Molecular and phenotypic reassessment of an infrequently used mouse model for spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) results from loss of the survival motor neuron 1 (SMN1) gene, with retention of its nearly identical homolog, SMN2. There is a direct correlation between disease severity and SMN2 copy number. Mice do not have a Smn2 gene, and thus cannot naturally replicate the disorder. However, two murine models of SMA have been generated using SMN2-BAC transgenic mice bred onto a mutant Smn background. In these instances mice die shortly after birth, have variable phenotypes within the same litter, or completely correct the SMA phenotype. Both models have been imported to The Jackson Laboratory for distribution to the research community. To ensure that similar results are obtained after importation to The Jackson Laboratory to what was originally reported in the literature, we have begun a molecular and phenotypic evaluation of these mouse models. Here we report our findings for the SMA mouse model that has been deposited by the Li group from Taiwan. These mice, JAX stock number TJL-005058, are homozygous for the SMN2 transgene, Tg(SMN2)2Hung, and a targeted Smn allele that lacks exon 7, Smn1^tm1Hung. Our findings are consistent with those reported originally for this line and clarify some of the original data. In addition, we have cloned and mapped the integration site for Tg(SMN2)2Hung to Chromosome 4, and provide a simple genotyping assay that is specific to the junction fragment. Finally, based upon the survival data from our genetic crosses, we suggest that this underused SMA model may be a useful compliment or alternative to the more commonly used “delta7” SMA mouse. We provide breeding schemes in which two genotypes of mice can be generated so that 50% of the litter will be SMA-like pups while 50% will be controls.     

Zinc-secreting Paneth cells studied by ZP fluorescence.

We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkühn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen.     

SHORT INTERNODES-like genes regulate shoot growth and xylem proliferation in Populus

? Genes controlling plant growth and form are of considerable interest, because they affect survival and productivity traits, and are largely unknown or poorly characterized. The SHORT INTERNODES(SHI) gene is one of a 10-member SHI-RELATED SEQUENCE (SRS) gene family in Arabidopsis that includes important developmental regulators. ? Using comparative sequence analysis of the SRS gene families in poplar and Arabidopsis, we identified two poplar proteins that are most similar to SHI and its closely related gene STYLISH1 (STY1). The two poplar genes are very similar in sequence and expression and are therefore probably paralogs with redundant functions. ? RNAi suppression of the two Populus genes enhanced shoot and root growth, whereas the overexpression of Arabidopsis SHI in poplar reduced internode and petiole length. The suppression of the two genes increased fiber length and the proportion of xylem tissue, mainly through increased xylem cell proliferation. The transgenic modifications were also associated with significant changes in the concentrations of gibberellins and cytokinin. ? We conclude that Populus SHI-RELATED SEQUENCE (SRS) genes play an important role in the regulation of vegetative growth, including wood formation, and thus could be useful tools for the modification of biomass productivity, wood quality or plant form.     

Window Area and Development Drive Spatial Variation in Bird-Window Collisions in an Urban Landscape

Collisions with windows are an important human-related threat to birds in urban landscapes. However, the proximate drivers of collisions are not well understood, and no study has examined spatial variation in mortality in an urban setting. We hypothesized that the number of fatalities at buildings varies with window area and habitat features that influence avian community structure. In 2010 we documented bird-window collisions (BWCs) and characterized avian community structure at 20 buildings in an urban landscape in northwestern Illinois, USA. For each building and season, we conducted 21 daily surveys for carcasses and nine point count surveys to estimate relative abundance, richness, and diversity. Our sampling design was informed by experimentally estimated carcass persistence times and detection probabilities. We used linear and generalized linear mixed models to evaluate how habitat features influenced community structure and how mortality was affected by window area and factors that correlated with community structure. The most-supported model was consistent for all community indices and included effects of season, development, and distance to vegetated lots. BWCs were related positively to window area and negatively to development. We documented mortalities for 16/72 (22%) species (34 total carcasses) recorded at buildings, and BWCs were greater for juveniles than adults. Based on the most-supported model of BWCs, the median number of annual predicted fatalities at study buildings was 3 (range = 0–52). These results suggest that patchily distributed environmental resources and levels of window area in buildings create spatial variation in BWCs within and among urban areas. Current mortality estimates place little emphasis on spatial variation, which precludes a fundamental understanding of the issue. To focus conservation efforts, we illustrate how knowledge of the structural and environmental factors that influence bird-window collisions can be used to predict fatalities in the broader landscape.     

Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2^‐/‐) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell‐types. Here, we describe Cxcr2^fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2^fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2^fl/fl mice were crossed to Mx‐Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic‐polycytidylic acid (poly(I:C)). CXCR2‐deficient neutrophils were observed in poly(I:C) treated Cxcr2^fl/fl::Mx‐Cre⁺ (Cxcr2‐CKO) mice, but not in poly(I:C) treated Cxcr2^f//+::Mx‐Cre⁺ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2‐CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2‐CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2^fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations. genesis 51:587–595. © 2013 Wiley Periodicals, Inc.     

Synaptonemal complex-dependent centromeric clustering and the initiation of synapsis in Drosophila oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.     

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.     

Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

Niemann–Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.     

DR5 as a reporter system to study auxin response in Populus

Key message

Auxin responsive promoter DR5 reporter system is functional in Populus to monitor auxin response in tissues including leaves, roots, and stems.

Abstract

We described the behavior of the DR5::GUS reporter system in stably transformed Populus plants. We found several similarities with Arabidopsis, including sensitivity to native and synthetic auxins, rapid induction after treatment in a variety of tissues, and maximal responses in root tissues. There were also several important differences from Arabidopsis, including slower time to maximum response and lower induction amplitude. Young leaves and stem sections below the apex showed much higher DR5 activity than did older leaves and stems undergoing secondary growth. DR5 activity was highest in cortex, suggesting high levels of auxin concentration and/or sensitivity in this tissue. Our study shows that the DR5 reporter system is a sensitive and facile system for monitoring auxin responses and distribution at cellular resolution in poplar.