RESUMEN
First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.
Asunto(s)
Anticuerpos Monoclonales/genética , Sistemas de Liberación de Medicamentos/métodos , Inmunización Pasiva/métodos , ARN Mensajero/administración & dosificación , ARN Mensajero/uso terapéutico , Animales , Enfermedades Transmisibles/terapia , Composición de Medicamentos/métodos , Humanos , Lípidos/química , Nanopartículas/química , Neoplasias/terapia , Toxinas Biológicas/inmunologíaRESUMEN
While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.
Asunto(s)
Inmunización Pasiva , ARN Mensajero/metabolismo , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Inmunoterapia Adoptiva , Caperuzas de ARN/química , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/genéticaRESUMEN
Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.
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Expresión Génica , Terapia Genética , ARN Mensajero/genética , Animales , Línea Celular , Índices de Eritrocitos , Eritropoyetina/sangre , Eritropoyetina/genética , Eritropoyetina/metabolismo , Genes Reporteros , Terapia Genética/métodos , Humanos , Lípidos/química , Macaca fascicularis , Ratones , Modelos Animales , Nanopartículas/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Mutación Silenciosa , Sus scrofaRESUMEN
Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA vaccines (RNActive® technology) with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by formulation into lipid nanoparticles. Methods described here include the synthesis, purification, and formulation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.
Asunto(s)
Vacunas de ARNm , Animales , Ratones , Humanos , ARN Mensajero/genética , ARN Mensajero/inmunología , Nanopartículas/química , Inmunogenicidad Vacunal , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/genética , Estabilidad del ARN , SARS-CoV-2/inmunología , SARS-CoV-2/genética , LiposomasRESUMEN
mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein expression, mRNA vaccines are capable of inducing a balanced immune response comprising both cellular and humoral immunity while not subject to MHC haplotype restriction. In addition, mRNA is an intrinsically safe vector as it is a minimal and only transient carrier of information that does not interact with the genome. Because any protein can be expressed from mRNA without the need to adjust the production process, mRNA vaccines also offer maximum flexibility with respect to development. Taken together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology platform. Here, we outline the current knowledge regarding different aspects that should be considered when developing an mRNA-based vaccine technology.
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Adyuvantes Inmunológicos , ARN Mensajero/inmunología , Vacunas Sintéticas/inmunología , Animales , Transporte Biológico , Expresión Génica , Humanos , ARN Mensajero/administración & dosificación , ARN Mensajero/metabolismoRESUMEN
Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.
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Endocitosis , Lisosomas/metabolismo , ARN Mensajero/metabolismo , Transporte Biológico , Carbocianinas , Caveolas/metabolismo , Células HEK293 , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Microscopía Fluorescente , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , Receptores Depuradores/metabolismoRESUMEN
Hereditary spastic paraplegia type 5 is a neurodegenerative disease caused by loss-of-function mutations in the CYP7B1 gene encoding the oxysterol 7-α-hydroxylase involved in bile acid synthesis in the liver. Lack of CYP7B1 leads to an accumulation of its oxysterol substrates, in particular 25-hydroxycholesterol and 27-hydroxycholesterol that are able to cross the blood-brain barrier and have neurotoxic properties. A potential therapeutic strategy for SPG5 is the replacement of CYP7B1 by administration of mRNA. Here, we studied the intravenous application of formulated mouse and human CYP7B1 mRNA in mice lacking the endogenous Cyp7b1 gene. A single-dose injection of either mouse or human CYP7B1 mRNA led to a pronounced degradation of oxysterols in liver and serum within 2 days of treatment. Pharmacokinetics indicate a single injection of human CYP7B1 mRNA to be effective in reducing oxysterols for at least 5 days. Repetitive applications of mRNA were safe for at least 17 days and resulted in a significant reduction of neurotoxic oxysterols not only in liver and serum but also to some extent in the brain. Our study highlights the potential to use mRNA as a novel therapy to treat patients with SPG5 disease.
RESUMEN
Murine hair follicle morphogenesis gives rise to four distinct pelage follicle types that produce hair shafts differing in length, the number of medulla columns, and the presence and number of bends. Recently, Igfbp5 was identified as the first molecular marker that distinguishes among different hair follicle types and shown to mark zigzag hairs. Further, it was demonstrated that Igfbp5 expression is modulated by FGF signalling. Here, we identify Krox20 as a molecular marker whose expression in the proximal follicle appears to be restricted to zigzag hair follicles. Gene transcription occurs in precursors and early differentiating cells of the medulla. Spatial and temporal expression of Krox20 and Igfbp5 seem to be tightly co-regulated in wildtype follicles. This correlation also holds in transgenic mice in which Igfbp5 expression is disturbed. Inspection of the Igfbp5 promoter reveals several putative binding sites for KROX20. In transfection studies, KROX20 strongly stimulates transcription from a 1.5kb Igfbp5 promoter fragment which is significantly reduced by site-directed mutagenesis of putative KROX20 binding sites, indicating a potential role of KROX20 in activating Igfbp5 expression. Our data suggest Krox20 as a nodal point of FGF and IGF signalling pathways controlling Igfbp5 expression which is associated with hair shaft differentiation and may generate the periodicity of the zigzag hair.
