Post-translational regulation of planarian regeneration.

Most mammals cannot easily overcome degenerative disease or traumatic injuries. In contrast, an innate ability to regenerate is observed across animal phyla. Freshwater planarians are amongst the organisms that are capable of stem cell-mediated whole-body regeneration and have served as an exemplary model to study how pluripotency is maintained and regulated in vivo. Here, we review findings on the role of post-translational modifications and the genes regulating phosphorylation, ubiquitylation, and chromatin remodeling in planarian regeneration. Furthermore, we discuss how technological advances for identifying cellular targets of these processes will fill gaps in our knowledge of the signaling mechanisms that underlie regeneration in planarians, which should inform how tissue repair can be stimulated in non-regenerative model organisms and in humans.

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo.

Wnt/ß-catenin signaling promotes regeneration after adult zebrafish spinal cord injury.

Unlike mammals, zebrafish can regenerate their injured spinal cord and regain control of caudal tissues. It was recently shown that Wnt/ß-catenin signaling is necessary for spinal cord regeneration in the larval zebrafish. However, the molecular mechanisms of regeneration may or may not be conserved between larval and adult zebrafish. To test this, we assessed the role of Wnt/ß-catenin signaling after spinal cord injury in the adult zebrafish. We show that Wnt/ß-catenin signaling is increased after spinal cord injury in the adult zebrafish. Moreover, overexpression of Dkk1b inhibited Wnt/ß-catenin signaling in the regenerating spinal cord of adult zebrafish. Dkk1b overexpression also inhibited locomotor recovery, axon regeneration, and glial bridge formation in the injured spinal cord. Thus, our data illustrate a conserved role for Wnt/ß-catenin signaling in adult and larval zebrafish spinal cord regeneration.

Macrophages modulate adult zebrafish tail fin regeneration.

Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema proliferation. Moreover, we also sought to detail molecular regulators of inflammation in adult zebrafish and identified Wnt/ß-catenin as a signaling pathway that regulates the injury microenvironment, inflammatory cell migration and macrophage phenotype. These results provide a cellular and molecular link between components of the inflammation response and regeneration in adult zebrafish.

Altered splicing of ATP6AP2 causes X-linked parkinsonism with spasticity (XPDS).

We report a novel gene for a parkinsonian disorder. X-linked parkinsonism with spasticity (XPDS) presents either as typical adult onset Parkinson's disease or earlier onset spasticity followed by parkinsonism. We previously mapped the XPDS gene to a 28 Mb region on Xp11.2-X13.3. Exome sequencing of one affected individual identified five rare variants in this region, of which none was missense, nonsense or frame shift. Using patient-derived cells, we tested the effect of these variants on expression/splicing of the relevant genes. A synonymous variant in ATP6AP2, c.345C>T (p.S115S), markedly increased exon 4 skipping, resulting in the overexpression of a minor splice isoform that produces a protein with internal deletion of 32 amino acids in up to 50% of the total pool, with concomitant reduction of isoforms containing exon 4. ATP6AP2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy, a pathway frequently affected in Parkinson's disease. Reduction of the full-size ATP6AP2 transcript in XPDS cells and decreased level of ATP6AP2 protein in XPDS brain may compromise V-ATPase function, as seen with siRNA knockdown in HEK293 cells, and may ultimately be responsible for the pathology. Another synonymous mutation in the same exon, c.321C>T (p.D107D), has a similar molecular defect of exon inclusion and causes X-linked mental retardation Hedera type (MRXSH). Mutations in XPDS and MRXSH alter binding sites for different splicing factors, which may explain the marked differences in age of onset and manifestations.

DREADDing the lateral habenula: a review of methodological approaches for studying lateral habenula function.

The lateral habenula (LHb) is part of the habenular complex in the dorsal diencephalon. The LHb is an important regulator of several neurotransmitter systems in the midbrain; disturbances in this regulation may contribute to mood disorders, abnormalities in cognition, drive, and addiction. Owing to the critical role this nucleus plays in modulating activity of midbrain nuclei, there has been a rapid increase in studies targeting the LHb in the recent years. In this review, we describe studies using traditional approaches to elucidate the function of this brain region, such as lesion, electrical and chemical stimulation, electrophysiology and in vivo microdialysis. We have selected a variety of illustrative studies to discuss each of these methods. Next, we describe studies using methods that are based upon recent advances in molecular biology techniques including recent results from our laboratory using the Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology. Using a Gi/o-coupled DREADD, we found that inhibition of the LHb reduces depression-like behavior in the forced swim test in a manner that suggests enhanced serotonergic activity. The emerging picture reveals that the LHb is likely to be a critical node in the network of subcortical nuclei that regulate aversive learning, motivation, stress responses, etc. We describe how recently developed methods have advanced the study of the LHb and are leading research of this brain region in promising new directions. This article is part of a Special Issue entitled Optogenetics (7th BRES).