The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear.
The 2 matches
- [1] § Results › Pioneer SAG neurons share a transcriptional profile with aLL progenitors ↔ WT_14_18_22_30_48hpf_SAG.Rmd, lines 431 to 467 · score 0.83 · fstl1a, pou2f2a.1, irx1b, greb1l, irx1a, itga5 · Report a mismatch
- [2] § Materials and methods › Single-cell RNA sequencing ↔ WT_14_18_22_30_48hpf_SAG.Rmd, lines 343 to 428 · score 0.73 · hoxb5b, FindAllMarkers, identity, logfc, threshold, Gene · Report a mismatch
Paper
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The authors' code
R Markdown · 478 lines · 14 KB · no license · 2 matches
- ---
- title: "nechiporuk_10x_analysis"
- author: "avn"
- date: "9/16/2025 - 2/16/2026"
- output:
- html_document:
- toc: yes
- toc_depth: 2
- toc_float: yes
- toc_collapsed: yes
- editor_options:
- chunk_output_type: console
- ---
- ```{r setup, include=FALSE}
- knitr::opts_chunk$set(echo = TRUE, warning = FALSE, fig.align = 'center', out.width = '90%')
- ```
- <style>
- body .main-container {
- max-width: 2160px;
- }
- </style>
- # Set up
- ## Load libraries
- ```{r}
- library(tidyverse)
- library(Seurat)
- library(patchwork)
- library(MAST)
- library(ggrepel)
- library(RColorBrewer)
- ```
- ## Load data
- ```{r}
- #download data sets from NCBI and rename each folder as follows
- # "WT_14h" = "GSM8216056",
- # "WT_18h" = "GSM8997253",
- # "WT_22h" = "GSM8222579",
- # "WT30h_1" = "GSM8244692",
- # "WT30h_2" = "GSM7708237",
- # "WT48h_1" = "GSM8222580"
- #14h data set
- data_folder1 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT_14h"
- WT14_data <- Read10X(data.dir = data_folder1)
- WT14 <- CreateSeuratObject(counts = WT14_data,
- project = "WT14", min.cells = 3, min.features = 200)
- #18h data set
- data_folder2 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT_18h"
- WT18_data <- Read10X(data.dir = data_folder2)
- WT18 <- CreateSeuratObject(counts = WT18_data,
- project = "WT18", min.cells = 3, min.features = 200)
- # #22h data set
- data_folder2 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT_22h"
- WT22_data <- Read10X(data.dir = data_folder2)
- WT22 <- CreateSeuratObject(counts = WT22_data,
- project = "WT22", min.cells = 3, min.features = 200)
- # #30h data set#1
- data_folder3 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT30h_1"
- WT_3_data <- Read10X(data.dir = data_folder3)
- WT30_1 <- CreateSeuratObject(counts = WT_3_data,
- project = "WT30_1", min.cells = 3, min.features = 200)
- # #30h data set#2
- data_folder4 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT30h_2"
- WT_4_data <- Read10X(data.dir = data_folder4)
- WT30_2 <- CreateSeuratObject(counts = WT_4_data,
- project = "WT30_2", min.cells = 3, min.features = 200)
- #48h data set
- data_folder5 <- "/Users/nechipor/R_scripts/WT_14_18_22_30_48hpf/WT48h_1"
- WT_5_data <- Read10X(data.dir = data_folder5)
- WT48 <- CreateSeuratObject(counts = WT_5_data,
- project = "WT48_1", min.cells = 3, min.features = 200)
- ```
- ```{r}
- data.combined <- merge(x = WT14, y = c(WT18, WT22, WT30_1, WT30_2, WT48), add.cell.ids = c("WT14", "WT18", "WT22", "WT30_1", "WT30_2", "WT48"), project = "data.combined")
- data.combined
- data.combined[['condition']] <- str_split([email hidden]$orig.ident, pattern = '_', simplify = TRUE)[,1]
- head(colnames(data.combined))
- table(data.combined$orig.ident)
- ```
- # 'Standard' Seurat analysis
- ## Assign mitochondrial percent
- *NOTE (NLC):* zebrafish mitochondrial genes have a lowercase prefix, using the regex pattern '^mt-' appeared to work, however only 13 mitchondrial genes are present.
- ```{r}
- data.combined[["percent.mt"]] <- PercentageFeatureSet(data.combined, pattern = "^mt-|^COX3$")
- # Visualize QC metrics as a violin plot
- VlnPlot(data.combined, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3, pt.size = 0)
- ```
- ## QC visualization
- *Note:* FeatureScatter is typically used to visualize feature-feature relationships, but can be used for anything calculated by the object, i.e. columns in object metadata, PC scores etc.