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Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Cabello/fisiología , Animales , Secuencia de Bases , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Expresión Génica , Heterogeneidad Genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras GenéticasRESUMEN
Developing effective mRNA vaccines poses certain challenges concerning mRNA stability and ability to induce sufficient immune stimulation and requires a specific panel of techniques for production and testing. Here, we describe the production of stabilized mRNA with enhanced immunogenicity, generated using conventional nucleotides only, by introducing changes to the mRNA sequence and by complexation with the nucleotide-binding peptide protamine (RNActive® technology). Methods described here include the synthesis, purification, and protamine complexation of mRNA vaccines as well as a comprehensive panel of in vitro and in vivo methods for evaluation of vaccine quality and immunogenicity.
Asunto(s)
Inmunogenicidad Vacunal/inmunología , ARN Mensajero/inmunología , Vacunas/inmunología , Animales , RatonesRESUMEN
The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.
Asunto(s)
Antitoxina Botulínica/inmunología , Botulismo/prevención & control , Inmunización Pasiva/métodos , Profilaxis Posexposición , ARN Mensajero/administración & dosificación , Vacunas Antirrábicas/inmunología , Rabia/prevención & control , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Antitoxina Botulínica/administración & dosificación , Antitoxina Botulínica/sangre , Botulismo/terapia , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas , ARN Mensajero/genética , ARN Mensajero/inmunología , Rabia/terapia , Vacunas Antirrábicas/administración & dosificación , Vacunas Antirrábicas/sangre , Virus de la Rabia/inmunologíaRESUMEN
The murine hair coat consists of four different hair types that are characterised by hair length, the number of medulla columns, and the presence and number of bends. The molecular mechanisms underlying the establishment and maintenance of distinct hair follicle fates are unknown. We identify Igfbp5 as the first molecular marker that distinguishes among different hair follicle types. High-resolution expression analysis revealed that its expression in the medulla of hair shafts is associated with the bend-forming zones of zigzag hairs. To directly examine the functional importance of segmental gene expression in the hair follicle, we have generated transgenic mice expressing Igfbp5 in differentiating keratinocytes of the medulla and inner root sheath. Ectopic expression of Igfbp5 resulted in the appearance of remarkable curvatures and thinning of hair shafts, two hallmarks of hair bends. Both effects and the natural bending process are under negative control of IGF signalling. Thus, our data identify Igfbp5 as a central regulator of hair shaft differentiation and hair type determination.
Asunto(s)
Cabello/crecimiento & desarrollo , Cabello/metabolismo , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Animales , Secuencia de Bases , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Marcadores Genéticos , Cabello/anomalías , Cabello/anatomía & histología , Folículo Piloso/anomalías , Folículo Piloso/anatomía & histología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Mutación , Transducción de SeñalRESUMEN
Mesenchymal-epithelial signalling between the dermal papilla and the hair matrix regulates cell proliferation and differentiation in mature hair follicles. The molecular basis of these interactions is largely unexplored. According to its expression in the dermal papilla, IGF-I is likely involved in reciprocal signalling. To examine its biological function in pelage follicles further, we generated transgenic mice that express Igf-I in the inner root sheath and the medulla using an involucrin promoter fragment. We demonstrate that Igf-I affects follicular proliferation, tissue remodelling, and the hair growth cycle, as well as folliclular differentiation. Transgenic skin temporarily lacks visible adipose tissue in telogen. The onset of the second, aberrant growth phase is markedly retarded. Transgenic guard hairs are significantly elongated and a small fraction of hair follicles is severely disoriented. The microscopic appearance of most hair shafts is altered and, strikingly, Igf-I transgenic mice lack hairs with a zigzag shape due to the suppression of hair shaft bending. All transgenic effects are partially compensated by ectopic expression of Igfbp3. Finally, Pdgfralpha was identified as the first molecular target that is affected in Igf-I transgenic mice. In summary, our data identify IGF-I signalling as an important mitogenic and morphogenetic regulator in hair follicle biology.
Asunto(s)
Folículo Piloso/crecimiento & desarrollo , Cabello/crecimiento & desarrollo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Animales , Diferenciación Celular/genética , Proliferación Celular , Cabello/anomalías , Cabello/citología , Folículo Piloso/anomalías , Folículo Piloso/citología , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de SeñalRESUMEN
Hair follicles display a unique pattern of cyclic growth and regression involving cell proliferation, differentiation and migration. The molecular details of these processes are largely unexplored. Global expression analyses on the basis of about 20,000 genes for each morphologically distinguishable stage of the hair cycle revealed unexpected complexities of and major temporal shifts in transcriptional programs involving about 13% of all genes. In particular, hundreds of genes characterise the pattern of genomic activity during regression and resting phases; selected genes can be used to monitor hair growth in mice. We demonstrate that temporal expression patterns predict gene expression domains within the hair follicle. Expression of insulin-like growth factor binding proteins and anti-angiogenic factors is associated with the regression phase of the hair cycle.
