Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft‐10 tsf‐1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper‐vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1‐20 mutants. The terminal flower formed in tfl1‐20 mutants converted to a hyper‐vegetative shoot in ft‐10 tsf‐1 mutants. Grafting ft‐10 tsf‐1 or ft‐10 tsf‐1 tfl1‐20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild‐type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft‐10 tsf‐1 mutants the vasculature‐specific misexpression of FT converted the hyper‐vegetative shoot to a normal inflorescence, and in the ft‐10 tsf‐1 tfl1‐20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.     

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.     

Ectodermal dysplasia-cutaneous syndactyly syndrome maps to chromosome 7p21.1-p14.3

Ectodermal dysplasia syndromes are genetically heterogeneous group of disorders involving one or more of the classical ectodermal appendages (hair, nail, teeth, sweat glands) in association with anomalies of other organs or systems. In the present study a novel form of ectodermal dysplasia syndrome, ectodermal dysplasia cutaneous syndactyly (EDCS), segregating in an autosomal recessive pattern in a Pakistani family was investigated. The clinical features of the affected individuals included large prominent ear pinnae, tooth enamel hypoplasia, hypoplastic nails, bilateral partial cutaneous syndactyly, hypotrichosis, palmoplantar keratoderma and hyperhidrosis. Through genetic linkage study, EDCS syndrome was mapped on human chromosome 7p21.1-p14.3 flanked by markers D7S488 and D7S817. A maximum two-point LOD score of 2.94 (θ = 0.00) was obtained at marker D7S2496 while a maximum multipoint LOD score of 3.07 was obtained with several markers along the disease-interval. This interval spans 19.80-cM, which corresponds to 13.74-Mbp according to the sequence-based physical map (Build 36.1). Sequence analysis of 27 candidate genes, located in the candidate interval, did not reveal any functional sequence variant.     

Rats’ learning of a new motor skill: Insight into the evolution of motor sequence learning

Recent behavioral and neural evidence has suggested that ethologically relevant sub-movements (movement primitives) are used by primates for more complex motor skill learning. These primitives include extending the hand, grasping an object, and holding food while moving it toward the mouth. In prior experiments with rats performing a reach-to-grasp-food task, we observed that especially during early task learning, rats appeared to have movement primitives similar to those seen in primates. Unlike primates, however, during task learning the rats performed these sub-movements in a disordered manner not seen in humans or macaques, e.g. with the rat chewing before placing the food pellet in its mouth. Here, in two experiments, we tested the hypothesis that for rats, learning this ecologically relevant skill involved learning to concatenate the sub-movements in the correct order. The results confirmed our initial observations, and suggested that several aspects of forepaw/hand use, taken for granted in primate studies, must be learned by rats to perform a logically connected and seemingly ecologically important series of sub-movements. We discuss our results from a comparative and evolutionary perspective.     

Identification of genetic loci involved in diabetes using a rat model of depression

While diabetic patients often present with comorbid depression, the underlying mechanisms linking diabetes and depression are unknown. The Wistar Kyoto (WKY) rat is a well-known animal model of depression and stress hyperreactivity. In addition, the WKY rat is glucose intolerant and likely harbors diabetes susceptibility alleles. We conducted a quantitative trait loci (QTL) analysis in the segregating F₂ population of a WKY × Fischer 344 (F344) intercross. We previously published QTL analyses for depressive behavior and hypothalamic-pituitary-adrenal (HPA) activity in this cross. In this study we report results from the QTL analysis for multiple metabolic phenotypes, including fasting glucose, post-restraint stress glucose, postprandial glucose and insulin, and body weight. We identified multiple QTLs for each trait and many of the QTLs overlap with those previously identified using inbred models of type 2 diabetes (T2D). Significant correlations were found between metabolic traits and HPA axis measures, as well as forced swim test behavior. Several metabolic loci overlap with loci previously identified for HPA activity and forced swim behavior in this F₂ intercross, suggesting that the genetic mechanisms underlying these traits may be similar. These results indicate that WKY rats harbor diabetes susceptibility alleles and suggest that this strain may be useful for dissecting the underlying genetic mechanisms linking diabetes, HPA activity, and depression.     

