Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Harvard University

The mammalian glycopeptide hormone receptors (GPHRs) are key regulators of reproductive development, and their homologs are widely distributed throughout the animal kingdom. The C. elegans genome encodes a single GPHR family member, FSHR-1, which shares equal identity to the FSH, LH, and TSH receptors from mammals.

The C. elegans glycopeptide hormone receptor ortholog, FSHR-1, regulates germline differentiation and survival.

Morphogens are long-range signaling molecules that pattern developing tissues in a concentration-dependent manner. The graded activity of morphogens within tissues exposes cells to different signal levels and leads to region-specific transcriptional responses and cell fates. In its simplest incarnation, a morphogen signal forms a gradient by diffusion from a local source and clearance in surrounding tissues. Responding cells often transduce morphogen levels in a linear fashion, which results in the graded activation of transcriptional effectors. The concentration-dependent expression of morphogen target genes is achieved by their different binding affinities for transcriptional effectors as well as inputs from other transcriptional regulators. Morphogen distribution and interpretation are the result of complex interactions between the morphogen and responding tissues. The response to a morphogen is dependent not simply on morphogen concentration but also on the duration of morphogen exposure and the state of the target cells. In this review, we describe the morphogen concept and discuss the mechanisms that underlie the generation, modulation, and interpretation of morphogen gradients.

Morphogen gradients: from generation to interpretation.

Biological systems involving short-range activators and long-range inhibitors can generate complex patterns. Reaction-diffusion models postulate that differences in signaling range are caused by differential diffusivity of inhibitor and activator. Other models suggest that differential clearance underlies different signaling ranges. To test these models, we measured the biophysical properties of the Nodal/Lefty activator/inhibitor system during zebrafish embryogenesis. Analysis of Nodal and Lefty gradients revealed that Nodals have a shorter range than Lefty proteins. Pulse-labeling analysis indicated that Nodals and Leftys have similar clearance kinetics, whereas fluorescence recovery assays revealed that Leftys have a higher effective diffusion coefficient than Nodals. These results indicate that differential diffusivity is the major determinant of the differences in Nodal/Lefty range and provide biophysical support for reaction-diffusion models of activator/inhibitor-mediated patterning.

Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system.

The graded distribution of morphogens underlies many of the tissue patterns that form during development. How morphogens disperse from a localized source and how gradients in the target tissue form has been under debate for decades. Recent imaging studies and biophysical measurements have provided evidence for various morphogen transport models ranging from passive mechanisms, such as free or hindered extracellular diffusion, to cell-based dispersal by transcytosis or cytonemes. Here, we analyze these transport models using the morphogens Nodal, fibroblast growth factor and Decapentaplegic as case studies. We propose that most of the available data support the idea that morphogen gradients form by diffusion that is hindered by tortuosity and binding to extracellular molecules.

Morphogen transport.

La distribution gradu&eacute;e des morphog&egrave;nes sous-tend bon nombre des mod&egrave;les de tissus qui se forment au cours du d&eacute;veloppement. Comment morphog&egrave;nes dispersent provenant d&#39;une source localis&eacute;e et comment d&eacute;grad&eacute;s sous la forme de tissu de cible a &eacute;t&eacute; d&eacute;battue pendant des d&eacute;cennies. Des &eacute;tudes r&eacute;centes d&#39;imagerie et mesures biophysiques ont fourni des donn&eacute;es pour les diff&eacute;rents mod&egrave;les de transport de capacit&eacute;s allant de m&eacute;canismes passifs, comme diffusion extracellulaire libre ou entrav&eacute;e, &agrave; base de cellules de la dispersion par transcytose ou cytonemes. Ici, nous analysons ces mod&egrave;les de transport utilisant les morphog&egrave;nes Nodal, facteur de croissance fibroblastique et Decapentaplegic comme &eacute;tudes de cas. Nous proposons que la plupart des donn&eacute;es disponibles a soutenir l&#39;id&eacute;e qu&#39;autocatalytiques d&eacute;grad&eacute;s forment par diffusion qui est entrav&eacute;e par la tortuosit&eacute; et liaison aux mol&eacute;cules extracellulaires.

Capacit&eacute;s transport.

La distribuzione graduata dei morfogeni &egrave; alla base di molti dei modelli di tessuto che si formano durante lo sviluppo. Come morfogeni disperdono da una sorgente localizzata e come gradienti in forma di tessuto bersaglio &egrave; stato sotto dibattito per decenni. Recenti studi di imaging e misurazioni biofisiche hanno fornito evidenze per vari modelli di trasporto morphogen che vanno da meccanismi passivi, come diffusione gratuita o intralciata extracellulare, alla dispersione basati su celle da transcytosis o cytonemes. Qui, vediamo di analizzare questi modelli di trasporto utilizzando i morfogeni Nodal, fattore di crescita dei fibroblasti e Decapentaplegic come casi di studio. Proponiamo che la maggior parte dei dati disponibili supportano l&#39;idea che formano morphogen gradienti di diffusione che &egrave; ostacolato dalla tortuosit&agrave; e associazione alle molecole extracellulari.

Katherine W. Rogers

Department of Molecular and Cellular Biology

Harvard University

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Optogenetic Signaling Activation in Zebrafish Embryos

Katherine W. Rogers

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular and Cellular Biology

Harvard University

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Optogenetic Signaling Activation in Zebrafish Embryos

Department of Molecular and Cellular Biology