- *Note:* zebrafish mitochondrial genes have a lowercase prefix,
- Run this chunk to manually set cutoffs:
- ```{r, eval = TRUE}
- nCount_RNA_limits <- c(1e3, 7.5e4)
- nFeature_RNA_limits <- c(1900, 9000)
- percent_mt_limits <- c(0, 5)
- ```
- ```{r}
- FeatureScatter(data.combined, feature1 = "nCount_RNA", feature2 = "percent.mt", group.by = "orig.ident")+
- geom_vline(xintercept = nCount_RNA_limits, linetype = 'dashed', size = 1.5)+
- geom_hline(yintercept = percent_mt_limits, linetype = 'dashed', size = 1.5)
- FeatureScatter(data.combined, feature1 = "nCount_RNA", feature2 = "nFeature_RNA", group.by = "orig.ident")+
- geom_vline(xintercept = nCount_RNA_limits, linetype = 'dashed', size = 1.5)+
- geom_hline(yintercept = nFeature_RNA_limits, linetype = 'dashed', size = 1.5)
- data.combined_metadata <- [email hidden]
- ggplot(data.combined_metadata, aes(x = nCount_RNA, y = nFeature_RNA))+
- geom_point(alpha = 0.1)+
- geom_density2d()+
- geom_vline(xintercept = nCount_RNA_limits, linetype = 'dashed', size = 1.5)+
- geom_hline(yintercept = nFeature_RNA_limits, linetype = 'dashed', size = 1.5)+
- scale_x_log10()
- ggplot(data.combined_metadata, aes(x = nCount_RNA/nFeature_RNA, y = nFeature_RNA))+
- geom_point(alpha = 0.1)+
- geom_density2d()+
- geom_hline(yintercept = nFeature_RNA_limits, linetype = 'dashed', size = 1.5)
- ```
- ## Subset data
- ```{r}
- #Display counts before pruning
- print('Cell count before pruning:')
- table([email hidden]$orig.ident)
- data.combined <- subset(data.combined, subset = nFeature_RNA > min(nFeature_RNA_limits) & nFeature_RNA < max(nFeature_RNA_limits) & nCount_RNA > min(nCount_RNA_limits) & nCount_RNA < max(nCount_RNA_limits) & percent.mt > min(percent_mt_limits) & percent.mt < max(percent_mt_limits))
- #Display counts after pruning
- print('Cell count after pruning:')
- table([email hidden]$orig.ident)
- ```
- ## Ancor and normalize data
- ```{r}
- data.combined.list <- SplitObject(data.combined, split.by = "orig.ident")
- data.combined.list <- lapply(X = data.combined.list, FUN = function(x) {
- x <- NormalizeData(x)
- x <- FindVariableFeatures(x, selection.method = "vst", nfeatures = 2000)
- })
- anchors <- FindIntegrationAnchors(object.list = data.combined.list, dims = 1:25)
- data.combined <- IntegrateData(anchorset = anchors, dims = 1:25)
- #saveRDS(data.combined, file = "outputs/14_18_22_30_48h.rds")
- ```
- ## Identify variable features and scale data
- ```{r}
- # Run the standard workflow for visualization and clustering
- #data.combined <- readRDS("outputs/14_18_22_30_48h.rds")
- DefaultAssay(data.combined) <- "integrated"
- data.combined <- ScaleData(data.combined, features = row.names(data.combined), verbose = FALSE)
- data.combined <- RunPCA(data.combined, npcs = 50, verbose = FALSE)
- DimPlot(data.combined, reduction = 'pca')
- ElbowPlot(data.combined, ndims = 50)+
- geom_vline(xintercept = 30.5)
- # t-SNE and Clustering
- data.combined <- RunUMAP(data.combined, reduction = "pca", dims = 1:40)
- data.combined <- FindNeighbors(data.combined, reduction = "pca", dims = 1:40)
- data.combined <- FindClusters(data.combined, resolution = 2)
- plot_14_48h <- DimPlot(data.combined, reduction = 'umap', label = T) + NoLegend()+
- coord_equal()
- ggsave("outputs/plot_14_48h.jpeg", plot_14_48h, dpi = 600)
- DimPlot(data.combined, reduction = 'umap', group.by = 'orig.ident')+
- coord_equal()
- ```
- ## color blind friendly UMAP
- ```{r}
- my_colors <- c("5c5cc2",
- "#a6bc3a",
- "#733fa1",
- "#59c66f",
- "#b43996",
- "#7cb041",
- "#847deb",
- "#c8aa34",
- "#482070",
- "#4cbe84",
- "#d370d1",
- "#4d7f29",
- "#de4782",
- "#43c29e",
- "#9c1a54",
- "#36dee6",
- "#902a0e",
- 'deepskyblue2',
- "#d28826",
- "#353f8d",
- "#9ca03c",
- "#81438d",
- "#7cbf76",
- "#6d195d",
- 'darkmagenta',
- "#851657",
- "#3a8142",
- "#e66bab",
- "#255719",
- "#c08adc",
- "#dcae57",
- "#5963ad",
- "#cb692b",
- "#7199e0",
- "#d5533b",
