Termination of cell-type specification gene programs by the miR-183 cluster determines the population sizes of low-threshold mechanosensitive neurons.

Touch and mechanical sensations require the development of several different kinds of sensory neurons dedicated to respond to certain types of mechanical stimuli. The transcription factor Shox2 (short stature homeobox 2) is involved in the generation of TRKB+ low-threshold mechanoreceptors (LTMRs), but mechanisms terminating this program and allowing alternative fates are unknown. Here, we show that the conditional loss of the miR-183-96-182 cluster in mouse leads to a failure of extinction of Shox2 during development and an increase in the proportion of Aδ LTMRs (TRKB+/NECAB2+) neurons at the expense of Aß slowly adapting (SA)-LTMRs (TRKC+/Runx3-) neurons. Conversely, overexpression of miR-183 cluster that represses Shox2 expression, or loss of Shox2, both increase the Aß SA-LTMRs population at the expense of Aδ LTMRs. Our results suggest that the miR-183 cluster determines the timing of Shox2 expression by direct targeting during development, and through this determines the population sizes of Aδ LTMRs and Aß SA-LTMRs.

The transcription factor Hmx1 and growth factor receptor activities control sympathetic neurons diversification.

The sympathetic nervous system relies on distinct populations of neurons that use noradrenaline or acetylcholine as neurotransmitter. We show that fating of the sympathetic lineage at early stages results in hybrid precursors from which, genetic cell-lineage tracing reveals, all types progressively emerge by principal mechanisms of maintenance, repression and induction of phenotypes. The homeobox transcription factor HMX1 represses Tlx3 and Ret, induces TrkA and maintains tyrosine hydroxylase (Th) expression in precursors, thus driving segregation of the noradrenergic sympathetic fate. Cholinergic sympathetic neurons develop through cross-regulatory interactions between TRKC and RET in precursors, which lead to Hmx1 repression and sustained Tlx3 expression, thereby resulting in failure of TrkA induction and loss of maintenance of Th expression. Our results provide direct evidence for a model in which diversification of noradrenergic and cholinergic sympathetic neurons is based on a principle of cross-repressive functions in which the specific cell fates are directed by an active suppression of the expression of transcription factors and receptors that direct the alternative fate.

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest.

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing.

Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases.IMPORTANCE Prompt and effective antimicrobial therapy is crucial for the management of patients with severe bacterial infections but is becoming increasingly difficult to provide due to emerging antibiotic resistance. The traditional methods for antibiotic susceptibility testing (AST) used in most clinical laboratories are reliable but slow with turnaround times of 2 to 3 days, which necessitates the use of empirical therapy with broad-spectrum antibiotics. There is a great need for fast and reliable AST methods that enable starting targeted treatment within a few hours to improve patient outcome and reduce the overuse of broad-spectrum antibiotics. The multiplex fluidic chip for phenotypic AST described in the present study may enable data on antimicrobial resistance within 2 to 4 h, allowing for an early initiation of appropriate antibiotic therapy.

Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control.

Despite the variety of physiological and target-related functions, little is known regarding the cellular complexity in the sympathetic ganglion. We explored the heterogeneity of mouse stellate and thoracic ganglia and found an unexpected variety of cell types. We identified specialized populations of nipple- and pilo-erector muscle neurons. These neurons extended axonal projections and were born among other neurons during embryogenesis, but remained unspecialized until target organogenesis occurred postnatally. Target innervation and cell-type specification was coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall, our results provide compelling evidence for a highly sophisticated organization of the sympathetic nervous system into discrete outflow channels that project to well-defined target tissues and offer mechanistic insight into how diversity and connectivity are established during development.