FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Immobilization of C. elegans with different concentrations of an anesthetic for time-lapse imaging of dynamic protein trafficking in neurons.

Here we compare the percentage of anterograde and retrograde trafficking events as well as the average velocity of these events in worms immobilized with microbeads or 0.5-7.5 mM tetramisole. Our results show that the percentage and average velocity of TIR-1 ::GFP moving events in the C. elegans AWC axons are not significantly different between worms immobilized with 7.5 mM tetramisole and other conditions. Our results suggest that 7.5 mM tetramisole, compared to 0.5 mM, 1 mM, and 2 mM tetramisole, does not have a significant effect on the axonal transport of TIR-1 ::GFP along the AWC axons.

Limited Hyperoxia-Induced Proliferative Retinopathy (LHIPR) as a Model of Retinal Fibrosis, Angiogenesis, and Inflammation.

The progression to fibrosis and traction in retinopathy of prematurity (ROP) and other ischemic retinopathies remains an important clinical and surgical challenge, necessitating a comprehensive understanding of its pathogenesis. Fibrosis is an unbalanced deposition of extracellular matrix components responsible for scar tissue formation with consequent tissue and organ impairment. Together with retinal traction, it is among the main causes of retinal detachment and vision loss. We capitalize on the Limited Hyperoxia Induced Retinopathy (LHIPR) model, as it reflects the more advanced pathological phenotypes seen in ROP and other ischemic retinopathies. To model LHIPR, we exposed wild-type C57Bl/6J mouse pups to 65% oxygen from P0 to P7. Then, the pups were returned to room air to recover until later endpoints. We performed histological and molecular analysis to evaluate fibrosis progression, angiogenesis, and inflammation at several time points, from 1.5 months to 9 months. In addition, we performed in vivo retinal imaging by optical coherence tomography (OCT) or OCT Angiography (OCTA) to follow the fibrovascular progression in vivo. Although the retinal morphology was relatively preserved, we found a progressive increase in preretinal fibrogenesis over time, up to 9 months of age. We also detected blood vessels in the preretinal space as well as an active inflammatory process, altogether mimicking advanced preretinal fibrovascular disease in humans.

CEPsh glia development is not required for general AWC identity or AWC asymmetry.

The Caenorhabditis elegans VAB-3/Pax6 homeodomain protein was previously shown to play a role in both the development of cephalic sheath (CEPsh) glia and asymmetric differentiation of AWC olfactory neuron subtypes AWC ON /AWC OFF . Here we show that vab-3 is not required for the specification of general AWC identity. We also show that some vab-3 mutant alleles with defective CEPsh glia development displayed wild-type AWC asymmetry. These results suggest that vab-3 has separable roles in CEPsh glia development and AWC asymmetry. Together, our results suggest that general AWC identity and AWC asymmetry are not dependent on the development of CEPsh glia.

Using a consensus acupoints regimen to explore the relationship between acupuncture sensation and lumbar spinal postoperative analgesia: A retrospective analysis of prospective clinical cooperation.

OBJECTIVE: This study evaluated the effectiveness of acupuncture treatment on postoperative pain in patients with degenerative lumbar spine disease, and explored the relationship between the postoperative analgesic effect of acupuncture and the sensation of acupuncture experienced by the patients. METHODS: This retrospective study analyzed the medical records of 97 patients who had undergone an operation by the same surgeon due to degenerative lumbar disease. These patients were divided into acupuncture group (n = 32), patient-controlled analgesia (PCA) group (n = 27), and oral analgesia group (n = 38) according to the different postoperative analgesic methods. During their hospitalization, patients completed daily evaluations of their pain using a visual analogue scale (VAS), and injection times of supplemental meperidine were recorded. Also, the Chinese version of the Massachusetts General Hospital Acupuncture Sensation Scale (C-MASS) was used in the acupuncture group. RESULTS: Each of the three treatment groups showed significant reductions in postoperative pain, as shown by reduced VAS scores. The acupuncture group, however, had less rebound pain (P < 0.05) than the other two groups. Both the acupuncture and PCA groups experienced acute analgesic effects that were superior to those in the oral analgesia group. In addition, the higher the C-MASS index on the second day after surgery, the lower the VAS score on the fourth day after surgery. There was also a significant difference in the "dull pain" in the acupuncture sensation. CONCLUSION: The results demonstrated that acupuncture was beneficial for postoperative pain and discomfort after simple surgery for degenerative spinal disease. It is worth noting that there was a disproportionate relevance between the patient's acupuncture sensation and the improvement of pain VAS score.

mNG-tagged mls-2 knock-in alleles in C. elegans.