- "#db81c4",
- "#a67724",
- "#9b4486",
- "#7b6a24",
- "#b23679",
- "#cca361",
- "#6b1b4b",
- "#dc8e5a",
- "#6f193d",
- "#d16a43",
- "#a14e7c",
- "#8d4d17",
- "#e480a8",
- "#701e0f",
- "#eb5573",
- "#74171f",
- "#e5836a",
- "#72182e",
- "#dd4e4f",
- "#b1486e",
- "#a3553a",
- "#ad264f",
- "#dd7a87",
- "#a73830",
- "#d55b75",
- "#9d323b",
- "#df6f70",
- "#a34656",
- "#c4394f")
- plot_14_48h <- DimPlot(data.combined, reduction = 'umap', label = F, pt.size = 0.1, cols = my_colors) + NoLegend()
- ggsave("outputs/plot_14_48h.jpeg", plot_14_48h, dpi = 600, width = 210,
- height = 140, units = "mm")
- ```
- # Map cranial sensory neurons
- ```{r}
- DefaultAssay(data.combined) <- "RNA"
- FeaturePlot(data.combined, features = c('irx1b', 'irx1a'), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("phox2bb",
- "phox2a"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("neurod4",
- "pax2a"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("isl2b",
- "onecut1"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("ngfrb",
- "ets1"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("uchl1",
- "gng13b"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("kitb",
- "cart3"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("vegfd",
- "trarg1b"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- FeaturePlot(data.combined, features = c("pvalb5",
- "dlx4b"), min.cutoff = 0,
- max.cutoff = NA, order = TRUE)+
- coord_equal()
- ```
- # Analysis of clusters
- ```{r}
- # all_markers <- FindAllMarkers(data.combined, only.pos = T, logfc.threshold = 0.2, min.diff.pct = 0.2)
- # write_csv(all_markers, "outputs/all_markers")
- ```
- ## 'Automatic' subset and analysis a particular cluster
- ```{r}
- #Set the subset cluster(s) that you're interested in
- subset_cluster_id <- c(5,17,24)
- DefaultAssay(data.combined) <- "integrated"
- ##
- ##The code below is designed to run automatically once the subset ids are selected above
- ##
- print('Running automated ~analysis~ on the following clusters:')
- print(subset_cluster_id)
- #Pull the subset
- data.combined_subset <- subset(data.combined, subset = seurat_clusters %in% subset_cluster_id)
- #stash the original cluster identities
- [email hidden]$original_seurat_clusters <- [email hidden]$seurat_clusters
- #Perform relatively hands-free analysis of the selected clusters
- data.combined_subset <- FindVariableFeatures(data.combined_subset)
- data.combined_subset <- RunPCA(data.combined_subset)
- ElbowPlot(data.combined_subset)
- data.combined_subset <- FindNeighbors(data.combined_subset, dims = 1:10)
- data.combined_subset <- FindClusters(data.combined_subset, resolution = 0.2)
- data.combined_subset <- RunUMAP(data.combined_subset, dims = 1:10)
- DimPlot(data.combined_subset, label = TRUE, label.size = 10, repel = 0.1, group.by = 'seurat_clusters')+
- ggtitle('New clusters')
- DimPlot(data.combined_subset, label = TRUE, label.size = 10, repel= 0.1, group.by = 'original_seurat_clusters')+
- ggtitle('Original cluster identities')
- my_colors <- c("#847deb",
- "#c8aa34",
- "#482070",
- "#4cbe84",
- "#d370d1",
- "#4d7f29")
- pLL_aLL_plot <- DimPlot(data.combined_subset, label = F, label.size = 10, pt.size = .5, repel = 0.1, group.by = 'seurat_clusters', cols = my_colors)
- ggsave("outputs/pLL_aLL_plot.jpeg", pLL_aLL_plot, dpi = 600, width = 100,
- height = 67, units = "mm")
- #Identify anterior and posterior lateral line clusters
- Idents(data.combined_subset) <- "seurat_clusters"
- LL_markers <- FindAllMarkers(data.combined_subset, only.pos = T, logfc.threshold = 0.2, min.diff.pct = 0.2)
- print (LL_markers %>% filter(cluster == 0) %>%
- select(gene) %>% slice_head(n = 10), row.names = FALSE)
- #select DE markers for dotplot
- feat_list <- list(aLL_prog = c("uacab","greb1l","shisal1a","dchs1a"),
- late_pLL_prog = c("hoxb5b","crabp1b","meis2a.1","hoxb1b"),
- pLL_neurons = c("rab6ba","prph","nacad","ntrk3a"),
