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The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to potentially damaging stimuli. The receptive field for each sensory neuron is defined by its dendritic arbor, but the mechanisms that shape dendritic architecture are incompletely understood. Using the model nociceptor, the PVD neuron in C. elegans, we determined that two types of PVD lateral branches project along the dorsal/ventral axis to generate the PVD dendritic arbor: (1) Pioneer dendrites that adhere to the epidermis, and (2) Commissural dendrites that fasciculate with circumferential motor neuron processes. Previous reports have shown that the LIM homeodomain transcription factor MEC-3 is required for all higher order PVD branching and that one of its targets, the claudin-like membrane protein HPO-30, preferentially promotes outgrowth of pioneer branches. Here, we show that another MEC-3 target, the conserved TFIIA-like zinc finger transcription factor EGL-46, adopts the alternative role of specifying commissural dendrites. The known EGL-46 binding partner, the TEAD transcription factor EGL-44, is also required for PVD commissural branch outgrowth. Double mutants of hpo-30 and egl-44 show strong enhancement of the lateral branching defect with decreased numbers of both pioneer and commissural dendrites. Thus, HPO-30/Claudin and EGL-46/EGL-44 function downstream of MEC-3 and in parallel acting pathways to direct outgrowth of two distinct classes of PVD dendritic branches.
Copyright © 2017 Elsevier Inc. All rights reserved.
Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.
Published by Oxford University Press on behalf of Nucleic Acids Research 2017.
Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.
After lesions of the somatosensory dorsal column (DC) pathway, the cortical hand representation can become unresponsive to tactile stimuli, but considerable responsiveness returns over weeks of post-lesion recovery. The reactivation suggests that preserved subthreshold sensory inputs become potentiated and axon sprouting occurs over time to mediate recovery. Here, we studied the recovery process in 3 squirrel monkeys, using high-resolution cerebral blood volume-based functional magnetic resonance imaging (CBV-fMRI) mapping of contralateral somatosensory cortex responsiveness to stimulation of distal finger pads with low and high level electrocutaneous stimulation (ES) before and 2, 4, and 6weeks after a mid-cervical level contralateral DC lesion. Both low and high intensity ES of digits revealed the expected somatotopy of the area 3b hand representation in pre-lesion monkeys, while in areas 1 and 3a, high intensity stimulation was more effective in activating somatotopic patterns. Six weeks post-lesion, and irrespective of the severity of loss of direct DC inputs (98%, 79%, 40%), somatosensory cortical area 3b of all three animals showed near complete recovery in terms of somatotopy and responsiveness to low and high intensity ES. However there was significant variability in the patterns and amplitudes of reactivation of individual digit territories within and between animals, reflecting differences in the degree of permanent and/or transient silencing of primary DC and secondary inputs 2weeks post-lesion, and their spatio-temporal trajectories of recovery between 2 and 6weeks. Similar variations in the silencing and recovery of somatotopy and responsiveness to high intensity ES in areas 3a and 1 are consistent with individual differences in damage to and recovery of DC and spinocuneate pathways, and possibly the potentiation of spinothalamic pathways. Thus, cortical deactivation and subsequent reactivation depends not only on the degree of DC lesion, but also on the severity and duration of loss of secondary as well as primary inputs revealed by low and high intensity ES.
Copyright © 2016 Elsevier Inc. All rights reserved.
INSM1 is a zinc-finger protein expressed throughout the developing nervous system in late neuronal progenitors and nascent neurons. In the embryonic cortex and olfactory epithelium, Insm1 may promote the transition of progenitors from apical, proliferative, and uncommitted to basal, terminally-dividing and neuron producing. In the otocyst, delaminating and delaminated progenitors express Insm1, whereas apically-dividing progenitors do not. This expression pattern is analogous to that in embryonic olfactory epithelium and cortex (basal/subventricular progenitors). Lineage analysis confirms that auditory and vestibular neurons originate from Insm1-expressing cells. In the absence of Insm1, otic ganglia are smaller, with 40% fewer neurons. Accounting for the decrease in neurons, delaminated progenitors undergo fewer mitoses, but there is no change in apoptosis. We conclude that in the embryonic inner ear, Insm1 promotes proliferation of delaminated neuronal progenitors and hence the production of neurons, a similar function to that in other embryonic neural epithelia. Unexpectedly, we also found that differentiating, but not mature, outer hair cells express Insm1, whereas inner hair cells do not. Insm1 is the earliest known gene expressed in outer versus inner hair cells, demonstrating that nascent outer hair cells initiate a unique differentiation program in the embryo, much earlier than previously believed.
Copyright © 2015 Elsevier B.V. All rights reserved.
