It would be hard to overstate the importance of a receptor called DAF-2 to our understanding of aging and longevity. Almost 30 years ago it was discovered that loss of the daf-2 gene doubles lifespan in the worm C. elegans (Kenyon et al., 1993), and a few years later it was reported that DAF-2 is the only insulin/IGF-1-like receptor in C. elegans (Kimura et al., 1997). These findings led to an explosion of research into aging and longevity, revealing an intricate insulin signaling pathway that coordinates the sensing of nutrient levels with the regulation of age-related decline. In particular, it was found that reduced insulin signaling extends lifespan and increases stress resistance in flies and mice (Clancy et al., 2001; Holzenberger et al., 2003). Moreover, mutations in some of the genes associated with this pathway were found in centenarians (Suh et al., 2008). And in worms it became clear that, in addition to longevity and age-related declines, DAF-2 is involved in the regulation of a wide range of biological processes, including development, reproduction, memory, and stress responses.
DAF-2 was originally discovered for its role in controlling the dauer stage – an alternative stage of development in which a larva goes into a type of stasis to help it survive harsh conditions (Riddle et al., 1981). A lack of DAF-2 causes C. elegans to enter dauer, as does a lack of a number of other kinases (Paradis and Ruvkun, 1998). An ongoing mystery is why C. elegans has just a single gene for an insulin receptor despite having 40 different insulin-like peptides (Pierce et al., 2001). Some of these peptides are agonists (that is, they activate the receptor) and others are antagonists (they inhibit the receptor).
Given three decades of extensive research into the insulin signaling pathway in C. elegans, it would be shocking to find new functions for DAF-2 at this point. However, in a new paper in eLife, Matthew Gill of the Scripps Research Institute and colleagues – including Bryan Martinez and Pedro Reis Rodrigues as joint first authors – report evidence for such a shock: the gene for DAF-2 can also express another, truncated isoform of this protein as a result of alternative splicing (Martinez et al., 2020). The truncated version, which is called DAF-2B, can still form dimers but, unlike the full-length version, it is not expected to be able to span the membrane: this suggests that the truncated form could be secreted.
Truncated insulin receptors that have extracellular ligand-binding domains, but lack intracellular signaling domains, have been reported in both Drosophila and mammals, and are known to sequester insulin peptides. However, in these cases the full-length receptors and the truncated receptors are expressed from separate genes. Martinez et al. found that although DAF-2B was expressed in neuronal cells, it accumulated in cells called coelomocytes (macrophage-like cells that attack invading organisms such as bacteria and viruses). These results suggest that DAF-2B can indeed be secreted, rather than being retained in the neurons in which it is expressed and spliced.
But what does this shortened form of DAF-2 do? The best-characterized functions of the insulin signaling pathway are dauer formation and lifespan regulation, so Martinez et al. used these phenotypes to study DAF-2B. They found that overexpressing DAF-2B increased dauer formation, slowed dauer exit, and increased lifespan, whereas a lack of DAF-2B had the opposite effect. Basically, the data suggest that the function of DAF-2B is essentially the opposite of the function of DAF-2.
Martinez et al. also explored the interactions between DAF-2B and insulin-like peptides that were either agonists or antagonists. Overexpression of two peptides that are agonists (DAF-28 and INS-6) reduced the dauer-promoting effects of DAF-2B. Conversely, the overexpression of a peptide that is an antagonist (INS-18) would be expected to promote dauer, but this effect was blunted when DAF-2B was also overexpressed. Additionally, the researchers found that a point mutation in the proposed insulin-binding domain resulted in a form of DAF-2B that exhibited reduced dauer formation. Together, these results suggest that DAF-2B binds and may sequester insulin-like peptides, and/or form dimers with DAF-2.
Of course, mysteries remain. Given that worms have dozens of insulin-like peptides (Pierce et al., 2001), which of these bind to DAF-2B, and under what circumstances? And if DAF-2B is secreted, why does it matter where it is expressed, unless there are highly localized interactions? Finally, the mechanism by which DAF-2B acts and its dimerization state is not entirely understood.
The discovery of the truncated version of DAF-2, and the fact that it essentially works in opposition to the full-length version, raises new questions and will change how we think about DAF-2's role in insulin signaling regulation of aging and longevity.
The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open-source GSE182997 datasets (three samples) provided by Fang et al., we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And, we furtherly performed single-cell spatial transcriptomic sequencing on postnatal day 1 mouse enthesis, in order to deconvolute bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes.
Development of the nervous system depends on signaling centers – specialized cellular populations that produce secreted molecules to regulate neurogenesis in the neighboring neuroepithelium. In some cases, signaling center cells also differentiate to produce key types of neurons. The formation of a signaling center involves its induction, the maintenance of expression of its secreted molecules, and cell differentiation and migration events. How these distinct processes are coordinated during signaling center development remains unknown. By performing studies in mice, we show that Lmx1a acts as a master regulator to orchestrate the formation and function of the cortical hem (CH), a critical signaling center that controls hippocampus development. Lmx1a co-regulates CH induction, its Wnt signaling, and the differentiation and migration of CH-derived Cajal–Retzius neurons. Combining RNAseq, genetic, and rescue experiments, we identified major downstream genes that mediate distinct Lmx1a-dependent processes. Our work revealed that signaling centers in the mammalian brain employ master regulatory genes and established a framework for analyzing signaling center development.