SUMMARYIn the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell type–specific differentiation programs. We identified developmental stage–specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to a terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of normal retina than was that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.

Hindlimb pain models developed in rats have been transposed to mice, but assumed sciatic nerve neuroanatomic similarities have not been examined. We compared sciatic nerve structural organization in mouse strains (C57BL/6J, DBA/2J, and B6129PF2/J) and rat strains (Wistar, Brown Norway, and Sprague-Dawley). Dissection and retrograde labeling showed mouse sciatic nerve origins predominantly from the third lumbar (L3) and L4 spinal nerves, unlike the L4 and L5 in rats. Proportionate contributions by each level differed significantly between strains in both mice and rats. Whereas all rats had six lumbar vertebrae, variable patterns in mice included mostly five vertebrae in DBA/2J, mostly six vertebrae in C57BL/6J, and a mix in B6129PF2/J. Mice with a short lumbar vertebral column showed a rostral shift in relative contributions to the sciatic nerve by L3 and L4. Ligation of the mouse L4 nerve created hyperalgesia similar to that in rats after L5 ligation, and motor changes were similar after mouse L4 and rat L5 ligation (foot cupping) and after mouse L3 and rat L4 ligation (flexion weakness). Thus, mouse L3 and L4 neural segments are anatomically and functionally homologous with rat L4 and L5 segments. Neuronal changes after distal injury or inflammation should be sought in the mouse L3 and L4 ganglia, and the spinal nerve ligation model in mice should involve ligation of the L4 nerve while L3 remains intact. Strain-dependent variability in segmental contributions to the sciatic nerve may account in part for genetic differences in pain behavior after spinal nerve ligation.

Pain is the leading cause of emergency department visits, hospitalizations, and daily suffering in individuals with sickle cell disease (SCD). The pathologic mechanisms leading to the perception of pain during acute RBC sickling episodes and development of chronic pain remain poorly understood and ineffectively treated. We provide the first study that explores nociceptor sensitization mechanisms that contribute to pain behavior in mice with severe SCD. Sickle mice exhibit robust behavioral hypersensitivity to mechanical, cold, and heat stimuli. Mechanical hypersensitivity is further exacerbated when hypoxia is used to induce acute sickling. Behavioral mechanical hypersensitivity is mediated in part by enhanced excitability to mechanical stimuli at both primary afferent peripheral terminal and sensory membrane levels. In the present study, inhibition of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) with the selective antagonist A-425619 reversed the mechanical sensitization at both primary afferent terminals and isolated somata, and markedly attenuated mechanical behavioral hypersensitivity. In contrast, inhibition of TRPA1 with HC-030031 had no effect on mechanical sensitivity. These results suggest that the TRPV1 receptor contributes to primary afferent mechanical sensitization and a substantial portion of behavioral mechanical hypersensitivity in SCD mice. Therefore, TRPV1-targeted compounds that lack thermoregulatory side effects may provide relief from pain in patients with SCD. (Blood. 2011;118(12):3376-3383) IntroductionSickle cell disease (SCD) is a major health care and socioeconomic problem that affects millions of people worldwide. In the United States alone, SCD affects Ͼ 80 000 people, the majority of whom are African American. Pain is the hallmark symptom of SCD and the leading cause of emergency department visits, hospitalizations, and daily suffering. 1 Patients suffer unpredictable, incapacitating acute pain episodes that are believed to result from red blood cell (RBC) sickling and "vaso-occlusion." However, many features of this pain are not explained by hemoglobin polymerization and vascular obstruction. Furthermore, individuals with SCD often develop chronic pain syndromes that are poorly understood and ineffectively treated. 1,2 The frequency and severity of pain is associated with increased mortality and profoundly erodes patients' quality of life. 3 Because they are often from minority and lower socioeconomic groups, SCD patients are commonly underserved and suboptimally treated.The pathologic mechanisms leading to the perception of pain during RBC sickling episodes and the transition from acute to chronic pain remain poorly understood. 1,2 Patient descriptors of SCD pain include neuropathic pain attributes such as "aching," "shooting," and "stabbing," as well as nociceptive pain qualities such as "sharp," "throbbing," and "pounding." These descriptors depict spontaneous pain or nonstimulus-evoked pain. 2,4 Furthermore, increased hospital admissions and reports of...

Spinal muscular atrophy is a genetic disorder caused by deletion of the survival motor neuron 1 (SMN1) gene that leads to loss of motor neurons in the spinal cord. Though motor neurons are selectively lost during SMA pathology, selective replacement of SMN in motor neurons does not lead to full rescue in mouse models. Due to the ubiquitous expression of SMN, it is likely that other cell types besides motor neurons are affected by its disruption and therefore may contribute to disease pathology. Here we show that astrocytes in SMAΔ7 mouse spinal cord and from SMA induced pluripotent stem cells (iPSCs) exhibit morphological and cellular changes indicative of activation prior to overt motor neuron loss. Furthermore, our in vitro studies show mis regulation of basal calcium and decreased response to ATP stimulation indicating abnormal astrocyte function. Together, these data show for the first time early disruptions in astrocytes that may contribute to SMA disease pathology.

Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1 among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter (<27 µm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPα promoter, enabling identification of CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4 revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC specifically target TRPA1. Additionally, few large neurons (≥27 µm diameter) responded to AITC (6%) or cinnamaldehyde (4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1 is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.