Mesoscale local functional organizations of the primate spinal cord are largely unknown. Using high-resolution fMRI at 9.4 T, we identified distinct interhorn and intersegment fMRI activation patterns to tactile versus nociceptive heat stimulation of digits in lightly anesthetized monkeys. Within a spinal segment, 8 Hz vibrotactile stimuli elicited predominantly fMRI activations in the middle part of ipsilateral dorsal horn (iDH), along with significantly weaker activations in ipsilateral (iVH) and contralateral (cVH) ventral horns. In contrast, nociceptive heat stimuli evoked widespread strong activations in the superficial part of iDH, as well as in iVH and contralateral dorsal (cDH) horns. As controls, only weak signal fluctuations were detected in the white matter. The iDH responded most strongly to both tactile and heat stimuli, whereas the cVH and cDH responded selectively to tactile versus nociceptive heat, respectively. Across spinal segments, iDH activations were detected in three consecutive segments in both tactile and heat conditions. Heat responses, however, were more extensive along the cord, with strong activations in iVH and cDH in two consecutive segments. Subsequent subunit B of cholera toxin tracer histology confirmed that the spinal segments showing fMRI activations indeed received afferent inputs from the stimulated digits. Comparisons of the fMRI signal time courses in early somatosensory area 3b and iDH revealed very similar hemodynamic stimulus-response functions. In summary, we identified with fMRI distinct segmental networks for the processing of tactile and nociceptive heat stimuli in the cervical spinal cord of nonhuman primates. Significance statement: This is the first fMRI demonstration of distinct intrasegmental and intersegmental nociceptive heat and touch processing circuits in the spinal cord of nonhuman primates. This study provides novel insights into the local functional organizations of the primate spinal cord for pain and touch, information that will be valuable for designing and optimizing therapeutic interventions for chronic pain management.
Copyright © 2015 the authors 0270-6474/15/3510493-10$15.00/0.
BACKGROUND - Anomalies of the meniscus are uncommon. These anomalous formations have been predominately described in the lateral compartment of the knee. Congenital abnormalities of the medial meniscus are rare.
METHODS - Chart and radiographic review of a single patient with a symptomatic congenital abnormality of the medial meniscus.
RESULTS - The patient was a 5-year-old boy with popliteal pterygium who developed painful snapping in the medial knee after anterior hemiepiphyseodesis to improve his knee extension. The patient had achieved full-knee extension from a preoperative 45-degree flexion contracture. The newly developed snapping was attributed to the hemiepiphyseodesis implants. After implant removal, the snapping persisted and was localized at the medial joint line. Through an arthrotomy, a medial meniscus abnormality was identified and excised with resolution of symptoms.
CONCLUSIONS - This report describes a symptomatic congenital abnormality of the medial meniscus in a child with popliteal pterygium. The patient was treated with excision of the anomalous structure with complete resolution of the symptoms. This is the first report of an intra-articular knee anomaly associated with popliteal pterygium syndrome.
Inter-areal and ipsilateral cortical responses to tactile stimulation have not been well described in human S1 cortex. By taking advantage of the high signal-to-noise ratio at 7 T, we quantified blood oxygenation level dependent (BOLD) response patterns and time courses to tactile stimuli on individual distal finger pads at a fine spatial scale, and examined whether there are inter-areal (area 3b versus area 1) and interhemispheric response differences to unilateral tactile stimulation in healthy human subjects. We found that 2-Hz tactile stimulation of individual fingertips evoked detectable BOLD signal changes in both contralateral and ipsilateral area 3b and area 1. Contralateral digit activations were organized in an orderly somatotopic manner, and BOLD responses in area 3b were more digit selective than those in area 1. However, the area of cortex that was responsive to stimulation of a single digit (stimulus-response field) was similar across areas. In the ipsilateral hemisphere, response magnitudes in both areas 3b and 1 were significantly weaker than those of the contralateral hemisphere. Digit activations exhibited no clear somatotopic organizational pattern in either area 3b or area 1, yet digit selectivity was retained in area 1 but not in area 3b. The observation of distinct digit-selective responses of contralateral area 3b versus area 1 supports a higher order function of contralateral area 1 in spatial integration. In contrast, ipsilateral cortices may play a less discriminative role in the perception of unilateral tactile sensation in humans.
Copyright © 2014 Wiley Periodicals, Inc.
Skin biopsies have primarily been used to study the non-myelinated nerve fibers of the epidermis in a variety of neuropathies. In this study, we have expanded the skin biopsy technique to glabrous, non-hairy skin to evaluate myelinated nerve fibers in the most highly prevalent peripheral nerve disease, diabetic polyneuropathy (DPN). Twenty patients with DPN (Type I, n = 9; Type II, n = 11) and 16 age-matched healthy controls (age 29-73) underwent skin biopsy of the index finger, nerve conduction studies (NCS), and composite neuropathy scoring. In patients with DPN, we found a statistically significant reduction of both mechanoreceptive Meissner corpuscles (MCs) and their afferent myelinated nerve fibers (p = 0.01). This myelinated nerve fiber loss was correlated with the decreased amplitudes of sensory/motor responses in NCS. This study supports the utilization of skin biopsy to quantitatively evaluate axonal loss of myelinated nerve fibers in patients with DPN.
© 2013 Peripheral Nerve Society.
Recombination-activating gene 1 (Rag1) and Rag2 enzymes are required for T cell receptor assembly and thymocyte development. The mechanisms underlying the transcriptional activation and repression of Rag1 and Rag2 are incompletely understood. The zinc-finger protein, Zfp608, represses Rag1 and Rag2 expression when expressed in thymocytes blocking T-cell maturation. Here we show that the related zinc-finger protein, Zfp609, is necessary for Rag1 and Rag2 expression in developing thymocytes. Zfp608 represses Rag1 and Rag2 expression indirectly by repressing the expression of Zfp609. Thus, the balance of Zfp608 and Zfp609 plays a critical role in regulating Rag1 and Rag2 expression, which may manifest itself not only during development of immature thymocytes into mature T cells but also in generation of the T-cell arm of the adaptive immune system, which does not fully develop until after birth.