- prolif_prog = c("her6","boc","ccnd2a", "pcna"),
- early_pLL_prog = c("dla","b4galnt3b","cdh1","neurod4"),
- aLL_neurons = c("ptgir","b3gat2","alcama","slc1a2b")
- )
- markers <- unique(unlist(feat_list))
- markers <- markers[markers %in% rownames(data.combined_subset)]
- Idents(data.combined_subset) <- "seurat_clusters"
- all_LL_dotplot <- DotPlot(
- data.combined_subset,
- features = markers,
- group.by = "seurat_clusters",
- cols="RdBu",
- assay = DefaultAssay(data.combined_subset)
- ) +
- labs(x = NULL, y = NULL) +
- guides(color = guide_colorbar(title = NULL), size = guide_legend(title = NULL)) +
- theme_bw() +
- theme(
- plot.title = element_blank(),
- strip.background = element_blank(),
- strip.text = element_blank()
- )+
- RotatedAxis()+ theme(axis.text.x = element_text(size = 18))
- ggsave("outputs/all_LL_dotplot.jpeg", all_LL_dotplot, dpi = 600, width = 300,
- height = 100, units = "mm")
- ```
- ```{r}
- #plot 18hpf
- Idents(data.combined_subset) <- "condition"
- cell_values <- c("WT18")
- data.combined_subset_18h <- subset(x = data.combined_subset, idents = cell_values, subset = seurat_clusters != 5, invert = FALSE)
- #DE analysis for aLL at 18 hpf
- Idents(data.combined_subset_18h) <- "seurat_clusters"
- aLL_markers <- FindAllMarkers(data.combined_subset_18h, only.pos = T, logfc.threshold = 0.2, min.diff.pct = 0.2)
- #write.csv(aLL_markers, "outputs/aLL_markers.csv", row.names = TRUE)
- #dot plot top markers
- DefaultAssay(data.combined_subset_18h) <- "RNA"
- ear_markers <- c("fstl1a", "greb1l", "irx1a", "irx1b", "itga5", "pou2f2a.1")
- dotplot_18h <- DotPlot(data.combined_subset_18h, features = ear_markers, dot.scale=10, cols="RdBu")+
- labs(x = NULL, y = NULL) +
- guides(color = guide_colorbar(title = NULL), size = guide_legend(title = NULL)) +
- theme_bw() +
- theme(
- plot.title = element_blank(),
- strip.background = element_blank(),
- strip.text = element_blank()
- )+
- RotatedAxis()+ theme(axis.text.x = element_text(size = 20))
- ggsave("outputs/dotplot_18h.jpeg", dotplot_18h, dpi = 600, width = 200,
- height = 100, units = "mm")
- #plot irx1a/b
- DefaultAssay(data.combined_subset_18h) <- "RNA"
- irx1 <- FeaturePlot(data.combined_subset_18h, features = c('irx1a', 'irx1b'), min.cutoff = 0.1, pt.size = 1,
- max.cutoff = NA, order = TRUE)
- ggsave("outputs/irx1.jpeg", irx1, dpi = 600, width = 300,
- height = 100, units = "mm")
- ```
- # SessionInfo
- ```{r}
- sessionInfo()
- ```
WT_14_18_22_30_48hpf_SAG.Rmd at commit f2c8b7a, no license · at the source
Overview
- Department of Medicine and Life Sciences, Universitat Pompeu Fabra-Parc de Recerca Biomèdica de Barcelona, Barcelona, Spain
- Department of Cell, Developmental & Cancer Biology, Oregon Health & Science University, Portland, OR, USA
Abstract
The nervous system wiring requires the precise coordination of axon guidance, neuronal migration, and target cell recognition. Here, we show that inner ear circuit formation relies on pioneer cells extending an axonal scaffold that selectively targets early-born hair cells. High spatiotemporal imaging of pioneer axons reveals how they navigate through the cranial environment, establishing dynamic cell–cell contacts with other axons to finally stabilize in target cells. These pioneer axons are required not only for follower axon growth but also for coordinated migration of follower neurons, revealing a cellular hierarchy underlying circuit assembly. We identify the chemokine Cxcl14 as a novel instructive guidance cue regulating pioneer axon extension, turning, and fasciculation at discrete cellular decision points. Loss of Cxcl14 disrupts axonal navigation, compromises synaptic organization in hair cells, and impairs mechanosensory-based behavior. Together, our findings establish a new chemokine-based mechanism linking pioneer axon guidance to early sensory circuit assembly necessary for building mechanosensory networks.
Reproduced under the paper's license (CC BY), from the paper cited above.
Repository
Its files are read in the Code ↔ Paper reader above, with 2 matches between paragraphs and lines of code.
anechipor/Rumbo_et_al_2026
f2c8b7aaa182c59a0689c69694c8759978609b28, 17 February 2026Availability: 1 check, the latest on 12 November 2026: the link answers
- 12 November 2026: the link answers
1 file
- WT_14_18_22_30_48hpf_SAG
.Rmd , R, 478 lines, 2 matches
The paper's code and data availability statement is in the Data section.
Tracing map
Proposed by the machine: these links were found in the paper and verified at the source, without human review. The map will receive a Zenodo DOI once one of the paper's authors has validated it with their ORCID.
What the map holds:
- 1 repository of the authors' code, each at its verified commit, with its license and how the link was found in the paper;
- 1 script, each with its path and the digest of its content;
- 2 matches between paragraphs of the paper and lines of the code (method lexical-v1);
- neither the text of the paper nor the code itself.
Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.
Data
Datasets cited
- geo:GSE266312, at NCBI GEO; found in the text, “Single-cell RNA sequencing”
Data availability
The data supporting the findings of this study are available within the published article and its online supplementary material. The data underlying Fig. 1 are openly available in GitHub at https://
Reproduced under the paper's license (CC BY), from the paper cited above.
Versions
The history of this record: each version stored by the harvester or made by a correction of its authors or of the maintainers of its code, and what changed in its facts. The texts of the paper (its abstract, its availability statements) are not part of it; versions that changed only those are not listed.
Version 1, 12 November 2026: the first record
Recorded: type, language, journal, volume, issue, pages, dates, 4 authors, 7 MeSH terms, 6 funders, 66 references.
Cite
This paper
Rumbo, M., Bañón, A., Nechiporuk, A. V., & Alsina, B. (2026). The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear. The Journal of cell biology, 226(2), e202604185. https://
BibTeX
@article{rumbo2026cxcl14
author = {Rumbo, Mireia and Bañón, Aitor and Nechiporuk, Alex V and Alsina, Berta},
title = {{The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear}},
journal = {The Journal of cell biology},
year = {2026},
month = nov,
volume = {226},
number = {2},
pages = {e202604185},
publisher = {The Rockefeller University Press},
issn = {0021-9525},
doi = {10.1083/
url = {https://
pmid = {43012745},
pmcid = {PMC13753874}
}
RIS
TY - JOUR
AU - Rumbo, Mireia
AU - Bañón, Aitor
AU - Nechiporuk, Alex V
AU - Alsina, Berta
TI - The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear
T2 - The Journal of cell biology
J2 - J Cell Biol
PY - 2026
DA - 2026/
VL - 226
IS - 2
SP - e202604185
SN - 0021-9525
PB - The Rockefeller University Press
DO - 10.1083/
UR - https://
LA - en
ER -
CSL-JSON
{
"id": "10.1083/
"type": "article-journal",
"title": "The Cxcl14 chemokine defines pioneer axon guidance and early circuit assembly in the inner ear",
"container-title": "The Journal of cell biology",
"author": [
{
"family": "Rumbo",
"given": "Mireia"
},
{
"family": "Bañón",
"given": "Aitor"
},
{
"family": "Nechiporuk",
"given": "Alex V"
},
{
"family": "Alsina",
"given": "Berta"
}
],
"container-title-short":
"volume": "226",
"issue": "2",
"page": "e202604185",
"DOI": "10.1083/
"PMID": "43012745",
"PMCID": "PMC13753874",
"ISSN": "0021-9525",
"publisher": "The Rockefeller University Press",
"URL": "https://
"language": "en",
"issued": {
"date-parts": [
[
2026,
11,
11
]
]
}
}
The tracing map gets a citation of its own once an author has validated it and it has a DOI.
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