Y. Albert Pan

LKB1 and SAD Kinases Define a Pathway Required for the Polarization of Cortical Neurons

The polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. We show here that the serine/threonine kinase LKB1, previously implicated in the establishment of epithelial polarity and control of cell growth, is required for axon specification during neuronal polarization in the mammalian cerebral cortex. LKB1 polarizing activity requires its association with the pseudokinase Stradalpha and phosphorylation by kinases such as PKA and p90RSK, which transduce neurite outgrowth-promoting cues. Once activated, LKB1 phosphorylates and thereby activates SAD-A and SAD-B kinases, which are also required for neuronal polarization in the cerebral cortex. SAD kinases, in turn, phosphorylate effectors such as microtubule-associated proteins that implement polarization. Thus, we provide evidence in vivo and in vitro for a multikinase pathway that links extracellular signals to the intracellular machinery required for axon specification.

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.

Multicolor Brainbow Imaging in Zebrafish

This protocol describes how to use the Brainbow strategy to label neurons in many different hues. The Brainbow system uses a random Cre/lox recombination to create varied combinations of red, blue, and green fluorescent proteins in each cell. The differences in color allow users to follow multiple cells, regardless of how closely they are positioned. This protocol describes how to use Brainbow imaging in zebrafish and provides examples of how to use color as a guide to trace axonal processes. We use the zebrafish trigeminal sensory ganglion as an example and discuss potential modifications for the general use of this technique.

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.

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.

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

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet β-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 β-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 β-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines β-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet β-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.

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. J. Comp. Neurol. 523:1639–1663, 2015.

SAD-A Potentiates Glucose-Stimulated Insulin Secretion as a Mediator of Glucagon-Like Peptide 1 Response in Pancreatic β Cells

ABSTRACT Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic β cells, which can be restored by glucagon-like peptide 1 (GLP-1), an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet β cells remain poorly understood. Here we investigated a role of SAD-A, an AMP-activated protein kinase (AMPK)-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretins effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS and further potentiated GLP-1s effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is expressed exclusively in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through cyclic AMP (cAMP)/Ca2+-dependent signaling pathways in islet β cells. Furthermore, we identified Thr443 as a key autoinhibitory phosphorylation site which mediates SAD-As effect on incretin response in islet β cells. Consequently, ablation of Thr443 significantly enhanced GLP-1s effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet β cells.

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.

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.