CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4.

The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

A critical role for the nuclear protein Akirin2 in the formation of mammalian muscle in vivo.

Evolutionarily conserved Akirin nuclear proteins interact with chromatin remodeling complexes at gene enhancers and promoters, and have been reported to regulate cell proliferation and differentiation. Of the two mouse Akirin genes, Akirin2 is essential during embryonic development, with known in vivo roles in immune system function and the formation of the cerebral cortex. Here we demonstrate that Akirin2 is critical for mouse myogenesis, a tightly regulated developmental process through which myoblast precursors fuse to form mature skeletal muscle fibers. Loss of Akirin2 in somitic muscle precursor cells via Sim1-Cre-mediated excision of a conditional Akirin2 allele results in neonatal lethality. Mutant embryos exhibit a complete lack of forelimb, intercostal, and diaphragm muscles due to extensive apoptosis and loss of Pax3-positive myoblasts. Severe skeletal defects, including craniofacial abnormalities, disrupted ossification, and rib fusions are also observed, attributable to lack of skeletal muscles as well as patchy Sim1-Cre activity in the embryonic sclerotome. We further show that Akirin2 levels are tightly regulated during muscle cell differentiation in vitro, and that Akirin2 is required for the proper expression of muscle differentiation factors myogenin and myosin heavy chain. Our results implicate Akirin2 as a major regulator of mammalian muscle formation in vivo.

Transient activation of microglia following acute alcohol exposure in developing mouse neocortex is primarily driven by BAX-dependent neurodegeneration.

Fetal alcohol exposure is the most common known cause of preventable mental retardation, yet we know little about how microglia respond to, or are affected by, alcohol in the developing brain in vivo. Using an acute (single day) model of moderate (3 g/kg) to severe (5 g/kg) alcohol exposure in postnatal day (P) 7 or P8 mice, we found that alcohol-induced neuroapoptosis in the neocortex is closely correlated in space and time with the appearance of activated microglia near dead cells. The timing and molecular pattern of microglial activation varied with the level of cell death. Although microglia rapidly mobilized to contact and engulf late-stage apoptotic neurons, apoptotic bodies temporarily accumulated in neocortex, suggesting that in severe cases of alcohol toxicity the neurodegeneration rate exceeds the clearance capacity of endogenous microglia. Nevertheless, most dead cells were cleared and microglia began to deactivate within 1-2 days of the initial insult. Coincident with microglial activation and deactivation, there was a transient increase in expression of pro-inflammatory factors, TNFα and IL-1ß, after severe (5 g/kg) but not moderate (3 g/kg) EtOH levels. Alcohol-induced microglial activation and pro-inflammatory factor expression were largely abolished in BAX null mice lacking neuroapoptosis, indicating that microglial activation is primarily triggered by apoptosis rather than the alcohol. Therefore, acute alcohol exposure in the developing neocortex causes transient microglial activation and mobilization, promoting clearance of dead cells and tissue recovery. Moreover, cortical microglia show a remarkable capacity to rapidly deactivate following even severe neurodegenerative insults in the developing brain.

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo.

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically-interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific vs. shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified PKC phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via MARCKS is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remains unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point, and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo.

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC) phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

γ-Protocadherins control synapse formation and peripheral branching of touch sensory neurons.

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn leads to fewer corticospinal synapses on dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and stepwise assembly of central mechanosensory circuitry.

Pharmacist-managed chronic obstructive pulmonary disease screening in a community setting.

OBJECTIVES: To implement a spirometry-based chronic obstructive pulmonary disease (COPD) screening in a community pharmacy chain, determine whether pharmacists can accurately perform spirometry screenings and interpret results, and determine whether performing screenings improved enrollment in smoking cessation programs. DESIGN: Prospective study. SETTING: Kroger pharmacies in the Cincinnati-Dayton Kroger Marketing Area and off-site screening events in Cincinnati, OH, from March to December 2010. PATIENTS: Consenting individuals older than 35 years who met inclusion and exclusion criteria. INTERVENTION: Specially trained community pharmacists administered a validated COPD screening questionnaire and performed spirometry. The results were interpreted, given to the patient, and faxed to the primary care physician. Any patient who was currently smoking was offered smoking cessation counseling. MAIN OUTCOME MEASURES: Spirometry technical quality and interpretation accuracy, screening questionnaire scores in relationship to spirometry results, number of patients enrolled in smoking cessation programs. RESULTS: Of the 185 patients, 10 were excluded due to inability to perform spirometry. After review, 174 (99%) of the spirometries were judged acceptable and 157 (90%) demonstrated reproducible results. The mean (+/-SD) score on the COPD Population Screener questionnaire was 2.3 ± 1.6 (range 0-8). Airflow limitation (defined as forced expiratory volume in 1 second/forced vital capacity < lower limit of normal) was detected in 16 (9%) of the patients. Although 12 (75%) of these patients were former or current smokers, only 3 (19%) were at increased risk for COPD based on their screening questionnaire scores. Of the nine current smokers who participated in a follow-up interview, two had successfully abstained from smoking for 6 months after the screening and five others had made an attempt to quit. CONCLUSION: This study demonstrated that pharmacists are able to perform accurate and reproducible spirometry in a community pharmacy setting.

