Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis.

Cellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP). The senescence biomarker p16INK4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16INK4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16INK4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16INK4a in murine and human models of chondrocyte aging. We observed that p16INK4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50-fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r2  = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin-dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16INK4a expression. Moreover, increased chondrocyte p16INK4a expression correlated with several SASP transcripts. Despite the relationship between p16INK4a expression and these features of senescence, somatic inactivation of p16INK4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16INK4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

Myocardial NF-κB activation is essential for zebrafish heart regeneration.

Heart regeneration offers a novel therapeutic strategy for heart failure. Unlike mammals, lower vertebrates such as zebrafish mount a strong regenerative response following cardiac injury. Heart regeneration in zebrafish occurs by cardiomyocyte proliferation and reactivation of a cardiac developmental program, as evidenced by induction of gata4 regulatory sequences in regenerating cardiomyocytes. Although many of the cellular determinants of heart regeneration have been elucidated, how injury triggers a regenerative program through dedifferentiation and epicardial activation is a critical outstanding question. Here, we show that NF-κB signaling is induced in cardiomyocytes following injury. Myocardial inhibition of NF-κB activity blocks heart regeneration with pleiotropic effects, decreasing both cardiomyocyte proliferation and epicardial responses. Activation of gata4 regulatory sequences is also prevented by NF-κB signaling antagonism, suggesting an underlying defect in cardiomyocyte dedifferentiation. Our results implicate NF-κB signaling as a key node between cardiac injury and tissue regeneration.

The genomic basis of adaptive evolution in threespine sticklebacks.

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

The genetics of adaptive shape shift in stickleback: pleiotropy and effect size.

The distribution of effect sizes of genes underlying adaptation is unknown (Orr 2005). Are suites of traits that diverged under natural selection controlled by a few pleiotropic genes of large effect (major genes model), by many independently acting genes of small effect (infinitesimal model), or by a combination, with frequency inversely related to effect size (geometric model)? To address this we carried out a quantitative trait loci (QTL) study of a suite of 54 position traits describing body shapes of two threespine stickleback species: an ancestral Pacific marine form and a highly derived benthic species inhabiting a geologically young lake. About half of the 26 detected QTL affected just one coordinate and had small net effects, but several genomic regions affected multiple aspects of shape and had large net effects. The distribution of effect sizes followed the gamma distribution, as predicted by the geometric model of adaptation when detection limits are taken into account. The sex-determining chromosome region had the largest effect of any QTL. Ancestral sexual dimorphism was similar to the direction of divergence, and was largely eliminated during freshwater adaptation, suggesting that sex differences may provide variation upon which selection can act. Several shape QTL are linked to Eda, a major gene responsible for reduction of lateral body armor in freshwater. Our results are consistent with predictions of the geometric model of adaptation. Shape evolution in stickleback results from a few genes with large and possibly widespread effects and multiple genes of smaller effect.

Constraints on utilization of the EDA-signaling pathway in threespine stickleback evolution.

Many traits evolve in parallel in widely separated populations. The evolutionary radiation of threespine sticklebacks provides a powerful model for testing the molecular basis of parallel evolution in vertebrates. Although marine sticklebacks are completely covered with bony armor plates, most freshwater populations have dramatic reductions in plates. Recent genetic studies have shown that major changes in armor patterning are likely due to regulatory alterations in the gene encoding the secreted signaling molecule ectodysplasin (EDA). In mammals, mutations in many different components of the EDA-signaling pathway produce similar changes in hair, teeth, sweat glands, and dermal bones. To test whether other genes in the EDA pathway also control natural variation in armor plates, we identified and mapped stickleback EDA Receptor (EDAR), the EDAR-Associated Death Domain adaptor, Tumor Necrosis Factor Receptor (TNFR) SuperFamily member 19, its adaptor TNFR-Associated Factor 6, and the downstream regulator nuclear factor kappa B Essential Modulator (NEMO). In contrast to the diversity of genes underlying ectodermal dysplasia disease phenotypes in humans, none of these EDA pathway components map to chromosomes previously shown to modify armor plates in natural populations, though EDAR showed a small but significant effect on plate number. We further investigated whether these genes exhibit differences in copy number, target size, or genomic organization that might make them less suitable targets for evolutionary change. In comparison with EDA, all these genes have smaller surrounding noncoding (putative regulatory) regions, with fewer evolutionarily conserved regions. We suggest that the presence of highly modular cis-acting control sequences may be a key factor influencing the likelihood that particular genes will serve as the basis of major phenotypic changes in nature.

Induction of the neural crest: a multigene process.

In the embryo, the neural crest is an important population of cells that gives rise to diverse derivatives, including the peripheral nervous system and the craniofacial skeleton. Evolutionarily, the neural crest is of interest as an important innovation in vertebrates. Experimentally, it represents an excellent system for studying fundamental developmental processes, such as tissue induction. Classical embryologists have identified interactions between tissues that lead to neural crest formation. More recently, geneticists and molecular biologists have identified the genes that are involved in these interactions; this recent work has revealed that induction of the neural crest is a complex multistep process that involves many genes.

Identification and characterization of a calcium channel gamma subunit expressed in differentiating neurons and myoblasts.

Transient elevations of intracellular calcium (calcium transients) play critical roles in many developmental processes, including differentiation. Although the factors that regulate calcium transients are not clearly defined, calcium influx may be controlled by molecules interacting with calcium channels, including channel regulatory subunits. Here, we describe the chick gamma4 regulatory subunit (CACNG4), the first such subunit to be characterized in early development. CACNG4 is expressed early in the cranial neural plate, and later in the cranial and dorsal root ganglia; importantly, the timing of this later expression correlates precisely with the onset of neuronal differentiation. CACNG4 expression is also observed in nonneuronal tissues undergoing differentiation, specifically the myotome and a subpopulation of differentiating myoblasts in the limb bud. Finally, within the distal cranial ganglia, we show that CACNG4 is expressed in placode-derived cells (prospective neurons), but also, surprisingly, in neural crest-derived cells, previously shown to form only glia in this location; contrary to these previous results, we find that neural crest cells can form neurons in the distal ganglia. Given the proposed role of CACNG4 in modulating calcium channels and its expression in differentiating cells, we suggest that CACNG4 may promote differentiation via regulation of intracellular calcium levels.