RESUMO
Reaction-diffusion models have been used as a paradigm for describing the de novo emergence of biological patterns such as stripes and spots. In many organisms, these initial patterns are typically refined and elaborated over the subsequent course of development. Here we study the formation of secondary hair follicle patterns in the skin of developing mouse embryos. We used the expression of sex-determining region Y box 2 to identify and distinguish the primary and secondary hair follicles and to infer the spatiotemporal dynamics of the follicle formation process. Quantitative analysis of the specific follicle patterns observed reveals a simple geometrical rule governing the formation of secondary follicles, and motivates an expansion-induction (EI) model in which new follicle formation is driven by the physical growth of the embryo. The EI model requires only one diffusible morphogen and provides quantitative, accurate predictions on the relative positions and timing of secondary follicle formation, using only the observed configuration of primary follicles as input. The same model accurately describes the positions of additional follicles that emerge from skin explants treated with an activator. Thus, the EI model provides a simple and robust mechanism for predicting secondary space-filling patterns in growing embryos.
Assuntos
Folículo Piloso/embriologia , Modelos Biológicos , Morfogênese/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal , Proteínas de Transporte/metabolismo , Simulação por Computador , Galactosídeos , Técnicas Histológicas , Indóis , CamundongosRESUMO
Primary human hepatocytes (PHHs) are widely used as an in vitro model to evaluate various aspects of human hepatic physiology and pathology. However, PHHs isolated from the human liver have very limited ability for ex vivo expansion in culture. Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice are proven to be an ideal bioincubator for repopulation of PHHs. The human liver chimeric FRG mouse is not only a humanized animal model for disease study and drug screening in vivo, but also a potential source of PHHs for cellular therapy. This chapter describes experimental protocols to generate chimeric FRG mice with humanized liver and to isolate PHHs from human liver chimeric FRG mice. Using these methods, PHHs can be expanded to more than 100-fold for harvesting.
Assuntos
Hepatócitos , Fígado , Animais , Quimera , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
BACKGROUND AND AIMS: Wilson's disease (WD) is an inherited disorder of copper metabolism with predominant hepatic manifestations. Left untreated, it can be fatal. Current therapies focus on treating copper overload rather than targeting the pathophysiology of copper-induced liver injuries. We sought to investigate whether liposome-encapsulated curcumin (LEC) could attenuate the underlying pathophysiology of WD in a mouse model of WD. APPROACH AND RESULTS: Subcutaneous administration in a WD mouse model with ATP7B knockout (Atp7b-/-) resulted in robust delivery of LEC to the liver as determined by in-vitro and in-vivo imaging. Treatment with LEC attenuated hepatic injuries, restored lipid metabolism and decreased hepatic inflammation and fibrosis, and thus hepatosplenomegaly in Atp7b-/- mice. Mechanistically, LEC decreased hepatic immune cell and macrophage infiltration and attenuated the hepatic up-regulation of p65 by preventing cellular translocation of high-mobility group box-1 (HMGB-1). Moreover, decreased translocation of HMGB1 was associated with reduced splenic CD11b+/CD43+/Ly6CHi inflammatory monocyte expansion and circulating level of proinflammatory cytokines. Nevertheless there was no change in expression of oxidative stress-related genes or significant copper chelation effect of LEC in Atp7b-/- mice. CONCLUSION: Our results indicate that treatment with subcutaneous LEC can attenuate copper-induced liver injury in an animal model of WD via suppression of HMGB1-mediated hepatic and systemic inflammation. These findings provide important proof-of-principle data to develop LEC as a novel therapy for WD as well as other inflammatory liver diseases.
Assuntos
Curcumina , Proteína HMGB1 , Degeneração Hepatolenticular , Adenosina Trifosfatases/metabolismo , Animais , Cobre/metabolismo , Curcumina/metabolismo , Curcumina/farmacologia , Modelos Animais de Doenças , Fibrose , Proteína HMGB1/metabolismo , Degeneração Hepatolenticular/tratamento farmacológico , Degeneração Hepatolenticular/genética , Degeneração Hepatolenticular/metabolismo , Inflamação/metabolismo , Lipossomos , Fígado/metabolismo , CamundongosRESUMO
BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD. METHODS: We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG). RESULTS: We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity. CONCLUSIONS: Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders. LAY SUMMARY: Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.
