V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Role of EZH2 in Uterine Gland Development.

Enhancer of zeste homolog 2 (EZH2) is a core component of polycomb repressive complex 2 that plays a vital role in transcriptional repression of gene expression. Conditional ablation of EZH2 using progesterone receptor (Pgr)-Cre in the mouse uterus has uncovered its roles in regulating uterine epithelial cell growth and stratification, suppressing decidual myofibroblast activation, and maintaining normal female fertility. However, it is unclear whether EZH2 plays a role in the development of uterine glands, which are required for pregnancy success. Herein, we created mice with conditional deletion of Ezh2 using anti-Mullerian hormone receptor type 2 (Amhr2)-Cre recombinase that is expressed in mesenchyme-derived cells of the female reproductive tract. Strikingly, these mice showed marked defects in uterine adenogenesis. Unlike Ezh2 Pgr-Cre conditional knockout mice, deletion of Ezh2 using Amhr2-Cre did not lead to the differentiation of basal-like cells in the uterus. The deficient uterine adenogenesis was accompanied by impaired uterine function and pregnancy loss. Transcriptomic profiling using next generation sequencing revealed dysregulation of genes associated with signaling pathways that play fundamental roles in development and disease. In summary, this study has identified an unrecognized role of EZH2 in uterine gland development, a postnatal event critical for pregnancy success and female fertility.

Hydrophobic and Hydrogel-Based Methods for Passive Tissue Clearing.

Optical tissue clearing enables the precise imaging of cellular and subcellular structures in whole organs and tissues without the need for physical tissue sectioning. By combining tissue clearing with confocal or lightsheet microscopy, 3D images can be generated of entire specimens for visualization and large-scale data analysis. Here we demonstrate two different passive tissue clearing techniques that are compatible with immunofluorescent staining and lightsheet microscopy: PACT, an aqueous hydrogel-based clearing method, and iDISCO+, an organic solvent-based clearing method.

Spleen tyrosine kinase facilitates neutrophil activation and worsens long-term neurologic deficits after spinal cord injury.

BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.

The complex evolution of the metazoan HSP70 gene family.

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of "constitutive HSP70s" and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of 125 full-length HSP70s from a broad range of phyla revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of genes from many invertebrate phyla, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Platyhelminthes, Rotifera, Nematoda, Porifera/Cnidaria, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of "HSP70" or "HSC70" in previous phylogenetic analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: "HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known." e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.

Passive Clearing and 3D Lightsheet Imaging of the Intact and Injured Spinal Cord in Mice.

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

CHD8short, a naturally-occurring truncated form of a chromatin remodeler lacking the helicase domain, is a potent transcriptional coregulator.

Chromodomain-Helicase-DNA binding protein 8 (CHD8) is a member of a large family of eukaryotic ATP-dependent chromatin remodeling complexes. Loss of function alleles of human chd8 are correlated with autism spectrum disorder. The CHD subfamily members contain a tandem pair of chromodomains that are adjacent to a centrally located Snf2-like helicase domain. An alternatively spliced variant mRNA of CHD8 was identified years ago in mammals that encode a truncated form of the protein, called Duplin, that lacks the helicase domain and everything else in the carboxyl direction. We are using zebrafish to explore the functions of CHD8, especially the truncated form that we refer to as CHD8short (CHD8S). The mRNA for CHD8S is expressed differentially during embryonic development. Using a PCR assay we detected expression of putative zebrafish chd8s mRNA that is barely detectable during early embryogenesis (shield stage at 6h), but increases markedly soon thereafter at 80-90% epiboly (9h) and bud stages (10h), with a return to low levels in 16-somite (17h) and 24hpf embryos. Except for high expression during the shield stage, steady-state levels of chd8l (long) mRNA are relatively constant during the same period of development. We subcloned both chd8l and chd8s cDNAs into expression vector plasmids for use in transient transfection experiments in zebrafish ZF4 cells. In some experiments the luciferase reporter gene was driven by a synthetic promoter that is responsive to activation by ZNF143 activator protein, a known interacting protein with CHD8 in mammalian cells. Whereas CHD8L was a modest coactivator, CHD8S was a potent coactivator, a surprising result since CHD8S is lacking a critical domain to function as a chromatin remodeler enzyme. CHD8S coactivator function is dependent on a region of the protein within the first 50 amino-terminal amino acids. In transient transfection experiments using a Lef1/ß-catenin reporter gene, CHD8S was a modest repressor, but deletion of 50 or more amino-terminal amino acids converted it to a coactivator. When synthetic chd8s mRNA was injected into zebrafish embryos in order to overexpress CHD8S, we observed significant brain disruption phenotypes.