Serotonin signaling by maternal neurons upon stress ensures progeny survival.

Germ cells are vulnerable to stress. Therefore, how organisms protect their future progeny from damage in a fluctuating environment is a fundamental question in biology. We show that in Caenorhabditis elegans, serotonin released by maternal neurons during stress ensures the viability and stress resilience of future offspring. Serotonin acts through a signal transduction pathway conserved between C. elegans and mammalian cells to enable the transcription factor HSF1 to alter chromatin in soon-to-be fertilized germ cells by recruiting the histone chaperone FACT, displacing histones, and initiating protective gene expression. Without serotonin release by maternal neurons, FACT is not recruited by HSF1 in germ cells, transcription occurs but is delayed, and progeny of stressed C. elegans mothers fail to complete development. These studies uncover a novel mechanism by which stress sensing by neurons is coupled to transcription response times of germ cells to protect future offspring.

An essential role for the nuclear protein Akirin2 in mouse limb interdigital tissue regression.

The regulation of interdigital tissue regression requires the interplay of multiple spatiotemporally-controlled morphogen gradients to ensure proper limb formation and release of individual digits. Disruption to this process can lead to a number of limb abnormalities, including syndactyly. Akirins are highly conserved nuclear proteins that are known to interact with chromatin remodelling machinery at gene enhancers. In mammals, the analogue Akirin2 is essential for embryonic development and critical for a wide variety of roles in immune function, meiosis, myogenesis and brain development. Here we report a critical role for Akirin2 in the regulation of interdigital tissue regression in the mouse limb. Knockout of Akirin2 in limb epithelium leads to a loss of interdigital cell death and an increase in cell proliferation, resulting in retention of the interdigital web and soft-tissue syndactyly. This is associated with perdurance of Fgf8 expression in the ectoderm overlying the interdigital space. Our study supports a mechanism whereby Akirin2 is required for the downregulation of Fgf8 from the apical ectodermal ridge (AER) during limb development, and implies its requirement in signalling between interdigital mesenchymal cells and the AER.

Akirin2 is essential for the formation of the cerebral cortex.

BACKGROUND: The proper spatial and temporal regulation of dorsal telencephalic progenitor behavior is a prerequisite for the formation of the highly-organized, six-layered cerebral cortex. Premature differentiation of cells, disruption of cell cycle timing, excessive apoptosis, and/or incorrect neuronal migration signals can have devastating effects, resulting in a number of neurodevelopmental disorders involving microcephaly and/or lissencephaly. Though genes encoding many key players in cortical development have been identified, our understanding remains incomplete. We show that the gene encoding Akirin2, a small nuclear protein, is expressed in the embryonic telencephalon. Converging evidence indicates that Akirin2 acts as a bridge between transcription factors (including Twist and NF-κB proteins) and the BAF (SWI/SNF) chromatin remodeling machinery to regulate patterns of gene expression. Constitutive knockout of Akirin2 is early embryonic lethal in mice, while restricted loss in B cells led to disrupted proliferation and cell survival. METHODS: We generated cortex-restricted Akirin2 knockouts by crossing mice harboring a floxed Akirin2 allele with the Emx1-Cre transgenic line and assessed the resulting embryos using in situ hybridization, EdU labeling, and immunohistochemistry. RESULTS: The vast majority of Akirin2 mutants do not survive past birth, and exhibit extreme microcephaly, with little dorsal telencephalic tissue and no recognizable cortex. This is primarily due to massive cell death of early cortical progenitors, which begins at embryonic day (E)10, shortly after Emx1-Cre is active. Immunostaining and cell cycle analysis using EdU labeling indicate that Akirin2-null progenitors fail to proliferate normally, produce fewer neurons, and undergo extensive apoptosis. All of the neurons that are generated in Akirin2 mutants also undergo apoptosis by E12. In situ hybridization for Wnt3a and Wnt-responsive genes suggest defective formation and/or function of the cortical hem in Akirin2 null mice. Furthermore, the apical ventricular surface becomes disrupted, and Sox2-positive progenitors are found to "spill" into the lateral ventricle. CONCLUSIONS: Our data demonstrate a previously-unsuspected role for Akirin2 in early cortical development and, given its known nuclear roles, suggest that it may act to regulate gene expression patterns critical for early progenitor cell behavior and cortical neuron production.