RESUMO
In animal retina, hedgehog expression drives waves of neurogenesis, but genetic programs that control its expression during retinal neurogenesis are poorly elucidated. We have previously reported that irx1a is required for propagation of the sonic hedgehog (shh) expression waves in developing zebrafish retina. Here, we found that irx2a is expressed in the developing retina and that knockdown of irx2a results in a retinal phenotype strikingly similar to that of irx1a morphants. The expression of irx2a in retina ganglion cells was shown to be irx1a- and ath5-dependent suggesting that irx1a and ath5 are transcriptional regulators of irx2a. Furthermore, irx2a expression could rescue impaired propagation of shh waves in irx1a morphants. Together, these observations suggest that Irx2 functions downstream of irx1a to control shh expression in the retina. We proposed a novel transcriptional cascade of ath5-irx1a-irx2a in the regulation of hedgehog waves during vertebrate retinal development.
Assuntos
Proteínas de Homeodomínio/metabolismo , Retina/embriologia , Células Ganglionares da Retina/citologia , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Transcriptional programs guide the specification of neural cell types in the developing nervous system. However, it is unclear whether such programs also control specific aspects of neural circuit function at maturity. In the mammalian retina, Vsx1 and Irx5 transcription factors are present in a subset of bipolar interneurons that convey signals from photoreceptors to ganglion cells. The biased expression of Vsx1 and Irx5 in hyperpolarizing OFF compared with depolarizing ON bipolar cells suggests that these transcription factors may selectively regulate signal processing in OFF circuits. To test this hypothesis, we generated mice lacking both Vsx1 and Irx5. Bipolar cells in these mice were morphologically normal, but the expression of cell-specific markers in some OFF but not ON bipolar cells was reduced or absent. To assess visual function in Vsx1(-/-)Irx5(-/-) retinas, we recorded light responses from ensembles of retinal ganglion cells (RGCs). We first identified functional RGC types in control mice and describe their response properties and adaptation to temporal contrast using a simple linear-nonlinear model. We found that space-time receptive fields of RGCs are unchanged in Vsx1(-/-)Irx5(-/-) mice compared with control retinas. In contrast, response threshold, gain, and range were lowered in a cell-type-specific manner in OFF but not ON RGCs in Vsx1(-/-)Irx5(-/-) retinas. Finally, we discovered that the ability to adapt to temporal contrast is greatly reduced in OFF RGCs in the double mutant, suggesting that Vsx1 and Irx5 control specific aspects of visual function in circuits of the mammalian retina.
Assuntos
Sensibilidades de Contraste/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Rede Nervosa/fisiologia , Retina/fisiologia , Fatores de Transcrição/genética , Animais , Células Cultivadas , Sensibilidades de Contraste/fisiologia , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estimulação Luminosa/métodos , Fatores de Transcrição/fisiologiaRESUMO
Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants. Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos. Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina, disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia and visual impairment in cadmium-treated embryos.
Assuntos
Cádmio/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/embriologia , Peixe-Zebra/embriologia , Animais , Retina/citologiaRESUMO
Neurogenesis in the compound eyes of Drosophila and the camera eyes of vertebrates spreads in a wave-like fashion. In both phyla, waves of hedgehog expression are known to drive the wave of neuronal differentiation. The mechanism controlling the propagation of hedgehog expression during retinogenesis of the vertebrate eye is poorly understood. The Iroquois homeobox genes play important roles in Drosophila eye development; they are required for the up-regulation of hedgehog expression during propagation of the morphogenetic furrow. Here, we show that the zebrafish Iroquois homolog irx1a is expressed during retinogenesis and knockdown of irx1a results in a retinal phenotype strikingly similar to those of sonic hedgehog (shh) mutants. Analysis of shh-GFP transgene expression in irx1a knockdown retinas revealed that irx1a is required for the propagation of shh expression through the retina. Transplantation experiments illustrated that the effects of irx1a on shh expression are both cell-autonomous and non-cell-autonomous. Our results reveal a role for Iroquois genes in controlling hedgehog expression during vertebrate retinogenesis.
