Urotensin II-related peptides, Urp1 and Urp2, control zebrafish spine morphology.

The spine provides structure and support to the body, yet how it develops its characteristic morphology as the organism grows is little understood. This is underscored by the commonality of conditions in which the spine curves abnormally such as scoliosis, kyphosis, and lordosis. Understanding the origin of these spinal curves has been challenging in part due to the lack of appropriate animal models. Recently, zebrafish have emerged as promising tools with which to understand the origin of spinal curves. Using zebrafish, we demonstrate that the urotensin II-related peptides (URPs), Urp1 and Urp2, are essential for maintaining spine morphology. Urp1 and Urp2 are 10-amino acid cyclic peptides expressed by neurons lining the central canal of the spinal cord. Upon combined genetic loss of Urp1 and Urp2, adolescent-onset planar curves manifested in the caudal region of the spine. Highly similar curves were caused by mutation of Uts2r3, an URP receptor. Quantitative comparisons revealed that urotensin-associated curves were distinct from other zebrafish spinal curve mutants in curve position and direction. Last, we found that the Reissner fiber, a proteinaceous thread that sits in the central canal and has been implicated in the control of spine morphology, breaks down prior to curve formation in mutants with perturbed cilia motility but was unaffected by loss of Uts2r3. This suggests a Reissner fiber-independent mechanism of curvature in urotensin-deficient mutants. Overall, our results show that Urp1 and Urp2 control zebrafish spine morphology and establish new animal models of spine deformity.

The backbone, or spine, is an integral part of the human body, providing support to our torsos so that we can sit, stand, bend and twist. If this structure does not form correctly, it can lead to pain, neurologic problems, and mobility issues. The spine normally has curves, but these can become deformed for many reasons, including genetic and muscular factors. There are also cases in which the cause of a spine distortion is unknown, such as in scoliosis (where the spine twists to the side), lordosis (where the lower part of the spine curves excessively), and kyphosis (where the upper part of the spine shows extreme curvature). The structure of the spine is laid out during embryonic development and maintained throughout life. Experiments in zebrafish have shown that a crucial element in preserving the shape of the spine is the flow of cerebrospinal fluid or CSF. Propelled by the movement of little 'hairs' at the surface of specialized cells, this liquid runs through our central nervous system along a cavity lined with neurons. These nerve cells produce Urp1 and Urp2, two short molecules (or peptides) built from the same components as proteins. In zebrafish embryos, lowering the levels of these peptides had previously been shown to cause early body deformities. But what role, if any, do Urp1 and Urp2 play in maintaining the shape of the spine in adult zebrafish? Bearce et al. set out to answer this question. First, they generated mutant zebrafish which did not carry either Urp1, Urp2 or both peptides. Contrary to previous findings, all three of these mutants developed normally as embryos. Once they were adults, zebrafish lacking Urp1 exhibited normal spines, while those lacking Urp2 had slightly deformed curves. However, zebrafish lacking both peptides had prominent curves in the tail-region of their spines, somewhat akin to lordosis in humans. This indicates that both peptides are necessary for adult spine structure, but work in a semi-redundant manner. Interestingly, the defects observed first appeared in adolescent fish and gradually worsened as they grew; many forms of human spinal abnormalities follow a similar trajectory. Bearce et al. also tested the role of the protein Uts2r3, a receptor for peptides which belong to the urotensin family (such as Urp1 and Urp2). Fish lacking this protein showed normal spine structure as embryos, but distorted spinal curves as adults, suggesting that Urp1 and Urp2 might control spine morphology by signaling via the Uts2r3 receptor. Together, Bearce et al.'s observations show that disturbing urotensin signaling leads to a lordosis-like condition in adult zebrafish, with evident deformities in the tail-region of the spine. Considering the broad similarities in structures between the zebrafish and the human spine, these results point to a possible involvement of urotensin signaling in spine distortion in humans. More studies using zebrafish will likely provide further insights into the principles that control the shape of the spine and what goes wrong when it breaks down.

Biallelic DAW1 variants cause a motile ciliopathy characterized by laterality defects and subtle ciliary beating abnormalities.

PURPOSE: The clinical spectrum of motile ciliopathies includes laterality defects, hydrocephalus, and infertility as well as primary ciliary dyskinesia when impaired mucociliary clearance results in otosinopulmonary disease. Importantly, approximately 30% of patients with primary ciliary dyskinesia lack a genetic diagnosis. METHODS: Clinical, genomic, biochemical, and functional studies were performed alongside in vivo modeling of DAW1 variants. RESULTS: In this study, we identified biallelic DAW1 variants associated with laterality defects and respiratory symptoms compatible with motile cilia dysfunction. In early mouse embryos, we showed that Daw1 expression is limited to distal, motile ciliated cells of the node, consistent with a role in left-right patterning. daw1 mutant zebrafish exhibited reduced cilia motility and left-right patterning defects, including cardiac looping abnormalities. Importantly, these defects were rescued by wild-type, but not mutant daw1, gene expression. In addition, pathogenic DAW1 missense variants displayed reduced protein stability, whereas DAW1 loss-of-function was associated with distal type 2 outer dynein arm assembly defects involving axonemal respiratory cilia proteins, explaining the reduced cilia-induced fluid flow in particle tracking velocimetry experiments. CONCLUSION: Our data define biallelic DAW1 variants as a cause of human motile ciliopathy and determine that the disease mechanism involves motile cilia dysfunction, explaining the ciliary beating defects observed in affected individuals.

Daw1 regulates the timely onset of cilia motility during development.

Motile cilia generate cell propulsion and extracellular fluid flows that are crucial for airway clearance, fertility and left-right patterning. Motility is powered by dynein arm complexes that are assembled in the cytoplasm then imported into the cilium. Studies in Chlamydomonas reinhardtii showed that ODA16 is a cofactor which promotes dynein arm import. Here, we demonstrate that the zebrafish homolog of ODA16, Daw1, facilitates the onset of robust cilia motility during development. Without Daw1, cilia showed markedly reduced motility during early development; however, motility subsequently increased to attain close to wild-type levels. Delayed motility onset led to differential effects on early and late cilia-dependent processes. Remarkably, abnormal body axis curves, which formed during the first day of development due to reduced cilia motility, self-corrected when motility later reached wild-type levels. Zebrafish larva therefore possess the ability to survey and correct body shape abnormalities. This work defines Daw1 as a factor which promotes the onset of timely cilia motility and can explain why human patients harboring DAW1 mutations exhibit significant laterality perturbations but mild airway and fertility complications.

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Left-right asymmetric heart jogging increases the robustness of dextral heart looping in zebrafish.

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.