CXCL14 as a Key Regulator of Neuronal Development: Insights from Its Receptor and Multi-Omics Analysis

CXCL14 is not only involved in the immune process but is also closely related to neurodevelopment according to its molecular evolution. However, what role it plays in neurodevelopment remains unclear. In the present research, we found that, by crossbreeding CXCL14+/− and CXCL14−/− mice, the number of CXCL14−/− mice in their offspring was lower than the Mendelian frequency; CXCL14−/− mice had significantly fewer neurons in the external pyramidal layer of cortex than CXCL14+/− mice; and CXCL14 may be involved in synaptic plasticity, neuron projection, and chemical synaptic transmission based on analysis of human clinical transcriptome data. The expression of CXCL14 was highest at day 14.5 in the embryonic phase and after birth in the mRNA and protein levels. Therefore, we hypothesized that CXCL14 promotes the development of neurons in the somatic layer of the pyramidal cells of mice cortex on embryonic day 14.5. In order to further explore its mechanism, CXCR4 and CXCR7 were suggested as receptors by Membrane-Anchored Ligand and Receptor Yeast Two-Hybrid technology. Through metabolomic techniques, we inferred that CXCL14 promotes the development of neurons by regulating fatty acid anabolism and glycerophospholipid anabolism.


Introduction
C-X-C motif chemokine ligand 14 (CXCL14), also known as BRAK, is a non-ELR (glutamic acid-leucine-arginine) chemokine with a broad spectrum of biological activities [1].The chemokine (C-X-C Motif) ligand 14 gene (also known as BRAK, BMAC, or Mip-2γ), located on human chromosome 5q31, is expressed as a 99 amino acid residue precursor protein, which is processed to a 77 amino acid mature protein with a molecular weight of 9.4 kDa, having a highly basic isoelectric point of 9.9 [2].It is the evolutionary ancient chemokine for a highly conserved sequence and its homeostatic roles, which suggests that CXCL14 has important functions in growth and development [3].CXCL14 plays important roles in immune surveillance, inflammation and tumor development by regulating cell migration [4].
Previous studies showed that CXCL14 is expressed at high levels in fetal, adult and adult mouse brain tissues, including the cortex, hippocampus and cerebellum.A recent study indicated that CXCL14 regulates the development of the microglia in the early cortex, and it is important for cell localization in early cortical stages [5].CXCL14 is expressed in GABAergic interneurons of the adult mouse dentate gyrus and inhibits GABAergic transmission to neural stem cells [6,7].In stark contrast, CXCL12 was shown to enhance the activity of gamma-aminobutyric acid (GABA) released by synapses [6].Moreover, recent of 8-week-old male CXCL14 −/− and female CXCL14 +/− mice, they produced a total of 59 mice, 42 mice of CXCL14 +/− and 17 mice of CXCL14 −/− , and the birth rate of CXCL14 −/− was 28.8%, which was significantly different from expected percentage of 50% (n = 59, p < 0.01).(B) Cortical neuron staining of CXCL14 +/− and CXCL14 −/− 8-week-old mice by Nissl staining.(a,b) were slice images of CXCL14 +/− 8-week-old mice cortexes; (c,d) were slice image of CXCL14 −/− 8-week-old mice cortexes.(Layer I: molecular layer; II: external granular layer; III: external pyramidal layer; IV: internal granular layer; V: external pyramidal layer; VI: Multiform Layer.).(C) The density of neurons in each layer of cerebral cortex of CXCL14 −/− and CXCL14 +/− mice.The neuron density in external pyramidal layer of CXCL14 −/− 8-week-old mice was significantly lower than that of their CXCL14 +/− sibs.The red line represents CXCL14 −/− mice.The black line represents CXCL14 +/− mice.(All data are expressed as mean ± SD, n = 3, **: p < 0.01).
In order to explore the effect of CXCL14 knockout on mice brains, slices of the parietal cortex of the brain of CXCL14 −/− and CXCL14 +/− mice were observed by Nissl staining.The results showed that the number of pyramidal cells in CXCL14 −/− mice was significantly lower than that in CXCL14 +/− mice of pyramidal cells in the external pyramidal layer (Figure 1B,C).These results suggest that CXCL14 has an effect on the cerebral cortex development of mice.

The Expression of CXCL14 in Spatial and Temporal
The expression of CXCL14 in the main organs (small intestine, brain, heart, kidney, spleen, stomach, liver, skin, lung, testis and ovary) from 8-week-old mice (n = 3) was detected by real-time quantitative PCR.The results showed that CXCL14 was highly expressed not only in the skin, but also in the brain and lung (Figure 2A).

