Parabiosis is the surgical union of two organisms resulting in the development of a single, shared circulatory system. When animals of different ages are conjoined (i.e. heterochronic parabiosis), blood-borne factors from the parabionts can affect the physiology of the other parabiont. This is manifested sometimes by beneficial, rejuvenating impact upon the older animal's tissues and organs (anti-geronic effect), and sometimes by younger animal's tissues regressing and appearing old-like (pro-geronic effect). These effects, and the ability to identify individual factors that could recapitulate pro- and anti-geronic effects, have made heterochronic parabiosis a very attractive approach to studying biology of aging and rejuvenation.cHowever, heterochronic parabiosis has not been widely used to investigate the aged immune system. An important question to be answered is whether the cellular defects involved in the aged immune system are due to intrinsic defects or if they can be rescued by extrinsic factors. Heterochronic parabiosis is ideal to test cellular migration patterns, interrogate the mechanisms driving migration defects that occur with aging, establish if these defects can be rejuvenated and identify molecules that are targets for intervention. Here, we provide evidence of the importance of reducing differences in the background genetics of different C57BL/6 substrains prior to parabiosis. This improvement allowed us to improve survival and confirm robust lymphocyte equilibration across secondary, but not primary, lymphoid tissues. We found no evidence for rejuvenation of the old immune cells, whereas results suggested that adult peripheral lymph nodes (pLN) lost mass and cellularity, potentially indicating the presence of a pro-geronic factor(s) in the old circulation that affects pLN function. Adult and old immune cells were present in equal frequencies in both adult and old secondary lymphoid tissues, indicating that there was no restriction of cellular migration due to the age of the cell or age of the tissue. The propensity of adult immune cells (i.e. large naïve compartment) to occupy lymph nodes and old immune cells (i.e. large memory compartment) to occupy bone marrow was retained following heterochronic parabiosis. Finally, parabiosis separation experiments illuminated the peripheral survival advantage of old T cells over adult T cells. These results highlight the power of heterochronic parabiosis in studying immune aging and provide hypothesis-generating data for future mechanistic studies of peripheral T cell maintenance with aging.

The ability of a neuron to alter its synaptic connections during development is essential to circuit assembly. Synapse remodeling or refinement has been observed in many species and many neuronal circuits, yet the mechanisms defining which neurons undergo remodeling are unclear. Moreover, the molecules that execute the process of remodeling are also obscure. To address this issue, we sought to identify targets of the transcription factor unc-55 COUP-TF, which acts as a cell-specific repressor of synapse remodeling in C. elegans. unc-55 COUP-TF is expressed in VD neurons, where it prevents synapse remodeling. DD neurons can remodel synapses because they do not express unc-55 COUP-TF. Ectopic expression of unc-55 COUP-TF in DD neurons prevents remodeling. We identified the transcription factor Hunchback-like hbl-1 as a target of UNC-55 COUP-TF repression. Differential expression of hbl-1 explains the cell-type specificity of remodeling. hbl-1 is expressed in the DD neurons that are capable of remodeling, and is not expressed in the VD neurons that do not remodel. In unc-55 mutants, hbl-1 expression increases in VD neurons where it promotes ectopic remodeling. Moreover, hbl-1 expression levels bidirectionally regulate the timing of DD remodeling, as increases in hbl-1 cause precocious remodeling while decreases in hbl-1 cause remodeling delays. Finally, hbl-1 coordinates heterochronic microRNA and neuronal activity pathways to regulate the timing of remodeling. Increases or decreases in circuit activity cause increases or decreases in hbl-1 expression, and consequently early or delayed remodeling. Thus, convergent regulation of hbl-1 expression defines a genetic mechanism that patterns activity-dependent synaptic remodeling across cell types and across developmental time. We identified other targets of UNC-55 COUP-TF regulation using gene expression profiling, and implicate some of these factors in the regulation of remodeling using functional genomic screens. Our work suggests roles for conserved networks of transcription factors in the regulation of remodeling. We propose a model in which hbl-1 and other targets of unc-55 COUP-TF transcriptional repression are responsible for regulating synapse remodeling in C. elegans.

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Methylglyoxal is a highly reactive dicarbonyl compound, which is widely distributed in food products and beverages, and is particularly high in Manuka honey. In addition to its antibacterial effects, methylglyoxal is also known as a major precursor of advanced glycation end products (AGEs), that produces altered macromolecules (such as proteins and DNA), leading to abnormal physiological changes. However, the effects of methylglyoxal on development is unclear. Thus, this study aimed to determine the role of methylglyoxal in this aspect using Caenorhabditis elegans (C. elegans). Treatment of methylglyoxal at 0.1 mM and 1 mM for 48 h significantly inhibited development of C. elegans and reduced pumping rate. Activity, measured by moving speed, was increased with 0.1 mM methylglyoxal, but reduced with 1 mM methylglyoxal. Lifespan of C. elegans was not influenced by methylglyoxal at 0.1 mM, but was shortened at 1 mM. Treatment methylglyoxal on the mutant, lin-41, which has a precocious phenotype, could alleviate the implication on wild-type worms. These results suggested that methylglyoxal significantly influenced the development of C. elegans through the heterochronic pathway.

Robustness is a ubiquitous property of biological systems, however, underlying mechanisms that help reinforce the optimal phenotypes despite environmental or physiological perturbations are poorly understood. C. elegans development consists of four larval stages (L1-L4) and well-characterized invariant cell lineages, within which the heterochronic pathway controls the order and timing of cell-fates. Environmental or physiological stress signals can slow or temporarily halt larval stage progression; remarkably, however, temporal cell-fate progression remains unaffected. We show that two widely conserved signaling pathways, insulin and TGF- β, that regulate C. elegans larval stage progression in response to starvation and crowding, respectively, also regulate a rewiring of the heterochronic pathway so that cell-fates remain temporally anchored to appropriate larval stages. This rewiring is mediated by the nuclear hormone receptor DAF-12, and it involves a shift from the reliance on let-7-family microRNAs to the reliance on LIN-46 for proper downregulation of the transcription factor, Hunchback-like-1 (HBL-1), which promotes L2 cell-fates and opposes L3 cell-fates. LIN-46 (which is a homolog of bacterial molybdopterin molybdenum transferase (moeA) and human gephyrin) post-translationally inhibits HBL-1 activity. LIN-46 expression is repressed by the RNA-binding protein LIN-28 at the early stages to permit HBL-1 activity and hence the proper execution of L2 cell-fates. Our results indicate that robustness mechanisms of temporal cell-fate progression in C. elegans involves 1) coordinated regulation of temporal cell-fates and larval stage progression and 2) collaboration between translational regulation exerted by microRNAs and post-translational regulation exerted by LIN-46 to coordinate HBL-1 downregulation with stage progression.