Zebrafish dscaml1 Deficiency Impairs Retinal Patterning and Oculomotor Function.

Down syndrome cell adhesion molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), whereas abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders.SIGNIFICANCE STATEMENTDscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders.

Multicolor lineage tracing using in vivo time-lapse imaging reveals coordinated death of clonally related cells in the developing vertebrate brain.

The global mechanisms that regulate and potentially coordinate cell proliferation & death in developing neural regions are not well understood. In particular, it is not clear how or whether clonal relationships between neural progenitor cells and their progeny influence the growing brain. We have developed an approach using Brainbow in the developing zebrafish to visualize and follow multiple clones of related cells in vivo over time. This allows for clear visualization of many dividing clones of cells, deep in proliferating brain regions. As expected, in addition to undergoing interkinetic nuclear migration and cell division, cells also periodically undergo apoptosis. Interestingly, cell death occurs in a non-random manner: clonally related cells are more likely to die in a progressive fashion than cells from different clones. Multiple members of an individual clone die while neighboring clones appear healthy and continue to divide. Our results suggest that clonal relationships can influence cellular fitness and survival in the developing nervous system, perhaps through a competitive mechanism whereby clones of cells are competing with other clones. Clonal cell competition may help regulate neuronal proliferation in the vertebrate brain.

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Chronic alcohol exposure induces muscle atrophy (myopathy) in zebrafish and alters the expression of microRNAs targeting the Notch pathway in skeletal muscle.

Muscle wasting is estimated to affect 40-60% of alcoholics, and is more common than cirrhosis among chronic alcohol abusers. The molecular and cellular mechanisms underlying alcohol-related musculoskeletal dysfunction are, however, poorly understood. Muscle-specific microRNAs (miRNAs) referred to as myoMirs are now known to play a key role in both myogenesis and muscle atrophy. Yet, no studies have investigated a role for myoMirs in alcohol-related skeletal muscle damage. We developed a zebrafish model of chronic ethanol exposure to better define the mechanisms mediating alcohol-induced muscle atrophy. Adult fish maintained at 0.5% ethanol for eight weeks demonstrated significantly reduced muscle fiber cross-sectional area (∼12%, P < 0.05) compared to fish housed in normal water. Zebrafish miRNA microarray revealed marked changes in several miRNAs with ethanol treatment. Importantly, miR-140, a miRNA that shows 100% sequence homology with miR-140 from both mouse and human, is decreased 10-fold in ethanol treated fish. miR-140 targets several members of the Notch signaling pathway such as DNER, JAG1, and Hey1, and PCR data show that both Hey1 and Notch 1 are significantly up-related (3-fold) in muscle of ethanol treated fish. In addition, miR-146a, which targets the Notch antagonist Numb, is elevated in muscle from ethanol-treated fish. Upregulation of Notch signaling suppresses myogenesis and maintains muscle satellite cell quiescence. These data suggest that miRNAs targeting Notch are likely to play important roles in alcohol-related myopathy. Furthermore, zebrafish may serve as a useful model for better understanding the role of microRNAs in alcohol-related tissue damage.

Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish.

Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a 'Brainbow' of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish.

SAD-A kinase controls islet ß-cell size and function as a mediator of mTORC1 signaling.

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet ß-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic ß-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet ß-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines ß-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet ß-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons.

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system.

Deficiency in the cell-adhesion molecule dscaml1 impairs hypothalamic CRH neuron development and perturbs normal neuroendocrine stress axis function.