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Expresión Génica/fisiología , Folículo Piloso/metabolismo , Inductores de la Angiogénesis/metabolismo , Animales , Diferenciación Celular/fisiología , Biología Computacional , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal/fisiología , Somatomedinas/metabolismo , Factores de TiempoRESUMEN
Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.
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Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , ARN/administración & dosificación , ARN/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunología , Humanos , Receptor Toll-Like 7/inmunología , Vacunación/métodosRESUMEN
Despite substantial improvements, influenza vaccine production-and availability-remain suboptimal. Influenza vaccines based on mRNA may offer a solution as sequence-matched, clinical-grade material could be produced reliably and rapidly in a scalable process, allowing quick response to the emergence of pandemic strains. Here we show that mRNA vaccines induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice and that the vaccine remains protective upon thermal stress. This vaccine format elicits B and T cell-dependent protection and targets multiple antigens, including the highly conserved viral nucleoprotein, indicating its usefulness as a cross-protective vaccine. In ferrets and pigs, mRNA vaccines induce immunological correlates of protection and protective effects similar to those of a licensed influenza vaccine in pigs. Thus, mRNA vaccines could address substantial medical need in the area of influenza prophylaxis and the broader realm of anti-infective vaccinology.
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Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Envejecimiento/inmunología , Animales , Animales Recién Nacidos , Linfocitos B/inmunología , Biotecnología , Protección Cruzada , Femenino , Hurones , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , ARN Viral/genética , ARN Viral/inmunología , Ratas , Ratas Endogámicas Lew , Sus scrofa , Linfocitos T/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The hair follicle attracted significant attention as a model for the investigation of diverse biological problems. Whereas its morphology and the structure of the hair shaft are known in detail, the molecular biology of this miniorgan is significantly less characterised. Many efforts focussed on the development of the hair follicle and its stem cell reservoir; by contrast, the follicular product, the hair, which is interesting not only in terms of cosmetics was neglected. This review highlights our current knowledge of the control of hair structure and shape with emphasis on mouse hair follicle biology and discusses continuing problems.
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Folículo Piloso/anatomía & histología , Folículo Piloso/fisiología , Cabello/fisiología , Animales , Predicción , Cabello/citología , Ratones , Modelos BiológicosRESUMEN
Canonical WNT signals play an important role in hair follicle development. In addition to being crucial for epidermal appendage initiation, they control the interfollicular spacing pattern and contribute to the spatial orientation and largely parallel alignment of hair follicles. However, owing to the complexity of canonical WNT signalling and its interconnections with other pathways, many details of hair follicle formation await further clarification. Here, we discuss the recently suggested reaction-diffusion (RD) mechanism of spatial hair follicle arrangement in the light of yet unpublished data and conclusions. They clearly demonstrate that the observed hair follicle clustering in dickkopf (DKK) transgenic mice cannot be explained by any trivial process caused by protein overexpression, thereby further supporting our model of hair follicle spacing. Furthermore, we suggest future experiments to challenge the RD model of spatial follicle arrangement.
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Folículo Piloso/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Folículo Piloso/citología , Humanos , Ratones , Ratones Transgénicos , Proteínas Wnt/genéticaRESUMEN
Axial patterning is a recurrent theme during embryonic development. To elucidate its fundamental principles, the hair follicle is an attractive model due to its easy accessibility and dispensability. Hair follicle asymmetry is evident from its angling and the localization of associated structures. However, axial patterning is not restricted to the follicle itself but also generates rotational hair shaft asymmetry which, for zigzag hairs, generates 3-4 bends that alternately point into opposite directions. Here we show by analyzing mutant and transgenic mice that WNT and ectodysplasin signaling are involved in the control of the molecular and morphological asymmetry of the follicle and the associated hair shaft, respectively. Asymmetry is affected by polarized WNT and ectodysplasin signaling in mature hair follicles. When endogenous signaling is impaired, molecular asymmetry is lost and mice no longer form zigzag hairs. Both signaling pathways affect the polarized expression of Shh which likely functions as a directional reference for hair shaft production in all follicles. We propose that this regulatory pathway also establishes follicular asymmetry during morphogenesis. Moreover, the identified molecular hierarchy offers a model for the periodic patterning of zigzag hairs mechanistically similar to mesodermal segmentation.
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Tipificación del Cuerpo/fisiología , Ectodisplasinas/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Folículo Piloso/embriología , Proteínas Hedgehog/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Cartilla de ADN , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in Situ , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , beta-Galactosidasa/metabolismoRESUMEN
Mathematical reaction-diffusion models have been suggested to describe formation of animal pigmentation patterns and distribution of epidermal appendages. However, the crucial signals and in vivo mechanisms are still elusive. Here we identify WNT and its inhibitor DKK as primary determinants of murine hair follicle spacing, using a combined experimental and computational modeling approach. Transgenic DKK overexpression reduces overall appendage density. Moderate suppression of endogenous WNT signaling forces follicles to form clusters during an otherwise normal morphogenetic program. These results confirm predictions of a WNT/DKK-specific mathematical model and provide in vivo corroboration of the reaction-diffusion mechanism for epidermal appendage formation.