CXCL14 Play Important Functions in the Cerebral Cortex
The top 1000 genes that have the most similar expression pattern to CXCL14 in cere bral cortex were mined by the "Similar Genes Detection" function in the GEPIA websit The neuron density in each layer of the cerebral cortex was analyzed in 8-week-old CXCL14 −/− and CXCL14 +/− mice (n = 3).Cerebral cortex parietal lobe tissues were collected at various embryonic stages (E10.5,E12.5, E14.5, E16.5, E18.5, E19.5), as well as at postnatal days 1, 14, and 56 (P1, P14, P56).The expression level of CXCL14 in the cerebral cortex was determined using quantitative PCR and Western blot analysis (n = 3).The results of both tests showed that the expression of CXCL14 began at embryonic day 10.5, reached a peak at embryonic day 14.5, and then gradually decreased and maintained a certain expression level (Figure 2B,C).The abnormal death time of CXCL14 knockout mice in the embryonic period was concentrated in the middle embryonic period (E10.5~E14.5),which was the most active period of cerebral cortex and central nervous system development.During this period, CXCL14 showed a high level of expression in the cerebral cortex, suggesting that CXCL14 was related to cortical neural development in embryonic mice.

CXCL14 Play Important Functions in the Cerebral Cortex
The top 1000 genes that have the most similar expression pattern to CXCL14 in cerebral cortex were mined by the "Similar Genes Detection" function in the GEPIA website from cerebral cortex dataset of GTEx database.The genes were ranked by Pearson's correlation coefficient (PCC).These genes were enriched in the anchoring junction, synapse, neuron projection, chemical synaptic transmission, and dendrite-related pathways (Figure 3A).The key genes related to neuron projection and dendrite, SLC6A1, GPM6A, RELN, CALB showed strong correlations with CXCL14 (Figure 3B, p < 0.001).

CXCL14 Play Important Functions in the Cerebral Cortex
The top 1000 genes that have the most similar expression pattern to CXCL14 in cere bral cortex were mined by the "Similar Genes Detection" function in the GEPIA websit from cerebral cortex dataset of GTEx database.The genes were ranked by Pearson's cor relation coefficient (PCC).These genes were enriched in the anchoring junction, synapse neuron projection, chemical synaptic transmission, and dendrite-related pathways (Fig ure 3A).The key genes related to neuron projection and dendrite, SLC6A1, GPM6A RELN, CALB showed strong correlations with CXCL14 (Figure 3B, p < 0.001).

There Is a Clear Interaction between CXCL14 and CXCR4 and CXCR7
Membrane-Anchored Ligand and Receptor Yeast Two-Hybrid (MALAR-Y2H) is a novel method for detecting the interaction between ligands and receptors.This method uses transmembrane peptide to anchor the ligand and the C-terminus of ubiquitin (Ubi4) residue C ub to the yeast cell membrane, while the other half of the ubiquitin residue N ub G is fused with the ligand and expressed on the cell membrane.When the ligand and receptor interact, the intracellular split C ub and N ub G reconstitute as a whole and activate ubiquitin that is recognized by ubiquitin-specific proteases (UBPs) and released GAL4 from the C-terminal of C ub , which triggers transcription of reporter genes (Figure 4A).
tor interact, the intracellular split Cub and NubG reconstitute as a whole and activate ubiquitin that is recognized by ubiquitin-specific proteases (UBPs) and released GAL4 from the C-terminal of Cub, which triggers transcription of reporter genes (Figure 4A).
The interaction between CXCL14 and CXCRn showed that CXCL14 interacted with CXCR4 and CXCR7 (Figure 4C), suggesting that the receptor of CXCL14 may be CXCR4 and CXCR7.The interaction between CXCL14 and CXCRn showed that CXCL14 interacted with CXCR4 and CXCR7 (Figure 4C), suggesting that the receptor of CXCL14 may be CXCR4 and CXCR7.

Multivariate Data Analysis from UPLC-MS
Ten serum samples were collected from both genotypic mice of CXCL14 +/− (W) and CXCL14 −/− (C).To enhance the analysis of ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS, LC-MS) data, the raw UPLC-HRMS data were imported into LC/MS and GC/MS Data Analysis (XCMS 3.4) software for peak data matching and alignment.Subsequently, 7252 peaks were retained (Tables S3 and S4).A total of 7252 peaks were imported into SIMCA-P 14.1 (Umetrics, Umea, Sweden) for multivariate statistical analysis, including principal components analysis (PCA), and orthogonal partial least-squares discriminant analysis (OPLS-DA).In Figure 5A, principal component analysis (PCA) showed that there was a tendency for intergroup separation.

