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.

On being the right shape: Roles for motile cilia and cerebrospinal fluid flow in body and spine morphology.

How developing and growing organisms attain their proper shape is a central problem of developmental biology. In this review, we investigate this question with respect to how the body axis and spine form in their characteristic linear head-to-tail fashion in vertebrates. Recent work in the zebrafish has implicated motile cilia and cerebrospinal fluid flow in axial morphogenesis and spinal straightness. We begin by introducing motile cilia, the fluid flows they generate and their roles in zebrafish development and growth. We then describe how cilia control body and spine shape through sensory cells in the spinal canal, a thread-like extracellular structure called the Reissner fiber, and expression of neuropeptide signals. Last, we discuss zebrafish mutants in which spinal straightness breaks down and three-dimensional curves form. These curves resemble the common but little-understood human disease Idiopathic Scoliosis. Zebrafish research is therefore poised to make progress in our understanding of this condition and, more generally, how body and spine shape is acquired and maintained through development and growth.

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.

Optimizing the Translational Value of Mouse Models of ALS for Dysphagia Therapeutic Discovery.

The goal of this study was to compare dysphagia phenotypes in low and high copy number (LCN and HCN) transgenic superoxide dismutase 1 (SOD1) mouse models of ALS to accelerate the discovery of novel and effective treatments for dysphagia and early amyotrophic lateral sclerosis (ALS) diagnosis. Clinicopathological features of dysphagia were characterized in individual transgenic mice and age-matched controls utilizing videofluoroscopy in conjunction with postmortem assays of the tongue and hypoglossal nucleus. Quantitative PCR accurately differentiated HCN-SOD1 and LCN-SOD1 mice and nontransgenic controls. All HCN-SOD1 mice developed stereotypical paralysis in both hindlimbs. In contrast, LCN-SOD1 mice displayed wide variability in fore- and hindlimb involvement. Lick rate, swallow rate, inter-swallow interval, and pharyngeal transit time were significantly altered in both HCN-SOD1 and LCN-SOD1 mice compared to controls. Tongue weight, tongue dorsum surface area, total tongue length, and caudal tongue length were significantly reduced only in the LCN-SOD1 mice compared to age-matched controls. LCN-SOD1 mice with lower body weights had smaller/lighter weight tongues, and those with forelimb paralysis and slower lick rates died at a younger age. LCN-SOD1 mice had a 32% loss of hypoglossal neurons, which differed significantly when compared to age-matched control mice. These novel findings for LCN-SOD1 mice are congruent with reported dysphagia and associated tongue atrophy and hypoglossal nucleus pathology in human ALS patients, thus highlighting the translational potential of this mouse model in ALS research.

Variation in traction forces during cell cycle progression.

BACKGROUND INFORMATION: Tissue morphogenesis results from the interplay between cell growth and mechanical forces. While the impact of geometrical confinement and mechanical forces on cell proliferation has been fairly well characterised, the inverse relationship is much less understood. Here, we investigated how traction forces vary during cell cycle progression. RESULTS: Cell shape was constrained on micropatterned substrates in order to distinguish variations in cell contractility from cell size increase. We performed traction force measurements of asynchronously dividing cells expressing a cell-cycle reporter, to obtain measurements of contractile forces generated during cell division. We found that forces tend to increase as cells progress through G1, before reaching a plateau in S phase, and then decline during G2. CONCLUSIONS: While cell size increases regularly during cell cycle progression, traction forces follow a biphasic behaviour based on specific and opposite regulation of cell contractility during early and late growth phases. SIGNIFICANCE: These results highlight the key role of cellular signalling in the regulation of cell contractility, independently of cell size and shape. Non-monotonous variations of cell contractility during cell cycle progression are likely to impact the mechanical regulation of tissue homoeostasis in a complex and non-linear manner.

Visualization and quantitation of spine deformity in zebrafish models of scoliosis by micro-computed tomography.

Zebrafish (Danio rerio) are increasingly used to investigate spine development, growth, and for studying the etiology of spinal deformity, such as scoliosis. Here, we present a micro-computed tomography-based pipeline for visualizing the zebrafish skeleton. We describe steps for sample preparation, imaging, data management, and processing. We then detail analysis of vertebral and spine morphology using open-source software. This protocol will be useful for scientists using zebrafish to understand spine development and disease. For complete details on the use and execution of this protocol, please refer to Bearce et al. (2022).1.

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.

Live Imaging of Cytoskeletal Dynamics in Embryonic Xenopus laevis Growth Cones and Neural Crest Cells.

