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1.
Nat Commun ; 11(1): 3377, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632100

RESUMO

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.


Assuntos
Glândulas Mamárias Humanas/metabolismo , Morfogênese/genética , Mutação , Comunicação Parácrina/genética , Biomimética/métodos , Linhagem Celular , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Glândulas Mamárias Humanas/irrigação sanguínea , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Fenótipo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
2.
Nat Commun ; 11(1): 3516, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665580

RESUMO

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Feminino , Humanos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/genética
3.
Nucleic Acids Res ; 48(15): 8374-8392, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619237

RESUMO

The core-promoter, a stretch of DNA surrounding the transcription start site (TSS), is a major integration-point for regulatory-signals controlling gene-transcription. Cellular differentiation is marked by divergence in transcriptional repertoire and cell-cycling behaviour between cells of different fates. The role promoter-associated gene-regulatory-networks play in development-associated transitions in cell-cycle-dynamics is poorly understood. This study demonstrates in a vertebrate embryo, how core-promoter variations define transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. Assessment of cell proliferation across zebrafish embryo segmentation, using the FUCCI transgenic cell-cycle-phase marker, revealed a spatial and lineage-specific separation in cell-cycling behaviour. To investigate the role differential promoter usage plays in this process, cap-analysis-of-gene-expression (CAGE) was performed on cells segregated by cycling dynamics. This analysis revealed a dramatic increase in tissue-specific gene expression, concurrent with slowed cycling behaviour. We revealed a distinct sharpening in TSS utilization in genes upregulated in slowly cycling, differentiating tissues, associated with enhanced utilization of the TATA-box, in addition to Sp1 binding-sites. In contrast, genes upregulated in rapidly cycling cells carry broad distribution of TSS utilization, coupled with enrichment for the CCAAT-box. These promoter features appear to correspond to cell-cycle-dynamic rather than tissue/cell-lineage origin. Moreover, we observed genes with cell-cycle-dynamic-associated transitioning in TSS distribution and differential utilization of alternative promoters. These results demonstrate the regulatory role of core-promoters in cell-cycle-dependent transcription regulation, during embryo-development.


Assuntos
Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sítios de Ligação/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Humanos , Morfogênese/genética , Fator de Transcrição Sp1/genética , TATA Box/genética , Peixe-Zebra/genética
4.
BMC Evol Biol ; 20(1): 92, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727367

RESUMO

BACKGROUND: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classification of Gastrostyla-like species, a taxonomically difficult group of hypotrichs with a common ventral cirral pattern but various dorsal and ontogenetic patterns, is poorly understood. Hence, systematic relationships within this group and with other taxa in the subclass Hypotrichia remain unresolved. RESULTS: 18S rRNA gene sequence of a new Gastrostyla-like taxon was obtained. Phylogenetic analyses based on the 18S rRNA gene sequences indicate that this ciliate represents a new genus that is closely related to Heterourosomoida and Kleinstyla within the oxytrichid clade of the Hypotrichia. However, the position of this cluster remains unresolved. All three genera deviate from the typical oxytrichids by their incomplete (or lack of) dorsal kinety fragmentation during morphogenesis. Morphology and morphogenesis of this newly discovered form, Heterogastrostyla salina nov. gen., nov. spec., are described. Heterogastrostyla nov. gen., is characterised as follows: more than 18 fronto-ventral-transverse cirri, cirral anlagen V and VI develop pretransverse cirri, and dorsal ciliature in Urosomoida-like pattern. CONCLUSIONS: Similar to the CEUU-hypothesis about convergent evolution of urostylids and uroleptids, we speculate that the shared ventral cirral patterns of Gastrostyla-like taxa might have resulted from convergent evolution.


