Probes for LONG

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice.Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group).We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05).Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

Transthyretin (TTR) distributes thyroxine in the cerebrospinal fluid of mammals. Choroid plexus epithelial cells produce and secrete TTR, and were long recognized as the only CNS source of TTR. However, research over the last years has reported neuronal-specific expression as well, but without a clear function. Recently, we found Ttr transcripts in cells of the adult mouse subventricular zone (SVZ), the largest neural stem cell (NSC) region, but the protein was undetectable. We therefore investigated in more detail what role TTR might play in the SVZ, and when. We mapped temporal-spatial Ttr expression by re-analysing publicly available single-cell RNA-Seq data obtained from dissected mouse SVZs at E14-E17-P2-P7-P20-P61. We observed a peak in Ttr expression in NSCs, neural progenitors and differentiating cells at postnatal day 7 (P7). That is one week prior to when thyroxine serum levels peak and T3 activates SVZ-NSCs that start generating neurons and glia at a constant rate. RNAscope on P7 brain sections confirmed that few Ttr transcripts are present in a many SVZ-progenitors, oligodendrocyte precursors and neuroblasts. Unexpectedly though, no protein was detectable using commercially available antibodies, signal amplification and appropriate controls. This might suggest TTR is rapidly secreted to affect nearby cells. To test this hypothesis, we prepared neurospheres from dissected SVZ-progenitors at P7. After 7 days of proliferation, cells were dissociated, and allowed to differentiate for 1 or 5 days. In parallel with controls, we treated them once at day 0 of differentiation with a low (2.5 µg/ml) or a high dose (25 µg/ml) of human recombinant TTR, or with 5 nM T3. Low TTR doses reduced cell mitosis at day 1, as did T3. After 5 days, we counted a 30% lower proportion of differentiated neuroblasts with the highest TTR dose. That proportion had dropped 3-fold in the presence of T3. Proportions of oligodendroglia after 5 days of differentiation were only significantly higher in T3 conditions. As a result, the neuron/glia balance shifted in favour of oligodendrogenesis under T3, and borderline-significantly following high TTR doses. Altogether, the murine SVZ represents a novel region containing cells that express Ttr, with a peak at P7, despite seeming absence of the protein itself, precluding deducing its exact role. Single-cell RNA-Seq on treated neurospheres could reveal how exogenous TTR affects intracellular pathways, and whether its action is TH-dependent or not. This can help unravelling the pathophysiology of familial amyloid polyneuropathy, in which misfolded TTR proteins cause neurodegeneration.