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The degenerative loss of muscle associated with aging leading to muscular atrophy is called sarcopenia. Currently, practicing regular physical exercise is the only efficient way to delay sarcopenia onset. Identification of therapeutic targets to alleviate the symptoms of aging requires in vivo model organisms of accelerated muscle degeneration and atrophy. The zebrafish undergoes aging, with hallmarks including mitochondrial dysfunction, telomere shortening, and accumulation of senescent cells. However, zebrafish age slowly, and no specific zebrafish models of accelerated muscle atrophy associated with molecular events of aging are currently available. We have developed a new genetic tool to efficiently accelerate muscle-fiber degeneration and muscle-tissue atrophy in zebrafish larvae and adults. We used a gain-of-function strategy with a molecule that has been shown to be necessary and sufficient to induce muscle atrophy and a sarcopenia phenotype in mammals: Atrogin-1 (also named Fbxo32). We report the generation, validation, and characterization of a zebrafish genetic model of accelerated neuromuscular atrophy, the atrofish. We demonstrated that Atrogin-1 expression specifically in skeletal muscle tissue induces a muscle atrophic phenotype associated with locomotion dysfunction in both larvae and adult fish. We identified degradation of the myosin light chain as an event occurring prior to muscle-fiber degeneration. Biological processes associated with muscle aging such as proteolysis, inflammation, stress response, extracellular matrix (ECM) remodeling, and apoptosis are upregulated in the atrofish. Surprisingly, we observed a strong correlation between muscle-fiber degeneration and reduced numbers of neuromuscular junctions in the peripheral nervous system, as well as neuronal cell bodies in the spinal cord, suggesting that muscle atrophy could underly a neurodegenerative phenotype in the central nervous system. Finally, while atrofish larvae can recover locomotive functions, adult atrofish have impaired regenerative capacities, as is observed in mammals during muscle aging. In the future, the atrofish could serve as a platform for testing molecules aimed at treating or alleviating the symptoms of muscle aging, thereby opening new therapeutic avenues in the fight against sarcopenia.

We describe a real-time (rt) PCR-based method of quantifying DNA damage, adapted from the long-run rtPCR method of DNA damage quantification (LORD-Q) developed by Lehle et al. (Nucleic Acids Res 42(6):e41, 2014 ). We show that semi-long run rtPCR, which generates amplicons half the length of those generated in LORD-Q, provides equivalent sensitivity for detecting low lesion frequencies, and better sensitivity for detecting high frequencies. The smaller amplicon size greatly facilitates PCR optimization and allows greater flexibility in the use of detection dyes, and a modified data analysis method simplifies the calculation of lesion frequency. The method was used to measure DNA damage in the nuclear and mitochondrial genomes of different tissues in zebrafish of different ages. We find that nuclear DNA damage generally increases with age, and that the amount of mitochondrial DNA damage varies substantially between tissues, increasing with age in liver and brain but not in heart or skeletal muscle, the latter having the highest levels of damage irrespective of age.

Krüppel-like factor 9 (Klf9) is a ubiquitously expressed transcription factor that is a feedforward regulator of multiple stress-responsive and endocrine signaling pathways. We previously described how loss of Klf9 function affects the transcriptome of zebrafish larvae sampled at a single time point 5 days post-fertilization (dpf). However, klf9 expression oscillates diurnally, and the sampled time point corresponded to its expression nadir. To determine if the transcriptomic effects of the klf9 −/− mutation vary with time of day, we performed bulk RNA-seq on 5 dpf zebrafish embryos sampled at three timepoints encompassing the predawn peak and midmorning nadir of klf9 expression. We found that while the major effects of the klf9 −/− mutation that we reported previously are robust to time of day, the mutation has additional effects that manifest only at the predawn time point. We used a published single-cell atlas of zebrafish development to associate the effects of the klf9 −/− mutation with different cell types and found that the mutation increased mRNA associated with digestive organs (liver, pancreas, and intestine) and decreased mRNA associated with differentiating neurons and blood. Measurements from confocally-imaged larvae suggest that overrepresentation of liver mRNA in klf9 −/− mutants is due to development of enlarged livers.

Objective Chronic early life stress can affect development of the neuroendocrine stress system, leading to its persistent dysregulation and consequently increased disease risk in adulthood. One contributing factor is thought to be epigenetic programming in response to chronic cortisol exposure during early development. We have previously shown that zebrafish embryos treated chronically with cortisol develop into adults with constitutively elevated whole-body cortisol and aberrant immune gene expression. Here we further characterize that phenotype by assessing persistent effects of the treatment on cortisol tissue distribution and dynamics, chromatin accessibility, and activities of glucocorticoid-responsive regulatory genes klf9 and fkbp5 . To that end cortisol levels in different tissues of fed and fasted adults were measured using ELISA, open chromatin in adult blood cells was mapped using ATAC-seq, and gene activity in adult blood and brain cells was measured using qRT-PCR. Results Adults derived from cortisol-treated embryos have elevated whole-body cortisol with aberrantly regulated tissue distribution and dynamics that correlate with differential activity of klf9 and fkbp5 in blood and brain.