Assuntos
Cilióforos/classificação , Classificação , Salinidade , Solo , Animais , Sequência de Bases , Núcleo Celular/genética , DNA Ribossômico/genética , Hypotrichida/classificação , Hypotrichida/genética , Funções Verossimilhança , Morfogênese/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Especificidade da Espécie
5.
PLoS Genet ; 16(6): e1008885, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559217

RESUMO

Regulation of cell junctions is crucial for the integrity of epithelial tissues and organs. Cell junctions also play roles in controlling cell proliferation for organ growth. Translationally controlled tumor protein (TCTP) is a conserved protein involved in growth control, but its role in cell junctions is unknown. Here we show that Drosophila Tctp directly interacts with the septate junction protein Coracle (Cora) to regulate epithelial integrity and organ growth. Tctp localizes together with Cora in the epidermis of the embryo. Loss of Cora reduces the level of Tctp in the epidermis but not vice versa. cora/+ or Tctp/+ single heterozygotes develop normally to adulthood. However, double heterozygotes for cora and Tctp mutations show severe disruption of epithelia causing synthetic lethality in the embryo. Double knockdown of Cora and Tctp in eye imaginal disc synergistically leads to disruption of the eye disc, resulting in a severe reduction or loss of eye and head. Conversely, double knockdown of Cora and Tctp in wing disc causes overgrowth as well as cell death. Inhibition of cell death under this condition causes hyperplastic growth of the wing disc. Tctp also shows direct and functional interaction with Cora-associated factors like Yurt and Na+/K+-ATPase. This study suggests that proper levels of Tctp and Cora are essential for the maintenance of the Cora complex and the integrity of epithelia. Our data also provide evidence that both Cora and Tctp are required to suppress overgrowth in developing wing.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Proliferação de Células/genética , Proteínas de Drosophila/genética , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Discos Imaginais/crescimento & desenvolvimento , Junções Intercelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Morfogênese/genética , Mutações Sintéticas Letais , Asas de Animais/metabolismo
6.
PLoS One ; 15(6): e0234375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555682

RESUMO

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.


Assuntos
Rim/anormalidades , Rim/efeitos dos fármacos , Quercetina/farmacologia , beta Catenina/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Técnicas de Cultura de Órgãos , Gravidez , Vimentina/metabolismo , beta Catenina/química , beta Catenina/genética
7.
PLoS Genet ; 16(6): e1008849, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32516352

RESUMO

Cohesin, a multisubunit protein complex, is required for holding sister chromatids together during mitosis and meiosis. The recruitment of cohesin by the sister chromatid cohesion 2/4 (SCC2/4) complex has been extensively studied in Saccharomyces cerevisiae mitosis, but its role in mitosis and meiosis remains poorly understood in multicellular organisms, because complete loss-of-function of either gene causes embryonic lethality. Here, we identified a weak allele of Atscc2 (Atscc2-5) that has only minor defects in vegetative development but exhibits a significant reduction in fertility. Cytological analyses of Atscc2-5 reveal multiple meiotic phenotypes including defects in chromosomal axis formation, meiosis-specific cohesin loading, homolog pairing and synapsis, and AtSPO11-1-dependent double strand break repair. Surprisingly, even though AtSCC2 interacts with AtSCC4 in vitro and in vivo, meiosis-specific knockdown of AtSCC4 expression does not cause any meiotic defect, suggesting that the SCC2-SCC4 complex has divergent roles in mitosis and meiosis. SCC2 homologs from land plants have a unique plant homeodomain (PHD) motif not found in other species. We show that the AtSCC2 PHD domain can bind to the N terminus of histones and is required for meiosis but not mitosis. Taken together, our results provide evidence that unlike SCC2 in other organisms, SCC2 requires a functional PHD domain during meiosis in land plants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose/genética , Dedos de Zinco PHD/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Técnicas de Silenciamento de Genes , Genoma de Planta/genética , Mutação com Perda de Função , Mitose/genética , Morfogênese/genética , Mutagênese , Plantas Geneticamente Modificadas , Polinização/genética , Sequenciamento Completo do Genoma
8.
Nature ; 582(7811): 246-252, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499648

RESUMO

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Hipotálamo/embriologia , Morfogênese , Animais , Diferenciação Celular , Linhagem da Célula , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Ectoderma/citologia , Ectoderma/metabolismo , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Ácido Glutâmico/metabolismo , Hipotálamo/metabolismo , Masculino , Camundongos , Morfogênese/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Receptores Imunológicos/metabolismo , Regulon/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismo
9.
BMC Med Genet ; 21(1): 97, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32380970

