Regenerative capacity of adult cortical thymic epithelial cells

Involution of the thymus is accompanied by a decline in the number of thymic epithelial cells (TECs) and a severely restricted peripheral repertoire of T-cell specificities. TECs are essential for T-cell differentiation; they originate from a bipotent progenitor that gives rise to cells of cortical (cTEC) and medullary (mTEC) phenotypes, via compartment-specific progenitors. Upon acute selective near-total ablation during embryogenesis, regeneration of TECs fails, suggesting that losses from the pool of TEC progenitors are not compensated. However, it is unclear whether this is also true for the compartment-specific progenitors. The decline of cTECs is a prominent feature of thymic involution. Because cTECs support early stages of T-cell development and hence determine the overall lymphopoietic capacity of the thymus, it is possible that the lack of sustained regenerative capacity of cTEC progenitor cells underlies the process of thymic involution. Here, we examine this hypothesis by cell-type-specific conditional ablation of cTECs. Expression of the human diphtheria toxin receptor (hDTR) gene under the regulatory influence of the chemokine receptor Ccx-ckr1 gene renders cTECs sensitive to the cytotoxic effects of diphtheria toxin (DT). As expected, DT treatment of preadolescent and adult mice led to a dramatic loss of cTECs, accompanied by a rapid demise of immature thymocytes. Unexpectedly, however, the cTEC compartment regenerated after cessation of treatment, accompanied by the restoration of T-cell development. These findings provide the basis for the development of targeted interventions unlocking the latent regenerative potential of cTECs to counter thymic involution.     

Deletion of a gene encoding an amino acid transporter in the midgut membrane causes resistance to a Bombyx parvo-like virus

Bombyx mori densovirus type 2 (BmDNV-2), a parvo-like virus, replicates only in midgut columnar cells and causes fatal disease. The resistance expressed in some silkworm strains against the virus is determined by a single gene, nsd-2, which is characterized as nonsusceptibility irrespective of the viral dose. However, the responsible gene has been unknown. We isolated the nsd-2 gene by positional cloning. The virus resistance is caused by a 6-kb deletion in the ORF of a gene encoding a 12-pass transmembrane protein, a member of an amino acid transporter family, and expressed only in midgut. Germ-line transformation with a wild-type transgene expressed in the midgut restores susceptibility, showing that the defective membrane protein is responsible for resistance. Cumulatively, our data show that the membrane protein is a functional receptor for BmDNV-2. This is a previously undescribed report of positional cloning of a mutant gene in Bombyx and isolation of an absolute virus resistance gene in insects.     

Current perspectives in zebrafish reverse genetics: moving forward.

Use of the zebrafish as a model of vertebrate development and disease has expanded dramatically over the past decade. While many articles have discussed the strengths of zebrafish forward genetics (the phenotype-driven approach), there has been less emphasis on equally important and frequently used reverse genetics (the candidate gene-driven approach). Here we review both current and prospective reverse genetic techniques that are applicable to the zebrafish model. We include discussion of pharmacological approaches, popular gain-of-function and knockdown approaches, and gene targeting strategies. We consider the need for temporal and spatial control over gain/loss of gene function, and discuss available and developing techniques to achieve this end. Our goal is both to reveal the current technical advantages of the zebrafish and to highlight those areas where work is still required to allow this system to be exploited to full advantage.     

Cytokine reporter mice in immunological research: perspectives and lessons learned

Cytokines are soluble messenger molecules with important regulatory functions throughout the immune system. 'Cytokine reporter' strains express marker molecules under control of elements from cytokine genes allowing for easy identification of their cellular sources. Such systems are well-accepted tools for research of cytokine function. The value of these strains lies in the ability to perform experiments relying on identification and isolation of live cytokine-expressing cells, provided that the reporter faithfully reflects the proper cytokine mRNA and protein production. As more diverse cell subsets are defined by their cytokine expression, the field has adapted with the generation of more sophisticated strains. In this review we summarize the evolution of cytokine detection methods and give examples of knowledge gained using cytokine reporter mice for cell types expressing interferon-γ and interleukin-4, -10 and -17. We also discuss current options for generating such reporter strains and their potential pitfalls.     

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.     

The Ars insulator facilitates I-sceI meganuclease-mediated transgenesis in the sea urchin embryo

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo. Developmental Dynamics 237:2475-2482, 2008. (c) 2008 Wiley-Liss, Inc.