Localization of synapsin-I and PSD-95 in developing postnatal rat cerebellar cortex.

The comparative localization of two prominent synaptic proteins, synapsin-I (Syn-I) and PSD-95, was investigated in slices of developing (P3-P21) rat cerebellar cortex using double- or triple-label fluorescence immunohistochemistry and confocal microscopy. During the first postnatal week, Syn-I and PSD-95 immunoreactive (IR) puncta were strongly concentrated in the Purkinje cell layer (PCL) where they circumscribed irregularly shaped PC somata, forming pericellular nests that likely correspond to early climbing fiber synapses. PSD-95 and Syn-I puncta also were found along the shafts and at the tips of growing PC dendrite branches labeled with calbindin. During the second postnatal week, synaptic puncta were lost from the PC layer, while many new puncta were added to the molecular layer (ML). At P10, about half of the PCs were circumscribed by PSD-95 or Syn-I puncta, whereas at P14 no PCs were circumscribed. By P14, PSD-95 and Syn-I became most strongly localized to many small puncta in the ML and to large clusters at mossy fiber rosettes in the glomerular layer (GL) where PSD-95 often encircled Syn-I clusters. Some large clusters in the GL contained only PSD-95 or Syn-I, but not both, suggesting differential growth or remodeling of pre- and post-synaptic structures. No PSD-95 staining of pre-synaptic terminal pinceau was observed during the first 3 weeks of postnatal development. Thus, in relation to PCs, there is a developmental shift in PSD-95 localization whereby, first, it is concentrated on PC cell bodies and short dendrites (P3-P7), then it is lost on PC cell bodies (P7-14) and becomes localized almost exclusively to PC dendrites (P14-P21).

Neural crest cell motility in valproic acid.

Neural crest cells (NCCs) exit the dorsal neural tube and migrate to sites where they form diverse tissues. Valproic acid (VPA) is an anticonvulsant drug that induces neural tube and related defects. Altered NCC migration and proliferation have been proposed as mechanisms of teratogenicity. We cultured neural tube segments from chick embryos in 0.75-3.0mM VPA. We used image analysis, proliferation assays, and fluorescence localization to investigate NCCs during VPA exposure. VPA inhibited attachment of explants and the number that produced migrating cells. VPA markedly decreased the proportion of cells migrating individually, promoting migration as epithelial sheets. VPA at 3mM decreased cellular spreading. Area and perimeter change per minute were reduced, but migration velocity was not. VPA at 2mM reduced proliferation 11% and 3mM arrested proliferation. Immunostaining of VPA-exposed explants revealed N-cadherin-positive cell boundaries within sheets, but independent NCCs did not stain. F-actin staining was reduced in independent NCCs. The data support a VPA mechanism involving interference with epithelial-mesenchymal transition.

Imaging microglia in brain slices and slice cultures.

Here we describe a method for imaging fluorescently labeled parenchymal microglia (MG) in excised neonatal or adult rodent brain tissue slices. Using multichannel confocal or two-photon time-lapse imaging, the approach affords real-time analyses of MG behaviors, including motility, migration, chemotaxis, proliferation, and phagocytosis in live brain tissues. The method is applicable to acutely prepared tissue slices from developing and adult rodents and to slice cultures derived from neonatal rodents, including transgenic and green fluorescent protein reporter mice. A variety of fluorescent tags can be used to study the structure and physiology of MG in these preparations. Moreover, bath application of reagents (such as ATP) can establish spatial and temporal gradients that induce chemokinesis- and chemotaxis-like MG migration in tissue slices. Thus, the approach is useful for dissecting the molecular basis of MG behaviors and testing whether candidate reagents alter MG behavior and function in semi-intact central nervous system tissue preparations.

Preparation of rodent hippocampal slice cultures.

INTRODUCTIONRodent organotypic hippocampal slice cultures (OHSCs) provide an outstanding preparation of central nervous system tissue for exploring the dynamic structural and physiological features of neuronal and glial cells within their native three-dimensional environments. It is a straightforward matter to obtain tissue slices from neonatal rodents. These slices culture well for periods up to several weeks and are easy to manipulate, allowing for a variety of in vitro experimental models. OHSCs provide good optical and physiological accessibility for studies involving live cell imaging, with high spatial and temporal resolution. This protocol is used to harvest tissues for both immunohistochemical labeling after fixation, and for confocal time-lapse imaging in live tissues labeled by a variety of fluorescent dyes or by biolistic or viral transfection.