The cytoskeleton is a dynamic, fundamental network that not only provides mechanical strength to maintain a cell's shape but also controls critical events like cell division, polarity, and movement. Thus, how the cytoskeleton is organized and dynamically regulated is critical to our understanding of countless processes. Live imaging of fluorophore-tagged cytoskeletal proteins allows us to monitor the dynamic nature of cytoskeleton components in embryonic cells. Here, we describe a protocol to monitor and analyze cytoskeletal dynamics in primary embryonic neuronal growth cones and neural crest cells obtained from Xenopus laevis embryos.

Corrigendum: Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

[This corrects the article DOI: 10.3389/fphys.2019.00431.].

Wolf-Hirschhorn Syndrome-Associated Genes Are Enriched in Motile Neural Crest Cells and Affect Craniofacial Development in Xenopus laevis.

Wolf-Hirschhorn Syndrome (WHS) is a human developmental disorder arising from a hemizygous perturbation, typically a microdeletion, on the short arm of chromosome four. In addition to pronounced intellectual disability, seizures, and delayed growth, WHS presents with a characteristic facial dysmorphism and varying prevalence of microcephaly, micrognathia, cartilage malformation in the ear and nose, and facial asymmetries. These affected craniofacial tissues all derive from a shared embryonic precursor, the cranial neural crest (CNC), inviting the hypothesis that one or more WHS-affected genes may be critical regulators of neural crest development or migration. To explore this, we characterized expression of multiple genes within or immediately proximal to defined WHS critical regions, across the span of craniofacial development in the vertebrate model system Xenopus laevis. This subset of genes, whsc1, whsc2, letm1, and tacc3, are diverse in their currently-elucidated cellular functions; yet we find that their expression demonstrates shared tissue-specific enrichment within the anterior neural tube, migratory neural crest, and later craniofacial structures. We examine the ramifications of this by characterizing craniofacial development and neural crest migration following individual gene depletion. We observe that several WHS-associated genes significantly impact facial patterning, cartilage formation, neural crest motility in vivo and in vitro, and can separately contribute to forebrain scaling. Thus, we have determined that numerous genes within and surrounding the defined WHS critical regions potently impact craniofacial patterning, suggesting their role in WHS presentation may stem from essential functions during neural crest-derived tissue formation.

Cytoskeletal social networking in the growth cone: How +TIPs mediate microtubule-actin cross-linking to drive axon outgrowth and guidance.

The growth cone is a unique structure capable of guiding axons to their proper destinations. Within the growth cone, extracellular guidance cues are interpreted and then transduced into physical changes in the actin filament (F-actin) and microtubule cytoskeletons, providing direction and movement. While both cytoskeletal networks individually possess important growth cone-specific functions, recent data over the past several years point towards a more cooperative role between the two systems. Facilitating this interaction between F-actin and microtubules, microtubule plus-end tracking proteins (+TIPs) have been shown to link the two cytoskeletons together. Evidence suggests that many +TIPs can couple microtubules to F-actin dynamics, supporting both microtubule advance and retraction in the growth cone periphery. In addition, growing in vitro and in vivo data support a secondary role for +TIPs in which they may participate as F-actin nucleators, thus directly influencing F-actin dynamics and organization. This review focuses on how +TIPs may link F-actin and microtubules together in the growth cone, and how these interactions may influence axon guidance. © 2016 Wiley Periodicals, Inc.

TIPsy tour guides: how microtubule plus-end tracking proteins (+TIPs) facilitate axon guidance.

The growth cone is a dynamic cytoskeletal vehicle, which drives the end of a developing axon. It serves to interpret and navigate through the complex landscape and guidance cues of the early nervous system. The growth cone's distinctive cytoskeletal organization offers a fascinating platform to study how extracellular cues can be translated into mechanical outgrowth and turning behaviors. While many studies of cell motility highlight the importance of actin networks in signaling, adhesion, and propulsion, both seminal and emerging works in the field have highlighted a unique and necessary role for microtubules (MTs) in growth cone navigation. Here, we focus on the role of singular pioneer MTs, which extend into the growth cone periphery and are regulated by a diverse family of microtubule plus-end tracking proteins (+TIPs). These +TIPs accumulate at the dynamic ends of MTs, where they are well-positioned to encounter and respond to key signaling events downstream of guidance receptors, catalyzing immediate changes in microtubule stability and actin cross-talk, that facilitate both axonal outgrowth and turning events.

TACC3 is a microtubule plus end-tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types.

Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end-tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.