Joaquín Letelier

Analysis of cellular behavior and cytoskeletal dynamics reveal a constriction mechanism driving optic cup morphogenesis

Contractile actomyosin networks have been shown to power tissue morphogenesis. Although the basic cellular machinery generating mechanical tension appears largely conserved, tensions propagate in unique ways within each tissue. Here we use the vertebrate eye as a paradigm to investigate how tensions are generated and transmitted during the folding of a neuroepithelial layer. We record membrane pulsatile behavior and actomyosin dynamics during zebrafish optic cup morphogenesis by live imaging. We show that retinal neuroblasts undergo fast oscillations and that myosin condensation correlates with episodic contractions that progressively reduce basal feet area. Interference with lamc1 function impairs basal contractility and optic cup folding. Mapping of tensile forces by laser cutting uncover a developmental window in which local ablations trigger the displacement of the entire tissue. Our work shows that optic cup morphogenesis is driven by a constriction mechanism and indicates that supra-cellular transmission of mechanical tension depends on ECM attachment. DOI: http://dx.doi.org/10.7554/eLife.15797.001

A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins

Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.The authors study the cis-regulatory evolution of the Shh locus in vertebrates. Using genomic editing and chromatin profiling, they conclude that paired fins emerged through the co-option of developmental programs for the median fins of gnathostomes.

The pigmented epithelium, a bright partner against photoreceptor degeneration

Abstract Sight depends on the intimate association between photoreceptors and pigment epithelial cells. The evolutionary origin of this cellular tandem can be traced back to the emergence of bilateral animals, at least 450 million years ago, as they define the minimal unit of the ancestral prototypic eye. Phototransduction is a demanding process from the energetic and homeostatic points of view, and not surprisingly photoreceptive cells are particularly susceptible to damage and degeneration. Here, we will examine the different ancillary roles that the pigmented cells play in the physiology and homeostasis of photoreceptors, linking each one of these processes to the most common hereditary retinal diseases. We will discuss the challenges and opportunities of recent therapeutic advances based on cell and gene replacement. The transition from animal models to clinical trials will be addressed for each one of the different therapeutic strategies with a special focus on those depending on retinal-pigmented epithelial cells. Finally, we will discuss the potential impact of combining CRISPR technologies with gene and cell therapy approaches, which – in the frame of the personalized medicine revolution – may constitute a leap forward in the treatment of retinal dystrophies.

Analysis of opo cis-regulatory landscape uncovers Vsx2 requirement in early eye morphogenesis

The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6_10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish.

Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation

Significance Evolution of organismal complexity and species diversity depends on the emergence of novel gene functions. Nevertheless, evolution rarely produces novelties from scratch but works on the weak promiscuous preexisting activities or appears by genomic tinkering. We provide evidence of how rearrangement of conserved regulatory blocks can act as a driving force for gene cooption and evolution of novel developmental mechanisms at the base of important ecological adaptations. We gain insight into a crucial system for segregation of neuronal progenitors within the hindbrain: the evolutionary origin of the actomyosin-dependent cell-sorting mechanism, with rac3b as a main effector. We unveil that the rac3b/rfng/sgca regulatory cluster—specifically expressed at boundaries—emerged by establishment of novel long-range cis-regulatory interactions, allowing the evolution of a backup regulatory mechanism for cell segregation. Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Fishing forward and reverse: Advances in zebrafish phenomics

Understanding how the genome instructs the phenotypic characteristics of an organism is one of the major scientific endeavors of our time. Advances in genetics have progressively deciphered the inheritance, identity and biological relevance of genetically encoded information, contributing to the rise of several, complementary omic disciplines. One of them is phenomics, an emergent area of biology dedicated to the systematic multi-scale analysis of phenotypic traits. This discipline provides valuable gene function information to the rapidly evolving field of genetics. Current molecular tools enable genome-wide analyses that link gene sequence to function in multi-cellular organisms, illuminating the genome-phenome relationship. Among vertebrates, zebrafish has emerged as an outstanding model organism for high-throughput phenotyping and modeling of human disorders. Advances in both systematic mutagenesis and phenotypic analyses of embryonic and post-embryonic stages in zebrafish have revealed the function of a valuable collection of genes and the general structure of several complex traits. In this review, we summarize multiple large-scale genetic efforts addressing parental, embryonic, and adult phenotyping in the zebrafish. The genetic and quantitative tools available in the zebrafish model, coupled with the broad spectrum of phenotypes that can be assayed, make it a powerful model for phenomics, well suited for the dissection of genotype-phenotype associations in development, physiology, health and disease.