RESUMO

BACKGROUND: Amelogenesis imperfecta (AI) is a highly heterogeneous group of hereditary developmental abnormalities which mainly affects the dental enamel during tooth development in terms of its thickness, structure, and composition. It appears both in syndromic as well as non-syndromic forms. In the affected individuals, the enamel is usually thin, soft, rough, brittle, pitted, chipped, and abraded, having reduced functional ability and aesthetics. It leads to severe complications in the patient, like early tooth loss, severe discomfort, pain, dental caries, chewing difficulties, and discoloration of teeth from yellow to yellowish-brown or creamy type. The study aimed to identify the disease-causing variant in a consanguineous family. METHODS: We recruited a consanguineous Pashtun family of Pakistani origin. Exome sequencing analysis was followed by Sanger sequencing to identify the pathogenic variant in this family. RESULTS: Clinical analysis revealed hypomaturation AI having generalized yellow-brown or creamy type of discoloration in affected members. We identified a novel nonsense sequence variant c.1192C > T (p.Gln398*) in exon-12 of SLC24A4 by using exome sequencing. Later, its co-segregation within the family was confirmed by Sanger sequencing. The human gene mutation database (HGMD, 2019) has a record of five pathogenic variants in SLC24A4, causing AI phenotype. CONCLUSION: This nonsense sequence variant c.1192C > T (p.Gln398*) is the sixth disease-causing variant in SLC24A4, which extends its mutation spectrum and confirms the role of this gene in the morphogenesis of human tooth enamel. The identified variant highlights the critical role of SLC24A4 in causing a rare AI type in humans.


Assuntos
Amelogênese Imperfeita/genética , Antiporters/genética , Cárie Dentária/genética , Predisposição Genética para Doença , Adulto , Amelogênese Imperfeita/epidemiologia , Amelogênese Imperfeita/patologia , Códon sem Sentido/genética , Cárie Dentária/epidemiologia , Cárie Dentária/patologia , Esmalte Dentário/metabolismo , Éxons/genética , Feminino , Humanos , Masculino , Morfogênese/genética , Paquistão/epidemiologia , Linhagem , Perda de Dente/genética , Perda de Dente/fisiopatologia , Sequenciamento Completo do Exoma , Adulto Jovem
10.
PLoS Comput Biol ; 16(4): e1007750, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251432

RESUMO

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
11.
PLoS One ; 15(4): e0231451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282855

RESUMO

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development.


Assuntos
Ecdisona/genética , Ecdisteroides/genética , Proteínas de Insetos/genética , Insetos/genética , Animais , Sistema Enzimático do Citocromo P-450/genética , Ecdisterona/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Insetos/crescimento & desenvolvimento , Hormônios Juvenis/genética , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Metamorfose Biológica/genética , Morfogênese/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , Caracteres Sexuais , Asas de Animais/crescimento & desenvolvimento
12.
Invest Ophthalmol Vis Sci ; 61(4): 9, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32293666

RESUMO

Purpose: Analysis of photoreceptor morphology and gene expression in mispatterned eyes of zebrafish growth differentiation factor 6a (gdf6a) mutants. Methods: Rod and cone photoreceptors were compared between gdf6a mutant and control zebrafish from larval to late adult stages using transgenic labels, immunofluorescence, and confocal microscopy, as well as by transmission electron microscopy. To compare transcriptomes between larval gdf6a mutant and control zebrafish, RNA-Seq was performed on isolated eyes. Results: Although rod and cone photoreceptors differentiate in gdf6a mutant zebrafish, the cells display aberrant growth and morphology. The cone outer segments, the light-detecting sensory endings, are reduced in size in the mutant larvae and fail to recover to control size at subsequent stages. In contrast, rods form temporarily expanded outer segments. The inner segments, which generate the required energy and proteins for the outer segments, are shortened in both rods and cones at all stages. RNA-Seq analysis provides a set of misregulated genes associated with the observed abnormal photoreceptor morphogenesis. Conclusions: GDF6 mutations were previously identified in patients with Leber congenital amaurosis. Here, we reveal a unique photoreceptor phenotype in the gdf6a mutant zebrafish whereby rods and cones undergo abnormal maturation distinct for each cell type. Further, subsequent development shows partial recovery of cell morphology and maintenance of the photoreceptor layer. By conducting a transcriptomic analysis of the gdf6a larval eyes, we identified a collection of genes that are candidate regulators of photoreceptor size and morphology.


