generating-retinal-injury-models-in-xenopus-tadpoles

Generating Retinal Injury Models in <em>Xenopus</em> Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

Université Paris-Saclay

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of  (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions  We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate  and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

Wnt proteins form a family of highly conserved secreted molecules that are critical mediators of cell-cell signaling during embryogenesis. Partial data on Wnt activity in different tissues and at different stages have been reported in frog embryos. Our objective here is to provide a coherent and detailed description of Wnt activity throughout embryo development. Using a transgenic Xenopus tropicalis line carrying a Wnt-responsive reporter sequence, we depict the spatial and temporal dynamics of canonical Wnt activity during embryogenesis. We provide a comprehensive series of in situ hybridization in whole-mount embryos and in cross-sections, from gastrula to tadpole stages, with special focus on neural tube, retina and neural crest cell development. This collection of patterns will thus constitute a valuable resource for developmental biologists to picture the dynamics of Wnt activity during development.

An atlas of Wnt activity during embryogenesis in Xenopus tropicalis.

A hallmark of Wnt/β-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in β-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/β-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/β-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.

Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/β-Catenin response driving Spemann organizer formation.

Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones.  is the most frequently mutated gene in this disease. We here developed  gene editing-based models of retinitis pigmentosa in two  species,  and , by using CRISPR/Cas9 technology. In both of them, loss of  function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in  and . While a significant proportion of Müller cells re-enter into the cell cycle in , their proliferation remains extremely limited in . This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.

Article	Year
PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation. Development (Cambridge, England)\| PubMed ID: 29038306	2017
A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates. PLoS biology\| PubMed ID: 29049289	2017
An atlas of Wnt activity during embryogenesis in Xenopus tropicalis. PloS one\| PubMed ID: 29672592	2018
Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/β-Catenin response driving Spemann organizer formation. Development (Cambridge, England)\| PubMed ID: 31036545	2019
CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between and . Cells\| PubMed ID: 35269429	2022

Caroline Borday

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