The implication of hinge 1 and hinge 4 in micro-dystrophin gene therapy for Duchenne muscular dystrophy

Human gene therapy

2022 Oct 31

Wasala, LP;Watkins, T;Wasala, N;Burke, M;Yue, Y;Lai, Y;Yao, G;Duan, D;
PMID: 36310439 | DOI: 10.1089/hum.2022.180

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by dystrophin deficiency. Dystrophin consists of the amino terminus, central rod domain with 24 spectrin-like repeats and four hinges (H), cysteine-rich domain, and carboxyl terminus. Several highly abbreviated micro-dystrophins are currently in clinical trials. They all carry H1 and H4. Here we investigated whether these two hinges are essential for micro-dystrophin function in murine DMD models. Three otherwise identical micro-dystrophins were engineered to contain H1 and/or H4 and were named H1/H4 (with both H1 and H4), ∆H1 (without H1), and ∆H4 (without H4). These constructs were packaged in adeno-associated virus serotype-9 and delivered to the tibialis anterior muscle of 3-month-old male mdx4cv mice (1E12 vector genome particles/muscle). Three months later, we detected equivalent micro-dystrophin expression in total muscle lysate. However, only H1/H4 and ∆H1 showed correct sarcolemmal localization. ∆H4 mainly existed as subsarcolemmal aggregates. H1/H4 and ∆H1, but not ∆H4, fully restored the dystrophin-associated protein complex and significantly improved the specific muscle force. Eccentric contraction-induced force decline was best protected by H1/H4, followed by ∆H1, but not by ∆H4. Next, we compared H1/H4 and ∆H1 in 6-week-old male mdx mice by intravenous injection (1E13 vector genome particles/mouse). Four months post-injection, H1/H4 significantly outperformed ∆H1 in extensor digitorum longus muscle force measurements but two constructs yielded comparable ECG improvements. We conclude that H4 is essential for micro-dystrophin function and H1 facilitates force production. Our findings will help develop next-generation micro-dystrophin gene therapy.

A Chimeric Antigen Receptor Targeting Malonaldehyde-modified Low-density lipoprotein Cholesterol Activates Regulatory T Cells in the Presence of Human Atherosclerotic Plaque

JVS-Vascular Science

2022 Jan 01

Shaligram, S;Lopez, JL;Lin, PY;Ho, P;Huang, A;
| DOI: 10.1016/j.jvssci.2022.05.021

Background: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to decrease the risk of myocardial infarction and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a chimeric antigen receptor (CAR) targeting malonaldehyde-modified low-density lipoprotein cholesterol (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. OxidizedLDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane, and CD28/CD3z cytoplasmic domains. CD4+ CD25+ CD127low/e Tregs were purified from human blood via fluorescent activated cell sorting and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. Autologous ox-CAR-Tregs were analyzed for activation after ex vivo coculture with carotid endarterectomy samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising oxCAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared with controls, based on CD71 expression, cytokine expression, and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using immunohistochemistry. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex vivo co-culture with atherosclerotic plaque. Conclusions: An optimized CAR targeting MDA-LDL activates Tregs Q10 when cultured with human atherosclerotic plaque ex vivo.

Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome

American journal of human genetics

2021 Oct 07

Mahyari, E;Guo, J;Lima, AC;Lewinsohn, DP;Stendahl, AM;Vigh-Conrad, KA;Nie, X;Nagirnaja, L;Rockweiler, NB;Carrell, DT;Hotaling, JM;Aston, KI;Conrad, DF;
PMID: 34626582 | DOI: 10.1016/j.ajhg.2021.09.001

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.

Rapid postmortem ventilation improves donor lung viability by extending the tolerable warm ischemic time after cardiac death in mice

American journal of physiology. Lung cellular and molecular physiology

2021 Jul 28

Yu, J;Xu, C;Lee, JS;Alder, JK;Wen, Z;Wang, G;Gil Silva, AA;Sanchez, PG;Pilewsky, JM;McDyer, JF;Wang, X;
PMID: 34318693 | DOI: 10.1152/ajplung.00011.2021

Uncontrolled donation after cardiac death (uDCD) contributes little to ameliorating donor lung shortage due to rapidly progressive warm ischemia after circulatory arrest. Here, we demonstrated non-hypoxia improves donor lung viability in a novel uDCD lung transplant model undergoing rapid ventilation after cardiac death and compared the evolution of ischemia-reperfusion injury in mice that underwent pulmonary artery ligation (PAL). The tolerable warm ischemia time at 37ºC was initially determined in mice using a modified PAL model. The donor lung following PAL was also transplanted into syngeneic mice and compared to those that underwent rapid ventilation or no ventilation at 37ºC prior to transplantation. Twenty-four hours following reperfusion, lung histology, PaO2/FIO2 ratio, and inflammatory mediators were measured. Four hours of PAL had little impact on PaO2/FIO2 ratio and acute lung injury score in contrast to significant injury induced by 5 hours of PAL. Four-hour PAL lungs showed an early myeloid-dominant inflammatory signature when compared to naïve lungs and substantially injured five-hour PAL lungs. In the context of transplantation, unventilated donor lungs showed severe injury after reperfusion, whereas ventilated donor lungs showed minimal changes in PaO2/FIO2 ratio, histologic score, and expression of inflammatory markers. Taken together, the tolerable warm ischemia time of murine lungs at 37oC can be extended by maintaining alveolar ventilation for up to 4 hours. Non-hypoxic lung warm ischemia-reperfusion injury shows an early transcriptional signature of myeloid cell recruitment and extracellular matrix proteolysis prior to blood-gas barrier dysfunction and significant tissue damage.

Biologic and behavioral associations of estrogen receptor alpha positivity in head and neck squamous cell carcinoma

Oral oncology

2021 Jul 22

Drake, V;Bigelow, E;Fakhry, C;Windon, M;Rooper, LM;Ha, P;Miles, B;Gourin, C;Mandal, R;Mydlarz, W;London, N;Vosler, PS;Yavvari, S;Troy, T;Waterboer, T;Eisele, DW;D'Souza, G;
PMID: 34304004 | DOI: 10.1016/j.oraloncology.2021.105461

Tumor HPV status is an established independent prognostic marker for oropharynx cancer (OPC). Recent studies have reported that tumor estrogen receptor alpha (ERα) positivity is also associated with prognosis independent of HPV. Little is known about the biologic and behavioral predictors of ERα positivity in head and neck squamous cell cancer (HNSCC). We therefore explored this in a multicenter prospective cohort study.Participants with HNSCC completed a survey and provided a blood sample. Tumor samples were tested for ERα using immunohistochemistry. ERα positivity was defined as ≥1%, standardized by the American Society of Clinical Oncology/College of American Pathologists in breast cancer. Characteristics were compared with χ2 and Fisher's exact test. Odds ratios (OR) were calculated using logistic regression.Of 318 patients with HNSCC, one third had ERα positive tumors (36.2%, n = 115). Odds of ERα expression were significantly increased in those with HPV-positive tumors (OR = 27.5, 95% confidence interval[CI] 12.1-62), smaller tumors (≤T2, OR = 3.6, 95% CI 1.9-7.1), male sex (OR = 2.0, 95% CI 1.1-3.6), overweight/obesity (BMI ≥ 25, OR = 1.9, 95% CI 1.1-3.3), and those married/living with a partner (OR = 1.7, 95% CI 1.0-3.0). In a multivariate model, HPV-positivity (aOR = 27.5, 95% CI 11.4-66) and small tumor size (≤T2, aOR = 2.2, 95% CI 1.0-4.8) remained independently associated with ERα status. When restricted to OPC (n = 180), tumor HPV status (aOR = 17.1, 95% CI 2.1-137) and small tumor size (≤T2, aOR = 4.0 95% CI 1.4-11.3) remained independently associated with ERα expression.Tumor HPV status and small tumor size are independently associated with ERα expression in HNSCC.

Spike Generators and Cell Signaling in the Human Auditory Nerve: An Ultrastructural, Super-Resolution, and Gene Hybridization Study

Frontiers in Cellular Neuroscience

2021 Mar 16

Liu, W;Luque, M;Li, H;Schrott-Fischer, A;Glueckert, R;Tylstedt, S;Rajan, G;Ladak, H;Agrawal, S;Rask-Andersen, H;
| DOI: 10.3389/fncel.2021.642211

Background: The human auditory nerve contains 30,000 nerve fibers (NFs) that relay complex speech information to the brain with spectacular acuity. How speech is coded and influenced by various conditions is not known. It is also uncertain whether human nerve signaling involves exclusive proteins and gene manifestations compared with that of other species. Such information is difficult to determine due to the vulnerable, “esoteric,” and encapsulated human ear surrounded by the hardest bone in the body. We collected human inner ear material for nanoscale visualization combining transmission electron microscopy (TEM), super-resolution structured illumination microscopy (SR-SIM), and RNA-scope analysis for the first time. Our aim was to gain information about the molecular instruments in human auditory nerve processing and deviations, and ways to perform electric modeling of prosthetic devices.Material and Methods: Human tissue was collected during trans-cochlear procedures to remove petro-clival meningioma after ethical permission. Cochlear neurons were processed for electron microscopy, confocal microscopy (CM), SR-SIM, and high-sensitive in situ hybridization for labeling single mRNA transcripts to detect ion channel and transporter proteins associated with nerve signal initiation and conductance.Results: Transport proteins and RNA transcripts were localized at the subcellular level. Hemi-nodal proteins were identified beneath the inner hair cells (IHCs). Voltage-gated ion channels (VGICs) were expressed in the spiral ganglion (SG) and axonal initial segments (AISs). Nodes of Ranvier (NR) expressed Nav1.6 proteins, and encoding genes critical for inter-cellular coupling were disclosed.Discussion: Our results suggest that initial spike generators are located beneath the IHCs in humans. The first NRs appear at different places. Additional spike generators and transcellular communication may boost, sharpen, and synchronize afferent signals by cell clusters at different frequency bands. These instruments may be essential for the filtering of complex sounds and may be challenged by various pathological conditions.

Oncolytic adenoviral H101 synergizes with radiation in cervical cancer cells

Current cancer drug targets

2021 Mar 07

Duan, Y;Bai, H;Li, X;Wang, D;Wang, Y;Cao, M;Zhang, N;Chen, H;Wang, Y;
PMID: 33687882 | DOI: 10.2174/1568009621666210308103541

A major challenge in cervical cancer radiotherapy is to tailor the radiation doses efficiently to both eliminate malignant cells and to reduce the side effects to normal tissue. Oncolytic adenoviral drug H101 is recently tested and approved for topical adjuvant treatment of several malignancies. This study is to evaluate the potential neoadjuvant radiotherapy benefits of H101 by testing the inhibitory function of H101 combined with radiation in different cervical cancer cells. Human cervical cancer cells C33a, SiHa, CaSki, and Hela were treated with varying concentrations of H101 alone or combined with radiation (2Gy or 4Gy). Cell viability and apoptosis were measured at indicated time intervals. HPV16 E6 and cellular p53 mRNA expression alteration were measured by qRT-PCR. RNA scope in-situ detect HPV E6 status. P53 protein alteration are detected by Western blot. Cell viability and apoptosis show the combination of a high dose of H101 (MOI=1000, 10000) with radiation yielded a synergistic anti-cancer effect in all tested cervical cancer cell lines (P<0.05), with the greatest effect achieved in HPV negative C33a cells (P<0.05). Low HPV16 viral load SiHa cell was more sensitive to combination therapy than high HPV16 viral load CaSki cell (P<0.05). The combined treatment could reduce HPV16 E6 expression and increase cellular P53 level compared to radiation alone in SiHa and CaSki (P<0.05). Oncolytic adenoviral H101 effectively enhances the antitumor efficacy of radiation in cervical cancer cells and may serve as a novel combination therapy for cervical cancer.

MALAT1 maintains the intestinal mucosal homeostasis in Crohn\'s disease via the miR-146b-5p-CLDN11/NUMB pathway

Journal of Crohn's & colitis

2021 Mar 02

Li, Y;Zhu, L;Chen, P;Wang, Y;Yang, G;Zhou, G;Li, L;Feng, R;Qiu, Y;Han, J;Chen, B;He, Y;Zeng, Z;Chen, M;Zhang, S;
PMID: 33677577 | DOI: 10.1093/ecco-jcc/jjab040

Intestinal homeostasis disorder is critical for developing Crohn's disease (CD). Maintaining mucosal barrier integrity is essential for intestinal homeostasis, preventing intestinal injury and complications. Among the remarkably altered long non-coding RNAs (lncRNAs) in CD, we aimed to investigate whether metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) modulated CD and consequent disruption of intestinal homeostasis. Microarray analyses on intestinal mucosa of CD patients and controls were performed to identify dysregulated lncRNAs. MALAT1 expression was investigated via qRT-PCR and its distribution in intestinal tissues was detected using BaseScope. Intestines from MALAT1 knockout mice with colitis were investigated using histological, molecular and biochemical approaches. Effects of intestinal epithelial cells transfected with MALAT1 lentiviruses and Smart Silencer, on monolayer permeability and apical junction complex (AJC) proteins were analysed. MiR-146b-5p were confirmed as a critical MALAT1 mediator in cells transfected with miR-146b-5p mimic/inhibitor and in colitis mice administered with agomir-146b-5p/antagomir-146b-5p. Interaction between MALAT1 and miR-146b-5p was predicted via bioinformatics and validated using Dual-luciferase reporter assay and Ago2-RIP. MALAT1 was aberrantly downregulated in the intestine mucosa of CD patients and mice with experimental colitis. MALAT1 knockout mice were hypersensitive to DSS-induced experimental colitis. MALAT1 regulated intestinal mucosal barrier and regained intestinal homeostasis by sequestering miR-146b-5p and maintaining the expression of the AJC proteins NUMB and CLDN11. Downregulation of MALAT1 contributed to the pathogenesis of CD by disrupting AJC. Thus, a specific MALAT1-miR-146b-5p-NUMB/CLDN11 pathway that plays a vital role in maintaining intestinal mucosal homeostasis may serve as a novel target for CD treatment.

Zinc Dependent Regulation of ZEB1 and YAP1 Co-activation Promotes EMT Plasticity and Metastasis in Pancreatic Cancer

Gastroenterology

2021 Jan 06

Liu, M;Zhang, Y;Yang, J;Zhan, H;Zhou, Z;Jiang, Y;Shi, X;Fan, X;Zhang, J;Luo, W;Fung, KA;Xu, C;Bronze, MS;Houchen, CW;Li, M;
PMID: 33421513 | DOI: 10.1053/j.gastro.2020.12.077

Pancreatic cancer is characterized by extensive metastasis. EMT plasticity plays a critical role in tumor progression and metastasis by maintaining the transition between EMT and MET states. Our aim is to understand the molecular events regulating metastasis and EMT plasticity in pancreatic cancer. The interactions between a cancer promoting zinc transporter ZIP4, a zinc dependent EMT transcriptional factor ZEB1, a co-activator YAP1, and integrin α3 (ITGA3) were examined in human pancreatic cancer cells, clinical specimens, spontaneous mouse models (KPC and KPCZ) and orthotopic xenografts, and 3D spheroid and organoid models. Correlations between ZIP4, miR-373, and its downstream targets were assessed by RNA in situ hybridization and IHC staining. The transcriptional regulation of ZEB1, YAP1, ITGA3 by ZIP4 was determined by ChIP, Co-IP and luciferase reporter assays. The Hippo pathway effector YAP1 is a potent transcriptional co-activator and forms a complex with ZEB1 to activate ITGA3 transcription through the YAP1/TEAD binding sites in human pancreatic cancer cells and KPC derived mouse cells. ZIP4 upregulated YAP1 expression via activation of miR-373 and inhibition of the YAP1 repressor LATS2. Furthermore, upregulation of ZIP4 promoted EMT plasticity, cell adhesion, spheroid formation and organogenesis both in human pancreatic cancer cells, 3D spheroid model, xenograft model, and spontaneous mouse models (KPC and KPCZ) through ZEB1/YAP1-ITGA3 signaling axis. We demonstrated that ZIP4 activates ZEB1 and YAP1 through distinct mechanisms. The ZIP4-miR-373-LATS2-ZEB1/YAP1-ITGA3 signaling axis has a significant impact on pancreatic cancer metastasis and EMT plasticity.

Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea

The Ocular Surface

2021 Jan 01

Li, D;Kim, S;Li, J;Gao, Q;Choi, J;Bian, F;Hu, J;Zhang, Y;Li, J;Lu, R;Li, Y;Pflugfelder, S;Miao, H;Chen, R;
| DOI: 10.1016/j.jtos.2020.12.004

Purpose This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution. Methods Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models. Results Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. Conclusions This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

Colonic epithelial-derived Selenoprotein P is the source for antioxidant-mediated protection in colitis-associated cancer

Gastroenterology

2021 Jan 01

Short, S;Pilat, J;Barrett, C;Reddy, V;Haberman, Y;Hendren, J;Marsh, B;Keating, C;Motley, A;Hill, K;Zemper, A;Washington, M;Shi, C;Chen, X;Wilson, K;Hyams, J;Denson, L;Burk, R;Rosen, M;Williams, C;
| DOI: 10.1053/j.gastro.2020.12.059

Background and Aims Patients with inflammatory bowel disease (IBD) demonstrate nutritional selenium deficiencies and are at greater risk for colon cancer. Previously, we determined that global reduction of the secreted antioxidant selenium-containing protein, Selenoprotein P (SELENOP), substantially increased tumor development in an experimental colitis-associated cancer (CAC) model. We next sought to delineate tissue-specific contributions of SELENOP to intestinal inflammatory carcinogenesis and define clinical context. Methods Selenop floxed mice crossed with Cre driver lines to delete Selenop from the liver, myeloid lineages, or intestinal epithelium were placed on an azoxymethane/dextran sodium sulfate (AOM/DSS) experimental CAC protocol. SELENOP loss was assessed in human ulcerative colitis (UC) organoids, and expression was queried in human and adult UC samples. Results Although large sources of SELENOP, both liver- and myeloid-specific Selenop deletion failed to modify AOM/DSS-mediated tumorigenesis. Instead, epithelial-specific deletion increased CAC tumorigenesis, likely due to elevated oxidative stress with a resulting increase in genomic instability and augmented tumor initiation. SELENOP was downregulated in UC colon biopsies and levels were inversely correlated with endoscopic disease severity and tissue S100A8 (calprotectin) gene expression. Conclusions While global selenium status is typically assessed by measuring liver-derived plasma SELENOP levels, our results indicate that the peripheral SELENOP pool is dispensable for CAC. Colonic epithelial SELENOP is the main contributor to local antioxidant capabilities. Thus, colonic SELENOP is the most informative means to assess selenium levels and activity in IBD patients and may serve as a novel biomarker for UC disease severity and identify patients most predisposed to CAC development.

Skin-dependent morphological and molecular maturation of specialized mechanosensory neurons (S34.005)

Wednesday, April 26

2023 Apr 25

Koutsioumpa, C;Santiago, C;Jacobs, K;Lehnert, B;Ginty, D;
| DOI: 10.1212/wnl.0000000000203780

Objective: Identification of the developmental steps leading to somatosensory neuron development. Background: Our sense of touch is essential for life and relies on Low-Threshold Mechanoreceptors (LTMRs). LTMR subtypes characterized by early embryonic expression of Ntrk2 (TrkB) and Ret exhibit distinct properties depending on the skin region they innervate - hairy skin or glabrous(hairless) skin. In glabrous skin, TrkB+ and Ret+ LTMRs form Meissner corpuscles, while in hairy skin they form longitudinal lanceolate endings around hair follicles. These morphological features reflect the physiological properties and specialized functions of these neurons. The developmental steps leading to glabrous and hairy skin LTMR properties are largely unknown, in particular whether they are genetically pre-specified or whether interactions with different target skin regions define their unique features. Design/Methods: Sparse genetic labeling experiments demonstrate that morphological specialization of glabrous- and hairy paw skin-innervating TrkB+ and Ret+ LTMRs arise at nearly identical times during postnatal development. Interestingly, we find that individual neurons that terminate along the border of glabrous and hairy skin, termed “border neurons”, exhibit branches that form both lanceolate endings and Meissner corpuscle endings. Additionally, transcriptomic profiling and RNAscope experiments show that neonatal glabrous skin-and hairy skin-innervating TrkB+ and Ret+ neurons are transcriptionally similar, although distinct from other DRG neuron types. Lastly, using mouse mutants that have either ectopic glabrous skin or ectopic hairy skin we find that neurons that innervate ectopic skin regions of these mutants form ending types (either lanceolate or Meissner corpuscle endings) in accordance with the ectopic skin type. Results: These findings support a model in which embryonic TrkB+ and Ret+ LTMRs are able to form either Meissner corpuscle or lanceolate endings, and that the skin target region differentially instructs morphological maturation of these LTMR types. Conclusions: This model implies that neuronal identity in the peripheral nervous system is flexibly determined by target tissue.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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