Krüppel-like factor 9 (Klf9) is a feedforward regulator of glucocorticoid receptor (GR) signaling. Here we show that in zebrafish klf9 is expressed with GR-dependent oscillatory dynamics in synchrony with fkbp5 , a GR target that encodes a negative feedback regulator of GR signaling. We found that fkbp5 transcript levels are elevated in klf9 –/– mutants and that Klf9 associates with chromatin at the fkbp5 promoter, which becomes hyperacetylated in klf9 –/ – mutants, suggesting that the GR regulates fkbp5 via an incoherent feedforward loop with klf9 . As both the GR and Fkbp5 are known to regulate metabolism, we asked how loss of Klf9 affects metabolic rate and gene expression. We found that klf9 –/– mutants have a decreased oxygen consumption rate (OCR) and upregulate glycolytic genes, the promoter regions of which are enriched for potential Klf9 binding motifs. Our results suggest that Klf9 functions downstream of the GR to regulate cellular glucocorticoid responsivity and metabolic homeostasis.

Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity.

Summary Aging in many animals is characterized by a failure to maintain tissue homeostasis and the loss of regenerative capacity. In this study, the ability to maintain tissue homeostasis and regenerative potential was investigated in sea urchins, a novel model to study longevity and negligible senescence. Sea urchins grow indeterminately, regenerate damaged appendages and reproduce throughout their lifespan and yet different species are reported to have very different life expectancies (ranging from 4 to more than 100 years). Quantitative analyses of cell proliferation and apoptosis indicated a low level of cell turnover in tissues of young and old sea urchins of species with different lifespans (Lytechinus variegatus,Strongylocentrotus purpuratus and Mesocentrotus franciscanus). The ability to regenerate damaged tissue was maintained with age as assessed by the regrowth of amputated spines and tube feet (motor and sensory appendages). Expression of genes involved in cell proliferation (pcna), telomere maintenance (tert) and multipotency (seawi and vasa) was maintained with age in somatic tissues. Immunolocalization of the Vasa protein to areas of the tube feet, spines, radial nerve, esophagus and a sub-population of circulating coelomocytes suggests the presence of multipotent cells that may play a role in normal tissue homeostasis and the regenerative potential of external appendages. The results indicate that regenerative potential was maintained with age regardless of lifespan, contrary to the expectation that shorter lived species would invest less in maintenance and repair.

The Symposium on the Comparative Biology of Tissue Repair, Regeneration and Aging, held 26 June to 28 June 2015 at the MDI Biological Laboratory in Salisbury Cove, Maine, brought together a diverse group of biologists with a common interest in understanding why regenerative capacity varies among animal species, why it is progressively lost in senescence, and how answers obtained from studies that address those questions might be applied in regenerative medicine.

Background The Runt DNA binding domain (Runx) defines a metazoan family of sequence-specific transcription factors with essential roles in animal ontogeny and stem cell based development. Depending on cis -regulatory context, Runx proteins mediate either transcriptional activation or repression. In many contexts Runx-mediated repression is carried out by Groucho/TLE, recruited to the transcriptional complex via a C-terminal WRPY sequence motif that is found encoded in all heretofore known Runx genes. Findings Full-length Runx genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans, including placozoans and sponges, the most basally branching members of that clade. No sequences with significant similarity to the Runt domain were found in the genome of the choanoflagellate Monosiga brevicollis , confirming that Runx is a metazoan apomorphy. A contig assembled from genomic sequences of the haplosclerid demosponge Amphimedon queenslandica was used to construct a model of the single Runx gene from that species, AmqRunx , the veracity of which was confirmed by expressed sequences. The encoded sequence of the Runx protein OscRunx from the homoscleromorph sponge Oscarella carmella was also obtained from assembled ESTs. Remarkably, a syntenic linkage between Runx and Supt3h , previously reported in vertebrates, is conserved in A. queenslandica . Whereas OscRunx encodes a C-terminal Groucho-recruitment motif, AmqRunx does not, although a Groucho homologue is found in the A. queenslandica genome. Conclusion Our results are consistent with the hypothesis that sponges are paraphyletic, and suggest that Runx-WRPY mediated recruitment of Groucho to cis -regulatory sequences originated in the ancestors of eumetazoans following their divergence from demosponges.