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Mammalian skin is innervated by diverse, unmyelinated C fibers that are associated with senses of pain, itch, temperature, or touch. A key developmental question is how this neuronal cell diversity is generated during development. We reported previously that the runt domain transcription factor Runx1 is required to coordinate the development of these unmyelinated cutaneous sensory neurons, including VGLUT3+low-threshold c-mechanoreceptors (CLTMs), MrgprD+polymodal nociceptors, MrgprA3+pruriceptors, MrgprB4+c-mechanoreceptors, and others. However, how these Runx1-dependent cutaneous sensory neurons are further segregated is poorly illustrated. Here, we find that the Runx1-dependent transcription factor geneZfp521is expressed in, and required for establishing molecular features that define, VGLUT3+CLTMs. Furthermore, Runx1 and Zfp521 form a classic incoherent feedforward loop (I-FFL) in controlling molecular identities that normally belong to MrgprD+neurons, with Runx1 and Zfp51 playing activator and repressor roles, respectively (in genetic terms). A knock-out ofZfp521allows prospective VGLUT3 lineage neurons to acquire MrgprD+neuron identities. Furthermore, Runx1 might form other I-FFLs to regulate the expression of MrgprA3 and MrgprB4, a mechanism preventing these genes from being expressed in Runx1-persistent VGLUT3+and MrgprD+neurons. The evolvement of these I-FFLs provides an explanation for how modality-selective sensory subtypes are formed during development and may also have intriguing implications for sensory neuron evolution and sensory coding.

Background: The cellular and molecular programs that control specific types of pain are poorly understood. We reported previously that the runt domain transcription factor Runx1 is initially expressed in most nociceptors and controls sensory neuron phenotypes necessary for inflammatory and neuropathic pain. Results: Here we show that expression of Runx1-dependent ion channels and receptors is distributed into two nociceptor populations that are distinguished by persistent or transient Runx1 expression. Conditional mutation of Runx1 at perinatal stages leads to preferential impairment of Runx1-persistent nociceptors and a selective defect in inflammatory pain. Conversely, constitutive Runx1 expression in Runx1-transient nociceptors leads to an impairment of Runx1-transient nociceptors and a selective deficit in neuropathic pain. Notably, the subdivision of Runx1-persistent and Runx1-transient nociceptors does not follow the classical nociceptor subdivision into IB4+ nonpeptidergic and IB4- peptidergic populations. Conclusion: Altogether, we have uncovered two distinct Runx1-dependent nociceptor differentiation programs that are permissive for inflammatory versus neuropathic pain. These studies lend support to a transcription factor-based distinction of neuronal classes necessary for inflammatory versus neuropathic pain.

Mrg class G-protein-coupled receptors (GPCRs) are expressed exclusively in sensory neurons in the trigeminal and dorsal root ganglia. Pharmacological activation of Mrg proteins is capable of modulating sensory neuron activities and elicits nociceptive effects. In this study, we illustrate a control mechanism that allows the Runx1 runt domain transcription factor to generate compartmentalized expression of these sensory GPCRs. Expression ofMrgA,MrgB, andMrgCsubclasses is confined to an “A/B/C” neuronal compartment that expresses Runx1 transiently (or does not express Runx1), whereasMrgDexpression is restricted to a “D” compartment with persistent Runx1 expression.Runx1is initially required for the expression of allMrggenes. However, during late development Runx1 becomes a repressor forMrgA/B/Cgenes. As a result,MrgA/B/Cexpression persists only in the Runx1−“A/B/C” compartment. In Δ446mice, in which Runx1 lacks the C-terminal repression domain, expression ofMrgA/B/Cgenes is dramatically expanded into the Runx1+“D” compartment.MrgDexpression, however, is resistant to Runx1-mediated repression in the “D” compartment. Therefore, the creation of Runx1+and Runx1−compartments, in conjunction with different responses ofMrggenes to Runx1-mediated repression, results in the compartmentalized expression ofMrgA/B/CversusMrgDgenes. Within theMrgA/B/Ccompartment,MrgB4-expressing neurons innervate exclusively the hairy skin. Here we found that Smad4, a downstream component of bone morphological protein-mediated signaling, is required selectively for the expression ofMrgB4. Our study suggests a new line of evidence that specification of sensory subtypes is established progressively during perinatal and postnatal development.

At the core of the mammalian circadian clock is a feedback loop in which the heterodimeric transcription factor CLOCK-Brain, Muscle Arnt-like-1 (BMAL1) drives expression of its negative regulators, periods (PERs) and cryptochromes (CRYs). Here, we provide evidence that CLOCK-Interacting Protein, Circadian (CIPC) is an additional negative-feedback regulator of the circadian clock. CIPC exhibits circadian regulation in multiple tissues, and it is a potent and specific inhibitor of CLOCK-BMAL1 activity that functions independently of CRYs. CIPC-CLOCK protein complexes are present in vivo, and depletion of endogenous CIPC shortens the circadian period length. CIPC is unrelated to known proteins and has no recognizable homologues outside vertebrates. Our results suggest that negative feedback in the mammalian circadian clock is divided into distinct pathways, and that the addition of new genes has contributed to the complexity of vertebrate clocks.

The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-α (TGF-α) as a likely SCN inhibitor of locomotion. TGF-α is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.

Membrane-associated guanylate kinases (MAGUKs) contain multiple protein-binding domains that allow them to assemble specific multiprotein complexes in particular regions of the cell. CASK/LIN-2, a MAGUK required for EGF receptor localization and signalling in Caenorhabditis elegans, contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3 and guanylate kinase-like domains. In adult rat brain, CASK is concentrated at neuronal synapses and binds to the cell-surface proteins neurexin and syndecan and the cytoplasmic proteins Mint/LIN-10 and Veli/LIN-7. Here we report that, through its guanylate kinase domain, CASK interacts with Tbr-1, a T-box transcription factor that is involved in forebrain development. CASK enters the nucleus and binds to a specific DNA sequence (the T-element) in a complex with Tbr-1. CASK acts as a coactivator of Tbr-1 to induce transcription of T-element containing genes, including reelin, a gene that is essential for cerebrocortical development. Our findings show that a MAGUK which is usually associated with cell junctions has a transcription regulation function.

Background The neurocircuits that process somatic sensory information in the dorsal horn of the spinal cord are still poorly understood, with one reason being the lack of Cre lines for genetically marking or manipulating selective subpopulations of dorsal horn neurons. Here we describe Tac2-Cre mice that were generated to express the Cre recombinase gene from the Tac2 locus. Tachykinin 2 (Tac2) encodes a neurotransmitter, neurokinin B (NKB). Results By crossing Tac2-Cre mice with ROSA26-tdTomato reporter mice, we directly visualized Tac2 lineage neurons in the dorsal root ganglia, the dorsal horn of the spinal cord, and many parts of the brain including the olfactory bulb, cerebral cortex, amygdala, hippocampus, habenula, hypothalamus, and cerebellum. This Tac2-Cre allele itself was a null allele for the Tac2 gene. Behavioral analyses showed that Tac2 homozygous null mice responded normally to a series of algogenic (pain-inducing) and pruritic (itch-inducing) stimuli. Conclusions Tac2-Cre mice are a useful tool to mark specific subsets of neurons in the sensory ganglia, the dorsal spinal cord, and the brain. These mice can also be used for future genetic manipulations to study the functions of Tac2-expressing neurons or the functions of genes expressed in these neurons.