Assuntos
Fator 6 de Diferenciação de Crescimento/genética , Morfogênese/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anormalidades , Animais , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Larva , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação/genética , Inclusão em Parafina , Reação em Cadeia da Polimerase em Tempo Real , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Peixe-Zebra/genética
13.
J Vis Exp ; (156)2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32116304

RESUMO

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.


Assuntos
Agrobacterium tumefaciens/genética , Genes de Plantas , Endogamia , Morfogênese/genética , Transformação Genética , Zea mays/embriologia , Zea mays/genética , DNA Bacteriano/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , Polinização , Zea mays/crescimento & desenvolvimento
14.
PLoS One ; 15(2): e0228225, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32012186

RESUMO

The Lower Rhombic Lip (LRL) is a transient neuroepithelial structure of the dorsal hindbrain, which expands from r2 to r7, and gives rise to deep nuclei of the brainstem, such as the vestibular and auditory nuclei and most posteriorly the precerebellar nuclei. Although there is information about the contribution of specific proneural-progenitor populations to specific deep nuclei, and the distinct rhombomeric contribution, little is known about how progenitor cells from the LRL behave during neurogenesis and how their transition into differentiation is regulated. In this work, we investigated the atoh1 gene regulatory network operating in the specification of LRL cells, and the kinetics of cell proliferation and behavior of atoh1a-derivatives by using complementary strategies in the zebrafish embryo. We unveiled that atoh1a is necessary and sufficient for specification of LRL cells by activating atoh1b, which worked as a differentiation gene to transition progenitor cells towards neuron differentiation in a Notch-dependent manner. This cell state transition involved the release of atoh1a-derivatives from the LRL: atoh1a progenitors contributed first to atoh1b cells, which are committed non-proliferative precursors, and to the lhx2b-neuronal lineage as demonstrated by cell fate studies and functional analyses. Using in vivo cell lineage approaches we revealed that the proliferative cell capacity, as well as the mode of division, relied on the position of the atoh1a progenitors within the dorsoventral axis. We showed that atoh1a may behave as the cell fate selector gene, whereas atoh1b functions as a neuronal differentiation gene, contributing to the lhx2b neuronal population. atoh1a-progenitor cell dynamics (cell proliferation, cell differentiation, and neuronal migration) relies on their position, demonstrating the challenges that progenitor cells face in computing positional information from a dynamic two-dimensional grid in order to generate the stereotyped neuronal structures in the embryonic hindbrain.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Metencéfalo/metabolismo , Morfogênese/genética , Rombencéfalo/crescimento & desenvolvimento , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Imageamento Tridimensional , Neurônios/citologia , Rombencéfalo/citologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
15.
Nat Commun ; 11(1): 816, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041947

RESUMO

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their G1 period. Here we demonstrate that a gradual increase in the concentration of the second messenger c-di-GMP determines precise gene expression during G1/S transition in Caulobacter crescentus. We show that c-di-GMP stimulates the kinase ShkA by binding to its central pseudo-receiver domain, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. Activation of the ShkA-dependent genetic program causes c-di-GMP to reach peak levels, which triggers S-phase entry and promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in precise control of ShkA-TacA activity, enabling G1/S-specific gene expression that coordinates cell cycle and morphogenesis.


Assuntos
Caulobacter crescentus/citologia , Caulobacter crescentus/genética , Ciclo Celular/genética , GMP Cíclico/análogos & derivados , Histidina Quinase/metabolismo , Morfogênese/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/metabolismo , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/química , Histidina Quinase/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteólise , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo
16.
Nat Commun ; 11(1): 1092, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107390

RESUMO

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, we performed an unbiased screen for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, we discovered that miR-34 mutants display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Terminações Pré-Sinápticas/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a Calmodulina/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Larva/crescimento & desenvolvimento , Morfogênese/genética , Mutação , Junção Neuromuscular/citologia , Junção Neuromuscular/crescimento & desenvolvimento
17.
Development ; 147(3)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32014865

RESUMO

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.