The Caenorhabditis elegans HMX/NKX MLS-2 transcription factor was previously shown to play sequential roles in AWC general identity and the stochastic AWCON/AWCOFF subtype choice during embryogenesis. Here we analyze the expression pattern of endogenous mls-2 during AWC development using mNeonGreen (mNG) knock-in strains. Similar to transgenic GFP::MLS-2, functional mNG::MLS-2 knock-in displayed nuclear localization in AWC precursor cells but was not observed in AWC during the later embryonic stage. These results suggest that the expression of mls-2 is below the detectable level and/or the stability of MLS-2 protein is very low in AWC cells.

Analysis of unc-62 expression pattern in C. elegans embryonic AWC neurons.

The Caenorhabditis elegans UNC-62 homothorax/Meis/TALE homeodomain protein functions sequentially to regulate general identity of the AWC olfactory neuron pair and the stochastic choice of asymmetric AWC subtypes during embryogenesis. Here we analyze the expression pattern of unc-62 during AWC development using an integrated unc-62::GFP fosmid rescuing transgene. UNC-62::GFP was not detected in AWC neurons in early or late embryos. These results are consistent with previous single-cell RNA sequencing data and also suggest an undetectable level of unc-62 expression and/or low stability of UNC-62 protein in AWC neurons during embryogenesis.

Synergistic roles of homeodomain proteins UNC-62 homothorax and MLS-2 HMX/NKX in the specification of olfactory neurons in Caenorhabditis elegans.

General identity of the Caenorhabditis elegans AWC olfactory neuron pair is specified by the OTX/OTD transcription factor CEH-36 and the HMG-box transcription factor SOX-2, followed by asymmetrical differentiation of the pair into two distinct subtypes, default AWCOFF and induced AWCON, through a stochastic signaling event. The HMX/NKX transcription factor MLS-2 regulates the expression of ceh-36 to specify general AWC identity. However, general AWC identity is lost in only one of the two AWC cells in the majority of mls-2 null mutants displaying defective general AWC identity, suggesting that additional transcription factors have a partially overlapping role with MLS-2 in the specification of general AWC identity. Here, we identify a role of unc-62, encoding a homothorax/Meis/TALE homeodomain protein, in the specification of general AWC identity. As in mls-2 null mutants, unc-62 null mutants showed an incomplete penetrance in loss of general AWC identity. However, unc-62; mls-2 double mutants display a nearly complete penetrance of identity loss in both AWC cells. Thus, unc-62 and mls-2 have a partially overlapping function in the specification of general AWC identity. Furthermore, our genetic results suggest that mls-2 and unc-62 act cell autonomously in promoting the AWCON subtype. Together, our findings reveal the sequential roles of the unc-62 and mls-2 pair in AWC development, specification of general AWC identity in early embryogenesis, and asymmetric differentiation of AWC subtypes in late embryogenesis.

A universal transportin protein drives stochastic choice of olfactory neurons via specific nuclear import of a sox-2-activating factor.

Stochastic neuronal cell fate choice involving notch-independent mechanisms is a poorly understood biological process. The Caenorhabditis elegans AWC olfactory neuron pair asymmetrically differentiates into the default AWCOFF and induced AWCON subtypes in a stochastic manner. Stochastic choice of the AWCON subtype is established using gap junctions and SLO BK potassium channels to repress a calcium-activated protein kinase pathway. However, it is unknown how the potassium channel-repressed calcium signaling is translated into the induction of the AWCON subtype. Here, we identify a detailed working mechanism of how the homeodomain-like transcription factor NSY-7, previously described as a repressor in the maintenance of AWC asymmetry, couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype through the identification of a unique imb-2 (transportin 1) allele. imb-2 loss-of-function mutants are not viable; however, we identify a viable imb-2 allele from an unbiased forward genetic screen that reveals a specific role of imb-2 in AWC olfactory neuron asymmetry. IMB-2 specifically drives nuclear import of NSY-7 within AWC neurons to transactivate the expression of the high mobility group (HMG)-box transcription factor SOX-2 for the specification of the AWCON subtype. This study provides mechanistic insight into how NSY-7 couples SLO BK potassium channels to transactivation of sox-2 expression for the induction of the AWCON subtype. Our findings also provide structure-function insight into a conserved amino acid residue of transportins in brain development and suggest its dysfunction may lead to human neurological disorders.

Engulfing cells promote neuronal regeneration and remove neuronal debris through distinct biochemical functions of CED-1.

Two important biological events happen coincidently soon after nerve injury in the peripheral nervous system in C. elegans: removal of axon debris and initiation of axon regeneration. But, it is not known how these two events are co-regulated. Mutants of ced-1, a homolog of Draper and MEGF10, display defects in both events. One model is that those events could be related. But our data suggest that they are actually separable. CED-1 functions in the muscle-type engulfing cells in both events and is enriched in muscle protrusions in close contact with axon debris and regenerating axons. Its two functions occur through distinct biochemical mechanisms; extracellular domain-mediated adhesion for regeneration and extracellular domain binding-induced intracellular domain signaling for debris removal. These studies identify CED-1 in engulfing cells as a receptor in debris removal but as an adhesion molecule in neuronal regeneration, and have important implications for understanding neural circuit repair after injury.

