Discoveries in structure and physiology of mechanically activated ion channels.

The ability to sense physical forces is conserved across all organisms. Cells convert mechanical stimuli into electrical or chemical signals via mechanically activated ion channels. In recent years, the identification of new families of mechanosensitive ion channels-such as PIEZO and OSCA/TMEM63 channels-along with surprising insights into well-studied mechanosensitive channels have driven further developments in the mechanotransduction field. Several well-characterized mechanosensory roles such as touch, blood-pressure sensing and hearing are now linked with primary mechanotransducers. Unanticipated roles of mechanical force sensing continue to be uncovered. Furthermore, high-resolution structures representative of nearly every family of mechanically activated channel described so far have underscored their diversity while advancing our understanding of the biophysical mechanisms of pressure sensing. Here we summarize recent discoveries in the physiology and structures of known mechanically activated ion channel families and discuss their implications for understanding the mechanisms of mechanical force sensing.

Evidence for developmentally programmed transdifferentiation in mouse esophageal muscle.

Transdifferentiation is a relatively rare phenomenon in which cells of one differentiated type and function switch to a second discrete identity. In vertebrate embryos, smooth muscle and skeletal muscle are distinct tissues that arise from separate compartments of the mesoderm. The musculature of the mouse esophagus was found to undergo a conversion from smooth muscle in the fetus to skeletal muscle during early postnatal development. The switch from smooth to skeletal muscle features the transitory appearance of individual cells expressing a mixed phenotype, which suggests that this conversion is a result of programmed transdifferentiation.

Regulation of neurotrophin-3 expression by epithelial-mesenchymal interactions: the role of Wnt factors.

Neurotrophins regulate survival, axonal growth, and target innervation of sensory and other neurons. Neurotrophin-3 (NT-3) is expressed specifically in cells adjacent to extending axons of dorsal root ganglia neurons, and its absence results in loss of most of these neurons before their axons reach their targets. However, axons are not required for NT-3 expression in limbs; instead, local signals from ectoderm induce NT-3 expression in adjacent mesenchyme. Wnt factors expressed in limb ectoderm induce NT-3 in the underlying mesenchyme. Thus, epithelial-mesenchymal interactions mediated by Wnt factors control NT-3 expression and may regulate axonal growth and guidance.

Making the pain connection.

Dorsal root ganglia (DRG) neurons include multiple types of sensory neurons with well-appreciated anatomical and physiological distinctions. In this issue of Neuron, Chen et al. adds to our molecular understanding of these differences by reporting that DRG11, a paired homeodomain transcription factor, is specifically required for the proper development of pain-sensing nociceptive neurons.

Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.

Spinal sensory ganglia have been shown to contain neuronal subpopulations with different functions and neurotrophin dependencies. Neurotrophins act, in large part, through Trk receptor tyrosine kinases: nerve growth factor (NGF) via TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) via TrkB, and neurotrophin-3 (NT-3) via TrkC. In the present paper, we use antibodies to TrkA, TrkB, and TrkC to characterize their expression patterns and to determine which subpopulations of cells are lost in mice lacking individual neurotrophins or Trk receptors. Despite previous reports of Trk receptor mRNAs in neural crest cells, we detect Trk receptor proteins only in neurons and not in neural crest cells or neuronal precursors. Comparisons of neonatal mice deficient in NT-3 or its cognate receptor TrkC have shown that there is a much greater deficiency in spinal sensory neurons in the former, suggesting that NT-3 may activate receptors in addition to TrkC. Using the same antibodies, we show that, during the major period of neurogenesis, NT-3 is required to maintain neurons that express TrkB in addition to those that express TrkC but is not essential for neurons expressing TrkA. Results also indicate that survival of cells expressing both receptors can be maintained by activation of either one alone. NT-3 can thus activate more than one Trk receptor in vivo, which when coexpressed are functionally redundant.

Roles of Wnt proteins in neural development and maintenance.

Many constituents of Wnt signaling pathways are expressed in the developing and mature nervous systems. Recent work has shown that Wnt signaling controls initial formation of the neural plate and many subsequent patterning decisions in the embryonic nervous system, including formation of the neural crest. Wnt signaling continues to be important at later stages of development. Wnts have been shown to regulate the anatomy of the neuronal cytoskeleton and the differentiation of synapses in the cerebellum. Wnt signaling has been demonstrated to regulate apoptosis and may participate in degenerative processes leading to cell death in the aging brain.

Trk receptors: mediators of neurotrophin action.

