Nuclear actin and myocardin-related transcription factors control disuse muscle atrophy through regulation of Srf activity.

Skeletal muscle atrophy is a debilitating process that is associated with a wide variety of conditions including inactivity, disease and aging. Here, we demonstrate that the actin, myocardin-related transcription factors and serum response factor (actin-Mrtf-Srf) pathway is specifically downregulated in the muscle atrophy that is induced through disuse in mice. We show in vivo that the abolition of mechanical signals leads to the rapid accumulation of G-actin in myonuclei and the export of the Srf coactivator Mrtf-A, resulting in a decrease of Mrtf-Srf-dependent transcription that contributes to atrophy. We demonstrate that inhibition of the actin-Mrtf-Srf axis through overexpression of nuclear non-polymerizable actin, through pharmacological inhibition of Mrtf-Srf and through muscle-specific Srf deletion worsens denervation-induced atrophy. Conversely, maintenance of high levels of activity of Srf or Mrtfs in denervated muscle, through overexpression of constitutively active derivatives, counteracts atrophy. Altogether, our data provide new mechanistic insights into the control of muscle mass upon disuse atrophy by the actin-Mrtf-Srf pathway, highlighting Srf as a key mediator of mechanotransduction in muscle.

A distal intergenic region controls pancreatic endocrine differentiation by acting as a transcriptional enhancer and as a polycomb response element.

Lineage-selective expression of developmental genes is dependent on the interplay between activating and repressive mechanisms. Gene activation is dependent on cell-specific transcription factors that recognize transcriptional enhancer sequences. Gene repression often depends on the recruitment of Polycomb group (PcG) proteins, although the sequences that underlie the recruitment of PcG proteins, also known as Polycomb response elements (PREs), remain poorly understood in vertebrates. While distal PREs have been identified in mammals, a role for positive-acting enhancers in PcG-mediated repression has not been described. Here we have used a highly efficient procedure based on lentiviral-mediated transgenesis to carry out in vivo fine-mapping of, cis-regulatory sequences that control lineage-specific activation of Neurog3, a master regulator of pancreatic endocrine differentiation. Our findings reveal an enhancer region that is sufficient to drive correct spacio-temporal expression of Neurog3 and demonstrate that this same region serves as a PRE in alternative lineages where Neurog3 is inactive.

Vesicular glutamate transporter 3 immunoreactivity is present in cholinergic basal forebrain neurons projecting to the basolateral amygdala in rat.

The basal forebrain (BF) plays a role in behavioral and cortical arousal, attention, learning, and memory. It has been suggested that cholinergic BF neurons co-release glutamate, and some cholinergic BF neurons have been reported to contain vesicular glutamate transporter 3 (VGLUT3). We examined the distribution and projections of BF cholinergic neurons containing VGLUT3, by using dual-label immunofluorescence for choline acetyltransferase (ChAT) and VGLUT3, in situ hybridization, and retrograde tracing. Neurons immunoreactive (+) or containing mRNAs for both ChAT and VGLUT3 were mainly localized to the ventral pallidum and more caudal BF regions; the co-immunoreactive neurons represented 31% of cholinergic neurons in the ventral pallidum and 5-9% more caudally. Examination of cholinergic axon terminals in known target areas of BF projections indicated that the basolateral amygdaloid nucleus contained numerous terminals co-immunoreactive for ChAT and VGLUT3, whereas sampled areas of the olfactory bulb, neocortex, hippocampus, reticular thalamic nucleus, and interpeduncular nucleus were devoid of double-labeled terminals. The basolateral amygdala is innervated by cholinergic BF neurons lacking low-affinity p75 nerve growth factor receptors; many ChAT+VGLUT3+ BF neurons were immunonegative to this receptor. Twenty-five to 79% of ChAT+VGLUT3+ neurons in different BF regions were retrogradely labeled from the basolateral amygdala, up to 52% (ventral pallidum) of the retrogradely labeled ChAT+ neurons were VGLUT3+, and the largest number of amygdala-projecting ChAT+VGluT3+ neurons was found in the ventral pallidum. These findings indicate that BF cholinergic neurons containing VGLUT3 project to the basolateral amygdala and suggest that these neurons might have the capacity to release both acetylcholine and glutamate.

Developmentally regulated expression of VGLUT3 during early post-natal life.