The corticotropin-releasing hormone (CRH)-expressing neurons in the hypothalamus are critical regulators of the neuroendocrine stress response pathway, known as the hypothalamic-pituitary-adrenal (HPA) axis. As developmental vulnerabilities of CRH neurons contribute to stress-associated neurological and behavioral dysfunctions, it is critical to identify the mechanisms underlying normal and abnormal CRH neuron development. Using zebrafish, we identified Down syndrome cell adhesion molecule like-1 (dscaml1) as an integral mediator of CRH neuron development and necessary for establishing normal stress axis function. In dscaml1 mutant animals, hypothalamic CRH neurons had higher crhb (the CRH homolog in zebrafish) expression, increased cell number, and reduced cell death compared to wild-type controls. Physiologically, dscaml1 mutant animals had higher baseline stress hormone (cortisol) levels and attenuated responses to acute stressors. Together, these findings identify dscaml1 as an essential factor for stress axis development and suggest that HPA axis dysregulation may contribute to the etiology of human DSCAML1-linked neuropsychiatric disorders.

Cre-Dependent Anterograde Transsynaptic Labeling and Functional Imaging in Zebrafish Using VSV With Reduced Cytotoxicity.

The small size and translucency of larval zebrafish (Danio rerio) have made it a unique experimental system to investigate whole-brain neural circuit structure and function. Still, the connectivity patterns between most neuronal types remain mostly unknown. This gap in knowledge underscores the critical need for effective neural circuit mapping tools, especially ones that can integrate structural and functional analyses. To address this, we previously developed a vesicular stomatitis virus (VSV) based approach called Tracer with Restricted Anterograde Spread (TRAS). TRAS utilizes lentivirus to complement replication-incompetent VSV (VSVΔG) to allow restricted (monosynaptic) anterograde labeling from projection neurons to their target cells in the brain. Here, we report the second generation of TRAS (TRAS-M51R), which utilizes a mutant variant of VSVΔG [VSV(M51R)ΔG] with reduced cytotoxicity. Within the primary visual pathway, we found that TRAS-M51R significantly improved long-term viability of transsynaptic labeling (compared to TRAS) while maintaining anterograde spread activity. By using Cre-expressing VSV(M51R)ΔG, TRAS-M51R could selectively label excitatory (vglut2a positive) and inhibitory (gad1b positive) retinorecipient neurons. We further show that these labeled excitatory and inhibitory retinorecipient neurons retained neuronal excitability upon visual stimulation at 5-8 days post fertilization (2-5 days post-infection). Together, these findings show that TRAS-M51R is suitable for neural circuit studies that integrate structural connectivity, cell-type identity, and neurophysiology.

Using Zebrafish to Elucidate Glial-Vascular Interactions During CNS Development.

An emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2+ and gfap+ zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2+ glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap+ glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap+ glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes.

Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, in vivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Effects of Constitutive and Acute Connexin 36 Deficiency on Brain-Wide Susceptibility to PTZ-Induced Neuronal Hyperactivity.

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: (a) does Cx36 deficiency affect seizure susceptibility, (b) does seizure-like activity affect Cx36 expression patterns, and (c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol [PTZ; a GABA(A) receptor antagonist] induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within 30 min. This Cx36 reduction persists after 1-h of recovery but recovered after 3-6 h. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events.

Deficiency in the endocytic adaptor proteins PHETA1/2 impairs renal and craniofacial development.

A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, pheta1 and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient.This article has an associated First Person interview with the first author of the paper.

Structural Neural Connectivity Analysis in Zebrafish With Restricted Anterograde Transneuronal Viral Labeling and Quantitative Brain Mapping.

The unique combination of small size, translucency, and powerful genetic tools makes larval zebrafish a uniquely useful vertebrate system to investigate normal and pathological brain structure and function. While functional connectivity can now be assessed by optical imaging (via fluorescent calcium or voltage reporters) at the whole-brain scale, it remains challenging to systematically determine structural connections and identify connectivity changes during development or disease. To address this, we developed Tracer with Restricted Anterograde Spread (TRAS), a novel vesicular stomatitis virus (VSV)-based neural circuit labeling approach. TRAS makes use of replication-incompetent VSV (VSVΔG) and a helper virus (lentivirus) to enable anterograde transneuronal spread between efferent axons and their direct postsynaptic targets but restricts further spread to downstream areas. We integrated TRAS with the Z-Brain zebrafish 3D atlas for quantitative connectivity analysis and identified targets of the retinal and habenular efferent projections, in patterns consistent with previous reports. We compared retinofugal connectivity patterns between wild-type and down syndrome cell adhesion molecule-like 1 (dscaml1) mutant zebrafish and revealed differences in topographical distribution. These results demonstrate the utility of TRAS for quantitative structural connectivity analysis that would be valuable for detecting novel efferent targets and mapping connectivity changes underlying neurological or behavioral deficits.

Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors.

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies.

Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.

A direct histological assay of axonal regeneration would have many advantages over currently available behavioral, electrophysiological, and radiometric assays. We show that peripheral sensory axons marked with the yellow fluorescent protein in transgenic mice can be viewed transcutaneously in superficial nerves. Degenerating and regenerating axons can be followed in live animals with a dissecting microscope and then, after fixation, studied at high resolution by confocal microscopy. Using this approach, we document differences in regenerative ability after nerve transection, crush injury, and crush injury after a previous "conditioning" lesion. We also show that the chemotherapeutic drug vincristine rapidly but transiently blocks regeneration and that the immunosuppressive drug FK506 modestly enhances regeneration. Moreover, FK506 nearly restores normal regenerative ability in animals treated with submaximal doses of vincristine. Because neuropathy is the major dose-limiting side effect of vincristine, we propose that its efficacy could be enhanced by coadministration of FK506 analogs that are neuroactive but not immunosuppressive.

Brainbow: new resources and emerging biological applications for multicolor genetic labeling and analysis.

Brainbow is a genetic cell-labeling technique where hundreds of different hues can be generated by stochastic and combinatorial expression of a few spectrally distinct fluorescent proteins. Unique color profiles can be used as cellular identification tags for multiple applications such as tracing axons through the nervous system, following individual cells during development, or analyzing cell lineage. In recent years, Brainbow and other combinatorial expression strategies have expanded from the mouse nervous system to other model organisms and a wide variety of tissues. Particularly exciting is the application of Brainbow in lineage tracing, where this technique has been instrumental in parsing out complex cellular relationships during organogenesis. Here we review recent findings, new technical improvements, and exciting potential genetic and genomic applications for harnessing this colorful technique in anatomical, developmental, and genetic studies.

Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods.

Non-invasive visualization of epidermal responses to injury using a fluorescent transgenic reporter.

We describe transgenic mice in which expression of a reporter, the yellow fluorescent protein (YFP), is locally activated in epidermal cells at sites of injury. YFP is detectable a day after injury; its levels peak within 3 d, and then decline over the subsequent week. Expression is also activated by a chemical irritant, and is suppressed by topical administration of hydrocortisone. These mice permit non-invasive time-lapse monitoring of responses to injury in vivo. They can be used to detect epidermal activation and to test agents that may provoke or attenuate epidermal responses.

SAD kinases sculpt axonal arbors of sensory neurons through long- and short-term responses to neurotrophin signals.

Extrinsic cues activate intrinsic signaling mechanisms to pattern neuronal shape and connectivity. We showed previously that three cytoplasmic Ser/Thr kinases, LKB1, SAD-A, and SAD-B, control early axon-dendrite polarization in forebrain neurons. Here, we assess their role in other neuronal types. We found that all three kinases are dispensable for axon formation outside of the cortex but that SAD kinases are required for formation of central axonal arbors by subsets of sensory neurons. The requirement for SAD kinases is most prominent in NT-3 dependent neurons. SAD kinases transduce NT-3 signals in two ways through distinct pathways. First, sustained NT-3/TrkC signaling increases SAD protein levels. Second, short-duration NT-3/TrkC signals transiently activate SADs by inducing dephosphorylation of C-terminal domains, thereby allowing activating phosphorylation of the kinase domain. We propose that SAD kinases integrate long- and short-duration signals from extrinsic cues to sculpt axon arbors within the CNS.