The four mammalian neurotrophins - NGF, BDNF, NT-3 and NT-4 - each bind and activate one or more of the Trk family of receptor tyrosine kinases. Through these receptors, neurotrophins activate many intracellular signaling pathways, including those controlled by Ras, the Cdc42/Rac/RhoG protein family, MAPK, PI3K and PLC-gamma, thereby affecting both development and function of the nervous system. During the past two years, several novel signaling pathways controlled by Trk receptors have been characterized, and it has become clear that membrane transport and sorting controls Trk-receptor-mediated signaling because key intermediates are localized to different membrane compartments. Three-dimensional structures of the Trk receptors, in one instance in association with a neurotrophin, have revealed the structural bases underlying specificity in neurotrophin signaling.

Locomotor networks are targets of modulation by sensory transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 channels.

It is well recognized that proprioceptive afferent inputs can control the timing and pattern of locomotion. C and Adelta afferents can also affect locomotion but an unresolved issue is the identity of the subsets of these afferents that encode defined modalities. Over the last decade, the transient receptor potential (TRP) ion channels have emerged as a family of non-selective cation conductances that can label specific subsets of afferents. We focus on a class of TRPs known as ThermoTRPs which are well known to be sensor receptors that transduce changes in heat and cold. ThermoTRPs are known to help encode somatosensation and painful stimuli, and receptors have been found on C and Adelta afferents with central projections onto dorsal horn laminae. Here we show, using in vitro neonatal mouse spinal cord preparations, that activation of both spinal and peripheral transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) afferent terminals modulates central pattern generators (CPGs). Capsaicin or menthol and cooling modulated both sacrocaudal afferent (SCA) evoked and monoaminergic drug-induced rhythmic locomotor-like activity in spinal cords from wild type but not TRPV1-null (trpv1(-/-)) or TRPM8-null (trpm8(-/-)) mice, respectively. Capsaicin induced an initial increase in excitability of the lumbar motor networks, while menthol or cooling caused a decrease in excitability. Capsaicin and menthol actions on CPGs involved excitatory and inhibitory glutamatergic mechanisms, respectively. These results for the first time show that dedicated pathways of somatosensation and pain identified by TRPV1 or TRPM8 can target spinal locomotor CPGs.

Identification and purification of a factor that binds to the Mlu I cell cycle box of yeast DNA replication genes.

In Saccharomyces cerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently.

Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice.

In developing mouse embryos, MyoD family regulatory genes are expressed specifically in muscle precursors and mature myofibers. This pattern, taken together with the well-established ability of MyoD family members to convert a variety of cell types to skeletal muscle, suggests a significant role for these genes in regulating skeletal myogenesis. The possibility that expression of these genes may be causally associated with segregation of the myogenic lineage from other mesodermal derivatives, or with the subsequent maintenance of muscle phenotypes at later times, raises the issue of how MyoD family genes are themselves regulated during development. In this work, we have initiated studies to identify DNA sequences that govern Myf-5 and MRF4 (herculin, myf-6) transcription. Myf-5 is the first of the MyoD family to be expressed in the developing mouse embryo, while MRF4 is the most abundantly expressed myogenic factor in postnatal animals. In spite of their strikingly divergent patterns of expression, Myf-5 and MRF4 are tightly linked in the mouse genome; their translational start codons are only 8.5 kilobases apart. Here, the 5' flanking regions of the mouse Myf-5 and MRF4 genes were separately linked to a bacterial beta-galactosidase (lacZ) gene, and these constructs were each used to produce several lines of transgenic mice. Transgene expression was monitored by X-gal staining of whole embryos and by in situ hybridization of embryo sections. For the Myf-5/lacZ lines, the most intense transgene expression was in the visceral arches and their craniofacial muscle derivatives, beginning at day 8.75 post coitum (p.c.). This correlates with endogenous Myf-5 expression in visceral arches.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome.

MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals.

The Drosophila gene tailless is expressed at the embryonic termini and is a member of the steroid receptor superfamily.

The zygotically active tailless (tll) gene plays a key role in the establishment of nonmetameric domains at the anterior and posterior poles of the Drosophila embryo. We have cloned the tll gene and show that it encodes a protein with striking similarity to steroid hormone receptors in both the DNA binding "finger" and ligand binding domains. tll RNA is initially expressed in embryos in two mirror-image symmetrical domains; this pattern then quickly resolves into a pattern consistent with the mutant phenotype: a posterior cap and an anterior dorsal stripe. That the tll gene may also play a role in the nervous system is suggested by its strong expression in the forming brain and transient expression in the peripheral nervous system.