Three subtypes of vesicular glutamate transporters, named VGLUT1-3, accumulate glutamate into synaptic vesicles. In this study, the post-natal expression of VGLUT3 was determined with specific probes and antiserums in the rat brain and compared with that of VGLUT1 and VGLUT2. The expression of VGLUT1 and VGLUT2 increases linearly during post-natal development. In contrast, VGLUT3 developmental pattern appears to have a more or less biphasic profile. A first peak of expression is centered around post-natal day 10 (P10) while the second one is reached in the adult brain. Between P1 and P15, VGLUT3 is observed in the frontal brain (striatum, accumbens, and hippocampus) and in the caudal brain (colliculi, pons and cerebellum). During a second phase extending from P15 to adulthood, the labeling of the caudal brain fades away. The adult pattern is reached at P21. We further analyzed the transient expression of VGLUT3 in the cerebellum and found it to correspond to a temporary expression in Purkinje cells. At P10 VGLUT3 immunoreactivity was present both in the soma and terminals of Purkinje cells (PC), where it colocalized with the vesicular inhibitory amino acid transporter (VIAAT). In agreement with data from the literature [Gillespie, D.C., Kim, G., Kandler, K., 2005. Inhibitory synapses in the developing auditory system are glutamatergic. Nat. Neurosci. 8, 332-338], our results suggest that during the first 2 weeks of post-natal life PC may have the potential to transiently release simultaneously GABA and glutamate.

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy.

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.

Highly restricted diversity of TCR delta chains of the amphibian Mexican axolotl (Ambystoma mexicanum) in peripheral tissues.

Gammadelta T cells localize at mammalian epithelial surfaces to exert both protective and regulatory roles in response to infections. We have previously characterized the Mexican axolotl (Ambystoma mexicanum) T cell receptor delta (TRD) chain. In this study, TRD repertoires in spleen, liver, intestine and skin from larvae, pre-adult and adult axolotls were examined and compared to the thymic TRD repertoire. A TRDV transcript without N/D diversity, TRDV1S1-TRDJ1, dominates the TRD repertoires until sexual maturation. In adult tissues, this canonical transcript is replaced by another dominant TRDV1S1-TRDJ1 transcript. In the thymus, these two transcripts are detected early in development. Our results suggest that gammadelta T cells that express the canonical TRDV1S1-TRDJ1 transcript emerge from the thymus and colonize the peripheral tissues, where they are selectively expanded by recurrent ligands. This particular situation is probably related to the neotenic state and the slow development of the axolotl. In thymectomized axolotls, TRD repertoires appear different from those of normal axolotls, suggesting that extrathymic gammadelta T cell differentiation could occur. Gene expression analysis showed the importance of the gut in T cell development.

Differential expression and regulation of the high-affinity choline transporter CHT1 and choline acetyltransferase in neurons of superior cervical ganglia.

Previous studies revealed that leukemia inhibitory factor (LIF) and retinoic acid (RA) induce a noradrenergic to cholinergic switch in cultured sympathetic neurons of superior cervical ganglia (SCG) by up-regulating the coordinate expression of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. Here, we examined the effect of both factors on high-affinity choline uptake (HACU) and on expression of the high-affinity choline transporter CHT1. We found that HACU and CHT1-mRNA levels are up-regulated by LIF and down-regulated by RA in these neurons. Thus, in contrast to LIF, RA differentially regulates the expression of the presynaptic cholinergic proteins. Moreover, we showed that untreated SCG neurons express HACU and CHT1-mRNAs at much higher levels than ChAT activity and transcripts. In intact SCG, CHT1-mRNAs are abundant and synthesized by the noradrenergic neurons themselves. This study provides the first example of CHT1 expression in neurons which do not use acetylcholine as neurotransmitter.

Structure, diversity and expression of the TCRdelta chains in the Mexican axolotl.

Mammals and birds have two major populations of T cells, based on the molecular composition and biological properties of their antigen receptors (TCR). alpha beta T cells recognize antigenic peptides linked to major histocompatibility complex (MHC) molecules, and gamma delta T cells recognize native peptide or non-peptide antigens independently of MHC. Very little is known about gamma delta T cells in ectothermic vertebrates. We have cloned and characterized the TCRdelta chains of an urodele amphibian, the Mexican axolotl (Ambystoma mexicanum). The Cdelta domain is structurally similar to its mammalian homologues and the transmembrane domain is very well conserved. Four of the six Valpha regions that can associate with Calpha (Valpha2, Valpha3, Valpha5 and Valpha6) can also associate with Cdelta, but no specific Vdelta regions were found. This suggests that the axolotl TRD locus is nested within the TRA locus, as in mammals, and that this organization has been present in all tetrapod vertebrates and in the common ancestor of Lissamphibians and mammals, for over 400 million years. Two Jdelta regions were identified, but no Ddelta segments were clearly recognized at the Vdelta-Jdelta junctions. This results in shorter and less variable CDR3 loops than in other vertebrates and the size range of the Vdelta-Jdelta junctions is similar to that of mammalian immunoglobulin light chains. Equivalent quantities of TRD mRNA were found in the lymphoid organs, and in the skin and the intestines of normal and thymectomized axolotls. The analysis of several Valpha/delta6-Cdelta and Vbeta7-Cbeta junctions showed that both the TCRdelta and the TCRbeta chains were limited in diversity in thymectomized axolotls.