Analysis of Endogenous Metabolites and Pathway
The differences in peak areas of several metabolites were associated with CXCL14 knockout.The content of palmitic acid (PA), alpha-linolenic acid (ALA) and linoleic acid in the serum of CXCL14 −/− mice is lower than that in CXCL14 +/− mice.The pathway analysis of differential metabolites was conducted using MetaboAnalyst 4.0 software.The color and size of circles depended on the p value and pathway impact value analyzed by MetaboAnalyst 4.0, respectively.The bigger and the closer to the top right corner of the The Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model established by positive ion mode data, R2, Q2 ≥ 0.5, is stable and reliable in Figure 5B,C (R2: represents the model interpretation rate; Q2: represents the predictive ability of the model; The closer R2 and Q2 are to 1, the more stable the model is Reliable.).With the OPLS-DA model VIP > 1 and p value < 0.05 as screening criteria, significant differential metabolites were screened.The variable importance in the projection (VIP) statistic of the first principal component of the OPLS-DA model (threshold > 1), coupled with the p-value of Student's t-test (threshold < 0.05), was used for selecting significant variables responsible for group separation.The results showed there were a total of 50 differential metabolites between W and C groups (Figure 5D).

Analysis of Endogenous Metabolites and Pathway
The differences in peak areas of several metabolites were associated with CXCL14 knockout.The content of palmitic acid (PA), alpha-linolenic acid (ALA) and linoleic acid in the serum of CXCL14 −/− mice is lower than that in CXCL14 +/− mice.The pathway analysis of differential metabolites was conducted using MetaboAnalyst 4.0 software.The color and size of circles depended on the p value and pathway impact value analyzed by MetaboAnalyst 4.0, respectively.The bigger and the closer to the top right corner of the plot represented the more important metabolites.Therefore, we have obtained the following important metabolic pathways of linoleic acid metabolism, taurine and hypotaurine metabolism, alpha-linolenic acid metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism.

