clinically-compatible system for manufacturing human esophageal mucosa. Our approach creates a versatile platform to accelerate human tissue manufacturing for future cell replacement therapies to treat human genetic defects and wounds.
RESUMEN
Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.
RESUMEN
BACKGROUND: Initiation of the hair follicle placode and its subsequent growth, maturation and cycling in post-natal skin requires signaling interactions between epithelial cells and adjacent dermal cells and involves Shh signaling via the primary cilium. Previous reports have implicated laminins in hair follicle epithelial invagination. RESULTS: Here we use a human BCC model system and mouse mutants to re-evaluate the role of laminin-511 in epithelial invagination in the skin. Blocking laminin 511 and 332 in BCCs maintains primary cilia and Shh signalling, but prevents invagination. Similarly, in laminin-511 and dermal beta-1 integrin mutants, dermal papilla development and primary cilia formation are normal. Dermal beta-1 integrin mutants have normal hair follicle development. CONCLUSIONS: Our data provides support for a primary role of laminin-511 promoting hair follicle epithelial downgrowth without affecting dermal primary cilia and Shh target gene induction.
Asunto(s)
Carcinoma Basocelular/metabolismo , Folículo Piloso/embriología , Folículo Piloso/crecimiento & desarrollo , Integrina beta1/metabolismo , Laminina/metabolismo , Animales , Biomarcadores/metabolismo , Carcinoma Basocelular/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Folículo Piloso/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Integrina beta1/genética , Laminina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis/fisiología , Trasplante de Neoplasias , Transducción de Señal/fisiología , Trasplante de Piel , Trasplante Heterólogo , KalininaRESUMEN
Tissue development results from lineage-specific transcription factors (TFs) programming a dynamic chromatin landscape through progressive cell fate transitions. Here, we define epigenomic landscape during epidermal differentiation of human pluripotent stem cells (PSCs) and create inference networks that integrate gene expression, chromatin accessibility, and TF binding to define regulatory mechanisms during keratinocyte specification. We found two critical chromatin networks during surface ectoderm initiation and keratinocyte maturation, which are driven by TFAP2C and p63, respectively. Consistently, TFAP2C, but not p63, is sufficient to initiate surface ectoderm differentiation, and TFAP2C-initiated progenitor cells are capable of maturing into functional keratinocytes. Mechanistically, TFAP2C primes the surface ectoderm chromatin landscape and induces p63 expression and binding sites, thus allowing maturation factor p63 to positively autoregulate its own expression and close a subset of the TFAP2C-initiated surface ectoderm program. Our work provides a general framework to infer TF networks controlling chromatin transitions that will facilitate future regenerative medicine advances.
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Linaje de la Célula , Cromatina/metabolismo , Epidermis/metabolismo , Redes Reguladoras de Genes , Factor de Transcripción AP-2/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Diferenciación Celular , Ectodermo/citología , Epigénesis Genética , Retroalimentación Fisiológica , Humanos , Queratinocitos/citología , Transcriptoma/genéticaRESUMEN
Human embryonic stem cell (hESC) differentiation promises advances in regenerative medicine1-3, yet conversion of hESCs into transplantable cells or tissues remains poorly understood. Using our keratinocyte differentiation system, we employ a multi-dimensional genomics approach to interrogate the contributions of inductive morphogens retinoic acid and bone morphogenetic protein 4 (BMP4) and the epidermal master regulator p63 (encoded by TP63)4,5 during surface ectoderm commitment. In contrast to other master regulators6-9, p63 effects major transcriptional changes only after morphogens alter chromatin accessibility, establishing an epigenetic landscape for p63 to modify. p63 distally closes chromatin accessibility and promotes accumulation of H3K27me3 (trimethylated histone H3 lysine 27). Cohesin HiChIP10 visualizations of chromosome conformation show that p63 and the morphogens contribute to dynamic long-range chromatin interactions, as illustrated by TFAP2C regulation11. Our study demonstrates the unexpected dependency of p63 on morphogenetic signaling and provides novel insights into how a master regulator can specify diverse transcriptional programs based on the chromatin landscape induced by exposure to specific morphogens.
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Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Queratinocitos/fisiología , Factores de Transcripción/fisiología , Tretinoina/farmacología , Proteínas Supresoras de Tumor/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Epidermis/efectos de los fármacos , Epidermis/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.
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Colágeno Tipo VII/genética , Colágeno Tipo VII/uso terapéutico , Epidermólisis Ampollosa Distrófica/terapia , Genes Recesivos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Animales , Secuencia de Bases , Epidermólisis Ampollosa Distrófica/genética , Predisposición Genética a la Enfermedad , Terapia Genética , Genoma Humano , Recombinación Homóloga/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Queratinocitos/patología , Ratones , Datos de Secuencia Molecular , Mutación/genética , Análisis de Secuencia de ADNRESUMEN
Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.
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Comunicación Celular/genética , Células Epiteliales/citología , Folículo Piloso/fisiología , Mesodermo/citología , Regeneración/genética , Transducción de Señal/genética , Animales , Femenino , Folículo Piloso/citología , Queratinocitos/citología , Queratinocitos/fisiología , Ratones , Ratones Desnudos , Piel/citologíaRESUMEN
Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, D(N) and D(C), and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy.
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Carcinoma Basocelular/metabolismo , Proteínas Oncogénicas/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Carcinoma Basocelular/etiología , Citoplasma/metabolismo , Regulación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Células 3T3 NIH , Proteínas Oncogénicas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/fisiología , Transactivadores , Factores de Transcripción/genética , Transfección , Xenopus , Proteína con Dedos de Zinc GLI1RESUMEN
Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.
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Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Carcinoma Basocelular/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células del Estroma/patología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Carcinoma Basocelular/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Epiteliales/citología , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Piel/citología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Sonic hedgehog (Shh) signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. Here we identify the actin-binding protein Missing in Metastasis (MIM) as a new Shh-responsive gene. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis.