A brainstem circuit for gravity-guided vertical navigation.

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish (Danio rerio) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.

A mutation in CCDC91, Homo sapiens coiled-coil domain containing 91 protein, cause autosomal-dominant acrokeratoelastoidosis.

Acrokeratoelastoidosis (AKE) is a rare autosomal dominant hereditary skin disease characterized by small, round-oval, flat-topped keratotic papules on the palms, soles and dorsal aspect of hands or feet. The causative gene for AKE remains unidentified. This study aims to identify the causative gene of AKE and explore the underlying biological mechanisms. A large, three-generation Chinese family exhibiting classic AKE symptoms was identified. A genome-wide linkage analysis and whole-exome sequencing were employed to determine the causative gene. shRNA knockdown in human skin fibroblasts and CRISPR/Cas9 knockout in HEK293T cells were utilized to assess gene functions in the progression of elastic fiber biosynthesis. The linkage analysis identified a susceptibility region between rs7296765 to rs10784618 on chromosome 12. Whole-exome sequencing confirmed a splicing mutation of 1101 + 1 G > A in the CCDC91 gene, resulting in exon 11 skipping and a subsequent 59-amino-acid-residue loss (residues L309-Q367del). Further functional analysis revealed distended Golgi cisternae, cytoplasmic vesicle accumulation, and lysosome presence. Immnunostaining of si-CCDC91-HSF cells demonstrated tropoelastin accumulation in the Golgi and abnormal extracellular aggregates. There are no significant changes in Fibrillin-1 microfibril assembly and lysyl oxidase activity. The findings strongly suggest that the protein product of the CCDC91 gene plays a crucial role in elastin transport. This discovery enhances our understanding of CCDC91's function and broadens the known pathogenic mechanisms of AKE.

Multisensory strategies for postural compensation after lateral line loss.

To control elevation underwater, aquatic vertebrates integrate multisensory information (e.g., vestibular, visual, proprioceptive) to guide posture and swim kinematics. Here we characterized how larval zebrafish changed posture and locomotive strategies after imposed instability (decreased buoyancy) in the presence and absence of visual cues. We discovered that larvae sank more after acute loss of lateral line (flow-sensing) hair cells. In response, larvae engaged different compensatory strategies, depending on whether they were in the light or dark. In the dark, larvae swam more frequently, engaging their trunk to steer their nose up and climb more effectively. However, in the light, larvae climbed more often, engaging both pectoral fins and trunk to elevate. We conclude that larvae sense instability and use vestibular and visual information as available to control posture and trajectory. Our work is a step towards understanding the multisensory neural computations responsible for control strategies that allow orientation and navigation in depth.

Determinants of motor neuron functional subtypes important for locomotor speed.

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

SAMPL is a high-throughput solution to study unconstrained vertical behavior in small animals.

Balance and movement are impaired in many neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics but without the throughput and scalability necessary to screen candidate genes/potential therapeutics. Here, we present a scalable apparatus to measure posture and locomotion (SAMPL). SAMPL includes extensible hardware and open-source software with real-time processing and can acquire data from D. melanogaster, C. elegans, and D. rerio as they move vertically. Using SAMPL, we define how zebrafish balance as they navigate vertically and discover small but systematic variations among kinematic parameters between genetic backgrounds. We demonstrate SAMPL's ability to resolve differences in posture and navigation as a function of effect size and data gathered, providing key data for screens. SAMPL is therefore both a tool to model balance and locomotor disorders and an exemplar of how to scale apparatus to support screens.

Scalable Apparatus to Measure Posture and Locomotion (SAMPL): a high-throughput solution to study unconstrained vertical behavior in small animals.

Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPL's hardware and acquisition software can acquire data from from D. melanogaster, C. elegans, and D. rerio as they move vertically. Next, we leverage SAMPL's throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPL's analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPL's ability to resolve differences in posture or vertical navigation as a function of affect size and data gathered -- key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for labs using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for labs looking process videographic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.

Tilt in Place Microscopy: a Simple, Low-Cost Solution to Image Neural Responses to Body Rotations.

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative-Tilt In Place Microscopy (TIPM), a low-cost and noninvasive way to measure neural activity following rapid changes in body orientation. Here, we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e., sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental time points. There, we observed global increases in response strength and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish the utility of TIPM to study the neural basis of balance, especially in developing animals.SIGNIFICANCE STATEMENT Vestibular sensation influences everything from navigation to interoception. Here, we detail a straightforward, validated, and nearly universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand on past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the response of the brain to destabilization, particularly across development.

Transforming growth factor-beta signaling modulates perineurial glial bridging following peripheral spinal motor nerve injury in zebrafish.

Spinal motor nerves are necessary for organismal locomotion and survival. In zebrafish and most vertebrates, these peripheral nervous system structures are composed of bundles of axons that naturally regenerate following injury. However, the cellular and molecular mechanisms that mediate this process are still only partially understood. Perineurial glia, which form a component of the blood-nerve barrier, are necessary for the earliest regenerative steps by establishing a glial bridge across the injury site as well as phagocytosing debris. Without perineurial glial bridging, regeneration is impaired. In addition to perineurial glia, Schwann cells, the cells that ensheath and myelinate axons within the nerve, are essential for debris clearance and axon guidance. In the absence of Schwann cells, perineurial glia exhibit perturbed bridging, demonstrating that these two cell types communicate during the injury response. While the presence and importance of perineurial glial bridging is known, the molecular mechanisms that underlie this process remain a mystery. Understanding the cellular and molecular interactions that drive perineurial glial bridging is crucial to unlocking the mechanisms underlying successful motor nerve regeneration. Using laser axotomy and in vivo imaging in zebrafish, we show that transforming growth factor-beta (TGFß) signaling modulates perineurial glial bridging. Further, we identify connective tissue growth factor-a (ctgfa) as a downstream effector of TGF-ß signaling that works in a positive feedback loop to mediate perineurial glial bridging. Together, these studies present a new signaling pathway involved in the perineurial glial injury response and further characterize the dynamics of the perineurial glial bridge.

The Msi1-mTOR pathway drives the pathogenesis of mammary and extramammary Paget's disease.

Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.

Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System.

During neural tube closure and spinal cord development, many cells die in both the central and peripheral nervous systems (CNS and PNS, respectively). However, myeloid-derived professional phagocytes have not yet colonized the trunk region during early neurogenesis. How apoptotic cells are removed from this region during these stages remains largely unknown. Using live imaging in zebrafish, we demonstrate that neural crest cells (NCCs) respond rapidly to dying cells and phagocytose cellular debris around the neural tube. Additionally, NCCs have the ability to enter the CNS through motor exit point transition zones and clear debris in the spinal cord. Surprisingly, NCCs phagocytosis mechanistically resembles macrophage phagocytosis and their recruitment toward cellular debris is mediated by interleukin-1ß. Taken together, our results reveal a role for NCCs in phagocytosis of debris in the developing nervous system before the presence of professional phagocytes.

Disseminated Bacille Calmette-Guérin infection in a patient with severe combined immunodeficiency caused by JAK3 gene mutation.

Bacille Calmette-Guérin (BCG), a live attenuated vaccine prepared using Mycobacterium bovis, can prevent tuberculosis in children and is routinely administered to infants in China and many other countries. A serious complication following vaccination is disseminated BCG infection. The risk is greatly increased in patients with severe combined immunodeficiency disease (SCID), a syndrome characterized by deficiency of both humoral and cellular immunity. We report a case of disseminated BCG infection in an infant with SCID caused by two novel janus kinase 3 (JAK3) gene mutations.

Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release.

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2-that is, 'smell' ('find-me' signals or sensing factors released by apoptotic cells), 'taste' (phagocyte-apoptotic cell contact) and 'ingestion' (corpse internalization)-activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells.

The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis.

Dense multicilia in higher vertebrates are important for luminal flow and the removal of thick mucus. To generate hundreds of basal bodies for multiciliogenesis, specialized terminally differentiated epithelial cells undergo massive centriole amplification. In proliferating cells, however, centriole duplication occurs only once per cell cycle. How cells ensure proper regulation of centriole biogenesis in different contexts is poorly understood. We report that the centriole amplification is controlled by two duplicated genes, Cep63 and Deup1. Cep63 regulates mother-centriole-dependent centriole duplication. Deup1 governs deuterosome assembly to mediate large-scale de novo centriole biogenesis. Similarly to Cep63, Deup1 binds to Cep152 and then recruits Plk4 to activate centriole biogenesis. Phylogenetic analyses suggest that Deup1 diverged from Cep63 in a certain ancestor of lobe-finned fishes during vertebrate evolution and was subsequently adopted by tetrapods. Thus, the Cep63 gene duplication has enabled mother-centriole-independent assembly of the centriole duplication machinery to satisfy different requirements for centriole number.