Early activation, motility, and homing of neonatal microglia to injured neurons does not require protein synthesis.

Neuronal injury in CNS tissues induces a rapid activation and mobilization of resident microglia (MG). It is widely assumed that changes in gene expression drive the morphological transformation of MG and regulate their mobilization during activation. Here, we used acutely excised neonatal rat brain slices to test whether the morphological transformation and homing of MG to injured neurons requires gene expression and de novo protein synthesis. Traumatic injury during excision of live brain tissue slices induces a rapid and transient translocation of a transcription factor, NF-kappaB, to nuclei in MG. This is followed within 4-8 h by an increase in immunolabeling for cell adhesion molecules and lysosomal proteins, accompanied by changes in cell morphology. Application of anisomycin, a protein synthesis inhibitor, prevents the increase in immunolabeling for markers of MG activation but not the morphological transformation. Confocal time-lapse imaging in live tissue slices indicates that MG cell motility (branch extension and retraction) and locomotion are unaffected by anisomycin at early postinjury time-points (<4 h), while at later time-points (4-8 h postinjury) MG locomotion but not motility is inhibited. Thus, activated MG rapidly localize to injured pyramidal neuron cell bodies by 4-h postinjury, even in the presence of anisomycin. Moreover, this early MG activation and homing to injured neurons is unaffected in tissue slices from beta2 integrin deficient mice. These results indicate that gene activation and new protein synthesis coincide with, but are not necessary for, the rapid morphological transformation and early migration-dependent homing of activated MG to injured neurons in CNS tissues.

Dendritic arbors of developing retinal ganglion cells are stabilized by beta 1-integrins.

The architecture of dendritic arbors is a defining characteristic of neurons and is established through a sequential but overlapping series of events involving process outgrowth and branching, stabilization of the global pattern, and synapse formation. To investigate the roles of cadherins and beta1-integrins in maintaining the global architecture of the arbor, we used membrane permeable peptides and transfection with dominant-negative constructs to disrupt adhesion molecule function in intact chick neural retina at a stage when the architecture of the ganglion cell (RGC) arbor is established but synapse formation is just beginning. Inactivation of beta1-integrins induces rapid dendrite retraction, with loss of dynamic terminal filopodia followed by resorption of major branches. Disruption of N-cadherin-beta-catenin interactions has no effect; however, dendrites do retract following perturbation of the juxtamembrane region of N-cadherin, which disrupts N-cadherin-mediated adhesion and initiates a beta1-integrin inactivating signal. Thus, developing RGC dendritic arbors are stabilized by beta1-integrin-dependent processes.

Juxtavascular microglia migrate along brain microvessels following activation during early postnatal development.

Some parenchymal microglia in mammalian brain tissues, termed "juxtavascular microglia," directly contact the basal lamina of blood vessels; however, the functional consequences of this unique structural relationship are unknown. Here we used a rat brain slice model of traumatic brain injury to investigate the dynamic behavior of juxtavascular microglia following activation. Juxtavascular microglia were identified by confocal 3D reconstruction in tissue slices stained with a fluorescent lectin (FITC-IB4) that labels both microglia and blood vessel endothelial cells. Immunolabeling confirmed that juxtavascular cells were true parenchymal microglia (OX42+, ED2-) and not perivascular cells or pericytes. Time-lapse imaging in live tissue slices revealed that activating juxtavascular microglia withdraw most extant branches but often maintain contact with blood vessels, usually moving to the surface of a vessel within 1-4 h. Subsequently, some microglia migrate along the parenchymal surface of vessels, moving at rates up to 40 microm/h. Activated juxtavascular microglia sometimes repeatedly extend veil-like protrusions into the surrounding tissue, consistent with a role in tissue surveillance. Juxtavascular cells were twice as likely as nonjuxtavascular cells to be locomotory by 10 h in vitro, suggesting an enhanced activation response. Moreover, 38% of all juxtavascular cells migrated along a vessel, whereas this was never observed for a nonjuxtavascular cell. These observations identify a mobile subpopulation (10%-30%) of parenchymal microglia that activate rapidly and are preferentially recruited to the surfaces of blood vessels following brain tissue injury. The dynamic and sustained interaction of microglia with brain microvessels may facilitate signaling between injured brain parenchyma and components of the blood-brain barrier or circulating immune cells of the blood in vivo.