Assuntos
Olho/embriologia , Proteínas de Homeodomínio/genética , Retina/embriologia , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Oftalmopatias/genética , Oftalmopatias/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Retina/citologia , Retina/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Iroquois homeobox (Irx) genes have been implicated in the specification and patterning of several organs in Drosophila and several vertebrate species. Misexpression studies of chick, Xenopus, and zebra fish embryos have demonstrated that Irx genes are involved in the specification of the midbrain-hindbrain boundary. All six murine Irx genes are expressed in the developing heart, suggesting that they might possess distinct functions during heart development, and a role for Irx4 in normal heart development has been recently demonstrated by gene-targeting experiments. Here we describe the generation and phenotypic analysis of an Irx2-deficient mouse strain. By targeted insertion of a lacZ reporter gene into the Irx2 locus, we show that lacZ expression reproduces most of the endogenous Irx2 expression pattern. Despite the dynamic expression of Irx2 in the developing heart, nervous system, and other organs, Irx2-deficient mice are viable, are fertile, and appear to be normal. Although chick Irx2 has been implicated in the development of the midbrain-hindbrain region, we show that Irx2-deficient mice develop a normal midbrain-hindbrain boundary. Furthermore, Irx2-deficient mice have normal cardiac morphology and function. Functional compensation by other Irx genes might account for the absence of a phenotype in Irx2-deficient mice. Further studies of mutant mice of other Irx genes as well as compound mutant mice will be necessary to uncover the functional roles of these evolutionarily conserved transcriptional regulators in development and disease.
Assuntos
Coração/embriologia , Proteínas de Homeodomínio/fisiologia , Mesencéfalo/embriologia , Rombencéfalo/embriologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Padronização Corporal/genética , Padronização Corporal/fisiologia , DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Desenvolvimento Embrionário e Fetal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Reporter , Proteínas de Homeodomínio/genética , Hibridização In Situ , Óperon Lac , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Serotonergic (5HT) neurons produce neurotransmitter serotonin, which modulates various neuronal circuits. The specification and differentiation of 5HT neurons require both extrinsic signals such as Shh and Fgf, as well as intrinsic transcription factors such as nkx2.2, mash1, phox2b, Gata2, and pet1. In this study, we show that iroquois homeodomain factor irx1a, but not irx1b, is expressed in the 5HT neurons. Knockdown of irx1a by antisense morpholino nucleotides reveals that it is a critical determinant for the differentiation of 5HT neurons in the hindbrain. However, irx1a morphants do not show a reduction of the progenitors of 5HT neurons. Hence, irx1a is not required for the initial specification but it is required for the complete differentiation of 5HT neurons.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Laranja de Acridina/farmacologia , Animais , Apoptose , Biologia do Desenvolvimento/métodos , Corantes Fluorescentes/farmacologia , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Modelos Biológicos , Neurônios/metabolismo , Neurotransmissores/metabolismo , Serotonina/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways (Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells.
Assuntos
Diferenciação Celular/genética , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/fisiologia , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/fisiologia , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Neuroglia/fisiologia , Fatores de Transcrição/genéticaRESUMO
Rhythmic cardiac contractions depend on the organized propagation of depolarizing and repolarizing wavefronts. Repolarization is spatially heterogeneous and depends largely on gradients of potassium currents. Gradient disruption in heart disease may underlie susceptibility to fatal arrhythmias, but it is not known how this gradient is established. We show that, in mice lacking the homeodomain transcription factor Irx5, the cardiac repolarization gradient is abolished due to increased Kv4.2 potassium-channel expression in endocardial myocardium, resulting in a selective increase of the major cardiac repolarization current, I(to,f), and increased susceptibility to arrhythmias. Myocardial Irx5 is expressed in a gradient opposite that of Kv4.2, and Irx5 represses Kv4.2 expression by recruiting mBop, a cardiac transcriptional repressor. Thus, an Irx5 repressor gradient negatively regulates potassium-channel-gene expression in the heart, forming an inverse I(to,f) gradient that ensures coordinated cardiac repolarization while also preventing arrhythmias.