Asymmetric development of the nervous system.

The human nervous system consists of seemingly symmetric left and right halves. However, closer observation of the brain reveals anatomical and functional lateralization. Defects in brain asymmetry correlate with several neurological disorders, yet our understanding of the mechanisms used to establish lateralization in the human central nervous system is extremely limited. Here, we review left-right asymmetries within the nervous system of humans and several model organisms, including rodents, Zebrafish, chickens, Xenopus, Drosophila, and the nematode Caenorhabditis elegans. Comparing and contrasting mechanisms used to develop left-right asymmetry in the nervous system can provide insight into how the human brain is lateralized. Developmental Dynamics 247:124-137, 2018. © 2017 Wiley Periodicals, Inc.

Mechanisms controlling diversification of olfactory sensory neuron classes.

Animals survive in harsh and fluctuating environments using sensory neurons to detect and respond to changes in their surroundings. Olfactory sensory neurons are essential for detecting food, identifying danger, and sensing pheromones. The ability to sense a large repertoire of different types of odors is crucial to distinguish between different situations, and is achieved through neuronal diversity within the olfactory system. Here, we review the developmental mechanisms used to establish diversity of olfactory sensory neurons in various model organisms, including Caenorhabditis elegans, Drosophila, and vertebrate models. Understanding and comparing how different olfactory neurons develop within the nervous system of different animals can provide insight into how the olfactory system is shaped in humans.

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

Stochastic left-right neuronal asymmetry in Caenorhabditis elegans.

Left-right asymmetry in the nervous system is observed across species. Defects in left-right cerebral asymmetry are linked to several neurological diseases, but the molecular mechanisms underlying brain asymmetry in vertebrates are still not very well understood. The Caenorhabditis elegans left and right amphid wing 'C' (AWC) olfactory neurons communicate through intercellular calcium signalling in a transient embryonic gap junction neural network to specify two asymmetric subtypes, AWCOFF (default) and AWCON (induced), in a stochastic manner. Here, we highlight the molecular mechanisms that establish and maintain stochastic AWC asymmetry. As the components of the AWC asymmetry pathway are highly conserved, insights from the model organism C. elegans may provide a window onto how brain asymmetry develops in humans.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Protective Effects of Costunolide against Hydrogen Peroxide-Induced Injury in PC12 Cells.

Oxidative stress-mediated cellular injury has been considered as a major cause of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The scavenging of reactive oxygen species (ROS) mediated by antioxidants may be a potential strategy for retarding the diseases' progression. Costunolide (CS) is a well-known sesquiterpene lactone, used as a popular herbal remedy, which possesses anti-inflammatory and antioxidant activity. This study aimed to investigate the protective role of CS against the cytotoxicity induced by hydrogen peroxide (H2O2) and to elucidate potential protective mechanisms in PC12 cells. The results showed that the treatment of PC12 cells with CS prior to H2O2 exposure effectively increased the cell viability. Furthermore, it decreased the intracellular ROS, stabilized the mitochondria membrane potential (MMP), and reduced apoptosis-related protein such as caspase 3. In addition, CS treatment attenuated the cell injury by H2O2 through the inhibition of phosphorylation of p38 and the extracellular signal-regulated kinase (ERK). These results demonstrated that CS is promising as a potential therapeutic candidate for neurodegenerative diseases resulting from oxidative damage and further research on this topic should be encouraged.

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

Ptpn22 and Cd2 Variations Are Associated with Altered Protein Expression and Susceptibility to Type 1 Diabetes in Nonobese Diabetic Mice.

By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.

Postmitotic diversification of olfactory neuron types is mediated by differential activities of the HMG-box transcription factor SOX-2.

Diversification of neuron classes is essential for functions of the olfactory system, but the underlying mechanisms that generate individual olfactory neuron types are only beginning to be understood. Here we describe a role of the highly conserved HMG-box transcription factor SOX-2 in postmitotic specification and alternative differentiation of the Caenorhabditis elegans AWC and AWB olfactory neurons. We show that SOX-2 partners with different transcription factors to diversify postmitotic olfactory cell types. SOX-2 functions cooperatively with the OTX/OTD transcription factor CEH-36 to specify an AWC "ground state," and functions with the LIM homeodomain factor LIM-4 to suppress this ground state and drive an AWB identity instead. Our findings provide novel insights into combinatorial codes that drive terminal differentiation programs in the nervous system and reveal a biological function of the deeply conserved Sox2 protein that goes beyond its well-known role in stem cell biology.