Assuntos
Endoderma/citologia , Transição Epitelial-Mesenquimal/fisiologia , Gastrulação/fisiologia , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem Celular , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Morfogênese/genética
18.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32005738

RESUMO

APSES-type transcription factors (TFs) have analogous and diverse functions in the regulation of fungal morphogenesis processes. However, little is known about these functions in microsclerotium formation. In this study, we characterized two orthologous APSES genes (MrStuA and MrXbp) in the entomopathogenic fungus Metarhizium rileyi Deletion of either MrStuA or MrXbp impaired dimorphic transition, conidiation, fungal virulence, and microsclerotium formation. Compared with the wild-type strain, ΔMrStuA and ΔMrXbp mutants were hypersensitive to thermal and oxidative stress. Furthermore, transcriptome sequencing analysis revealed that MrStuA and MrXbp independently regulate their own distinctive subsets of signaling pathways during dimorphic transition and microsclerotium formation, but they also show an overlapping regulation of genes during these two distinct morphogenesis processes. These results provide a global insight into vital roles of MrStuA and MrXbp in M. rileyi and aid in dissection of the interacting regulatory mechanisms of dimorphism transition and microsclerotium development.IMPORTANCE Transcription factors (TFs) are core components of the signaling pathway and play an important role in transcriptional regulation of gene expression during fungal morphogenesis processes. A prevailing theory suggests an interplay between different TFs regulating microsclerotial differentiation; however, the persisting issue remains that these interplay mechanisms are not clear. Here, we analyzed two members of the APSES-type TFs in Metarhizium rileyi using a gene deletion strategy and transcriptome analysis. Mutants were significantly impaired in microsclerotium formation and dimorphic transition. Transcriptome analysis provided evidence for interacting regulatory mechanisms by the two TFs in microsclerotium formation and dimorphic transition. Furthermore, we investigated their overlapping roles in mediating the expression of genes required for different fungal morphogenesis processes. Characterization of TFs in this study will aid in dissecting the interplay between regulatory mechanisms in fungal morphogenesis processes.


Assuntos
Proteínas Fúngicas/genética , Metarhizium/genética , Fatores de Transcrição/genética , Proteínas Fúngicas/metabolismo , Metarhizium/crescimento & desenvolvimento , Morfogênese/genética , Fatores de Transcrição/metabolismo
19.
Gene ; 731: 144338, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31923576

RESUMO

The primary feather follicles are universal skin appendages widely distributed in the skin of feathered birds. The morphogenesis and development of the primary feather follicles in goose skin remain largely unknown. Here, the induction of primary feather follicles in goose embryonic skin (pre-induction vs induction) was investigated by de novo transcriptome analyses to reveal 409 differentially expressed genes (DEGs). The DEGs were characterized to potentially regulate the de novo formation of feather follicle primordia consisting of placode (4 genes) and dermal condensate (12 genes), and the thickening of epidermis (5 genes) and dermal fibroblasts (17 genes), respectively. Further analyses enriched DEGs into GO terms represented as cell adhesion and KEGG pathways including Wnt and Hedgehog signaling pathways that are highly correlated with cell communication and molecular regulation. Six selected Wnt pathway genes were detected by qPCR with up-regulation in goose skin during the induction of primary feather follicles. The localization of WNT16, SFRP1 and FRZB by in situ hybridization showed weak expression in the primary feather primordia, whereas FZD1, LEF1 and DKK1 were expressed initially in the inter-follicular skin and feather follicle primordia, then mainly restricted in the feather primordia. The spatial-temporal expression patterns indicate that Wnt pathway genes DKK1, FZD1 and LEF1 are the important regulators functioned in the induction of primary feather follicle in goose skin. The dynamic molecular changes and specific gene expression patterns revealed in this report provide the general knowledge of primary feather follicle and skin development in waterfowl, and contribute to further understand the diversity of hair and feather development beyond the mouse and chicken models.


Assuntos
Plumas/embriologia , Gansos , Genes Controladores do Desenvolvimento , Folículo Piloso/embriologia , Morfogênese/genética , Pele/embriologia , Animais , Embrião de Galinha , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Plumas/metabolismo , Gansos/embriologia , Gansos/genética , Gansos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes Controladores do Desenvolvimento/genética , Folículo Piloso/metabolismo , Pele/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(3): 1552-1558, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31900360

RESUMO

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Elementos Facilitadores Genéticos/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Sítios de Ligação , Padronização Corporal , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Gástrula/fisiologia , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Morfogênese/genética , Morfogênese/fisiologia , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
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