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1.
Annu Rev Cell Dev Biol ; 36: 339-357, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-33021822

RESUMEN

Reconstitution is an experimental strategy that seeks to recapitulate biological events outside their natural contexts using a reduced set of components. Classically, biochemical reconstitution has been extensively applied to identify the minimal set of molecules sufficient for recreating the basic chemistry of life. By analogy, reconstitution approaches to developmental biology recapitulate aspects of developmental events outside an embryo, with the goal of revealing the basic genetic circuits or physical cues sufficient for recreating developmental decisions. The rapidly growing repertoire of genetic, molecular, microscopic, and bioengineering tools is expanding the complexity and precision of reconstitution experiments. We review the emerging field of synthetic developmental biology, with a focus on the ways in which reconstitution strategies and new biological tools have enhanced our modern understanding of fundamental questions in developmental biology.


Asunto(s)
Biología Evolutiva , Biología Sintética , Linaje de la Célula , Tamaño de la Célula , Humanos , Factores de Tiempo
2.
Proc Natl Acad Sci U S A ; 121(36): e2400677121, 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39190357

RESUMEN

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.


Asunto(s)
Proteínas Hedgehog , Transducción de Señal , Animales , Humanos , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Difusión , Proteínas Hedgehog/metabolismo , Morfogénesis , Imagen Individual de Molécula/métodos , Ratones
3.
Proc Natl Acad Sci U S A ; 116(41): 20605-20611, 2019 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-31511420

RESUMEN

Nucleosomes are the fundamental structural unit of chromatin. In addition to stabilizing the DNA polymer, nucleosomes are modified in ways that reflect and affect gene expression in their vicinity. It has long been assumed that nucleosomes can transmit memory of gene expression through their covalent posttranslational modifications. An unproven assumption of this model, which is essential to most models of epigenetic inheritance, is that a nucleosome present at a locus reoccupies the same locus after DNA replication. We tested this assumption by nucleating a synthetic chromatin domain in vivo, in which ∼4 nucleosomes at an arbitrary locus were covalently labeled with biotin. We tracked the fate of labeled nucleosomes through DNA replication, and established that nucleosomes present at a locus remembered their position during DNA replication. The replication-associated histone chaperones Dpb3 and Mcm2 were essential for nucleosome position memory, and in the absence of both Dpb3 and Mcm2 histone chaperone activity, nucleosomes did not remember their position. Using the same approach, we tested the model that transcription results in retrograde transposition of nucleosomes along a transcription unit. We found no evidence of retrograde transposition. Our results suggest that nucleosomes have the capacity to transmit epigenetic memory across mitotic generations with exquisite spatial fidelity.


Asunto(s)
Replicación del ADN , Nucleosomas/genética , Procesamiento Proteico-Postraduccional , ARN de Hongos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina , Chaperonas de Histonas , Histonas/genética , Histonas/metabolismo , ARN de Hongos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
4.
Proc Natl Acad Sci U S A ; 111(29): 10598-603, 2014 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-24994903

RESUMEN

We present the use of recently developed live imaging methods to examine the dynamic regulation of even-skipped (eve) stripe 2 expression in the precellular Drosophila embryo. Nascent transcripts were visualized via MS2 RNA stem loops. The eve stripe 2 transgene exhibits a highly dynamic pattern of de novo transcription, beginning with a broad domain of expression during nuclear cycle 12 (nc12), and progressive refinement during nc13 and nc14. The mature stripe 2 pattern is surprisingly transient, constituting just ∼15 min of the ∼90-min period of expression. Nonetheless, this dynamic transcription profile faithfully predicts the limits of the mature stripe visualized by conventional in situ detection methods. Analysis of individual transcription foci reveals intermittent bursts of de novo transcription, with duration cycles of 4-10 min. We discuss a multistate model of transcription regulation and speculate on its role in the dynamic repression of the eve stripe 2 expression pattern during development.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Transcripción Genética , Animales , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Proteínas de Homeodominio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo
5.
bioRxiv ; 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38746265

RESUMEN

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extra-cellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, and morphogens can only overcome the barrier by passing through a membrane-unconfined state. Under this model, SCUBE1 promotes Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and discovered novel knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.

6.
Science ; 355(6330): 1184-1187, 2017 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-28302853

RESUMEN

In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility in old cells. This process is thought to reflect a decrease in the activity of the NAD+ (oxidized nicotinamide adenine dinucleotide)-dependent deacetylase Sir2. We tested whether Sir2 becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the heterochromatic HML and HMR loci was not lost during aging. Old cells could initiate a mating response; however, they were less sensitive to mating pheromone than were young cells because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old cells. We propose that aging phenotypes previously attributed to loss of heterochromatin silencing are instead caused by aggregation of the Whi3 cell cycle regulator.


Asunto(s)
Senescencia Celular/fisiología , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genes del Tipo Sexual de los Hongos , Heterocromatina/metabolismo , Agregado de Proteínas , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Senescencia Celular/genética , Péptidos/genética , Péptidos/fisiología , Dominios Proteicos/genética , Dominios Proteicos/fisiología , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Puntos de Control de la Fase S del Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo
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