Adaptive Autoimmunity and Foxp3-Based Immunoregulation in Zebrafish

Background

Jawed vertebrates generate their immune-receptor repertoire by a recombinatorial mechanism that has the potential to produce harmful autoreactive lymphocytes. In mammals, peripheral tolerance to self-antigens is enforced by Foxp3⁺ regulatory T cells. Recombinatorial mechanisms also operate in teleosts, but active immunoregulation is thought to be a late incorporation to the vertebrate lineage.

Methods/Principal Findings

Here we report the characterization of adaptive autoimmunity and Foxp3-based immunoregulation in the zebrafish. We found that zebrafish immunization with an homogenate of zebrafish central nervous system (zCNS) triggered CNS inflammation and specific antibodies. We cloned the zebrafish ortholog for mammalian Foxp3 (zFoxp3) which induced a regulatory phenotype on mouse T cells and controlled IL-17 production in zebrafish embryos.

Conclusions/Significance

Our findings demonstrate the acquisition of active mechanisms of self-tolerance early in vertebrate evolution, suggesting that active regulatory mechanisms accompany the development of the molecular potential for adaptive autoimmunity. Moreover, they identify the zebrafish as a tool to study the molecular pathways controlling adaptive immunity.     

X-linked and lineage-dependent inheritance of coping responses to stress

Coping—or how one routinely deals with stress—is a complex behavioral trait with bearing on chronic disease and susceptibility to psychiatric disorders. This complexity is a result of not only underlying multigenic factors, but also important non-genetic ones. The defensive burying (DB) test, although originally developed as a test of anxiety, can accurately measure differences in coping strategies by assaying an animals behavioral response to an immediate threat with ethological validity. Using offspring derived from reciprocal crosses of two inbred rat strains differing in DB behaviors, we provide convergent phenotypic and genotypic evidence that coping styles are inherited in an X-linked fashion. We find that first-generation (F₁) males, but not females, show maternally derived coping styles, and second-generation (F₂) females, but not males, show significant differences in coping styles when separated by grandmaternal lineage. By using a linear modeling approach to account for covariate effects (sex and lineage) in QTL analysis, we map three quantitative trait loci (QTL) on the X Chromosome (Chr) (Coping-1, Approach-1, and Approach-2) associated with coping behaviors in the DB paradigm. Distinct loci were associated with different aspects of coping, and their effects were modulated by both the sex and lineage of the animals, demonstrating the power of the general linear modeling approach and the important interplay of allelic and non-allelic factors in the inheritance of coping behaviors.     

High-affinity hnRNP A1 binding sites and duplex-forming inverted repeats have similar effects on 5' splice site selection in support of a common looping out and repression mechanism

High-affinity binding sites for the hnRNP A1 protein stimulate the use of a distal 5' splice site in mammalian pre-mRNAs. Notably, strong A1-mediated shifts in splice site selection are not accompanied by equivalent changes in the assembly of U1 snRNP-containing complexes on competing 5' splice sites. To explain the above results, we have proposed that an interaction between hnRNP A1 molecules bound to high-affinity sites loops out the internal 5' splice site. Here, we present additional evidence in support of the looping out model. First, replacing A1 binding sites with sequences that can generate a loop through RNA duplex formation activates distal 5' splice site usage in an equivalent manner. Second, increasing the distance between the internal 5' splice site and flanking A1 binding sites does not compromise activation of the distal 5' splice site. Similar results were obtained with pre-mRNAs carrying inverted repeats. Using a pre-mRNA containing only one 5' splice site, we show that splicing is repressed when flanked by two high-affinity A1 binding sites or by inverted repeats, and that inactivation of the internal 5' splice site is sufficient to elicit a strong increase in the use of the distal donor site. Our results are consistent with the view that the binding of A1 to high-affinity sites promotes loop formation, an event that would repress the internal 5' splice site and lead to distal 5' splice site activation.     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.