Discussion
CXCL14 is a chemotactic cytokine belonging to a small molecule-secreted protein superfamily.Studies showed that it mainly regulates cell migration, which plays an important role in immune surveillance, inflammation and cancer [14,15].
However, in research on the molecular evolution of the CXC chemokine family, the CXC chemokine family was formed before the emergence of vertebrates [16].Four branches were generated through two genome duplications and developed into genes common to vertebrates-CXCL14 and CXCL12 [17].Based on the timing of the two genomic repetition events, chemokines appear during the formation of the higher nervous system before the formation of the higher immune system.That suggests that ancestral genes in this family were initially involved in the genesis and development of the nervous system.It was previously thought that the chemokine family mainly plays the role of chemotaxis of immune cells, but CXCL14 seems to be an exception, which does not participate in the regulation of the innate immune response of immune cells [3].So it is speculated that CXCL14 has always maintained the neurodevelopmental functions of its ancestral genes, but has not further acquired other functions.
The research showed that the birth rate of CXCL14 −/− mice was about half the expected Mendelian ratio (Figure 1A) [18].As CXCL14 −/− female mice mated with CXCL14 −/− male mice, pregnancy characteristics and abdominal bulge can be observed on days 10-15 after mating.Seven of 10 female mice stopped gestation after 10-15 days of mating.One of the mice was unable to conceive (Tables S1 and S2).Therefore, CXCL14 gene knockout is the main cause of mouse embryonic death, which is presumed to be related to development.In view of its evolutionary characteristics in neurodevelopment, on the one hand, CXCL14 −/− mice had significantly fewer neurons in layer III of the cerebral cortex (external pyramidal layer) than CXCL14 −/+ mice by Nissle staining (Figure 1B,C).On the other hand, the analysis of CXCL14 expression in various organs of mice also indicated that CXCL14 expression was higher in the brain (Figure 2A).These two pieces of evidence confirmed that CXCL14 was involved in the development of neurons.
In order to explore the influence of CXCL14 on the brain development phase, we focused on CXCL14 expression on the cerebral cortex parietal lobe tissue of mice at different embryonic development times by Q-PCR and Western blot.The result showed that the expression of CXCL14 increased significantly at E14.5 during embryonic development.This inferred that CXCL14 plays an important role in brain development around E14.5 period, which meant the death of CXCL14 knockout mice is likely to be concentrated in this period (Figure 2B,C and Tables S1 and S2).
The neocortex develops from L4 to L3 in an inside-first, outside-last principle concentrated on E14-E15 [19].In early development before neurogenesis, progenitor cells are found in the ventricular zone of the neural tube.The subventricular zone continues to divide and multiply.Then, after the completion of division, these immature neurons migrate radially to the appropriate parts of the cortex.Starting from E10.5 in fetal mice, the generated cortical neurons first form the cortical layer and subsequently migrate over.New neurons divide the preplate layer into the superficial marginal zone and the deeper subplate [20].With the continuous development of neurogenesis, neuronal precursor cells undergo asymmetric division and differentiation to produce various types of projection neurons on E11.5 [21].The neurons in the deeper subplate region formed first, followed by layer (L6, L5) neurons on E12.Eventually, upper layer (L4, L3, L2) neurons are formed during E13-E16 [22].Therefore, according to our results, CXCL14 is involved in the development of the cerebral cortex of E14-E15 from L4 to L3, which results in the development of neurons in the somatic layer of the pyramidal cells.When CXCL14 of mice was knocked out, it caused abnormal development of neurons in the external pyramidal layer of the pyramidal cells.
The biological function of similar expression pattern genes with CXCL14 in the cerebral cortex was analyzed by GO analysis.The correlated pathways, such as anchoring junction, synapse formation, neuron projection, chemical synaptic transmission, and dendrite formation, were necessary stages in the neuron development process (Figure 3A,B).This suggests that the function of CXCL14 may be involved in one or more of these processes.Of particular note is the calcium-binding protein Calbindin 2(Calb2), an early developmental marker of midbrain dopamine cells.The same as CXCL14, it was the highest expression on embryonic day 14.5 (E14.5).
The absence of CXCL14 caused the reduction of neurons in the somatic external pyramidal layer, which was probably related to the migration and differentiation function of neurons.In the nervous system, chemokines and their receptors are not only inductively expressed in inflammation, but also constitutively expressed in physiological situations.It plays an important regulatory role in the differentiation, migration, apoptosis, proliferation and activation of neurons.
CXCL14 interacted with CXCR4 and CXCR7 in experiments of MALAR-Y2H (Figure 4B,C).Coincidentally, CXCR4 and CXCR7 also are receptors of CXCL12, whose function has been confirmed in the neuro-chemotactic field [23].The mainstream view considers that CXCR7 can regulate the function of CXCR4 by forming heteropolymers with CXCR4 [24].It has been suggested that CXCL14 was a natural inhibitor of the CXCL12-CXCR4 signal axis by interacting with CXCR4 [25].CXCR4 was also highly expressed during embryonic brain development.It could trigger the PI3K/AKT signaling pathway, which regulates cell proliferation and differentiation by activating D-cyclins [26,27].Other evidence suggests that CXCR4 activation caused the MAPK signaling pathway to activate the ERK1/2 and JAK/STAT, promoting the growth and differentiation of neurons [28][29][30][31].In this experiment, the number of cortical neurons decreased at E13.5, while CXCL12 knockout mice showed abnormal aggregation of neurons from E13-E14 in the embryonic stage.
Metabolomics was used to explore the mechanism of CXCL14 on neuronal growth.Most of the differential metabolites pointed to the pathway of glycerophospholipid anabolism and fatty acid anabolism, in which CXCL14 played a particularly significant role in regulating alpha-linolenic acid and linoleic acid (Figure 5A-C).
Polyunsaturated fatty acids (PUFAs) are divided into two types of unsaturated fatty acids, one is the Ω-3 (Omega-3) series of polyunsaturated fatty acids based on alphalinolenic Acid (ALA), which can be metabolized into Docosahexaenoic Acid (DHA); The other is the Ω-6 (Omega-6) series of polyunsaturated fatty acids based on linoleic acid, which can synthesize gamma-linolenic acid and arachidonic acid (Figure 6E) [32,33].During embryonic development, non-esterified polyunsaturated fatty acids can bind to the ligand domain of nuclear receptors and promote neurogenesis, neuronal differentiation and synaptic plasticity [34,35].In addition, arachidonic acid and docosahexaenoic acid are the main long-chain polyunsaturated fatty acids (LCPUFA) in the membranes of brain cells, which can increase the length and number of branches of synapses [36][37][38].The levels of Palmitic acid, linoleic acid and alpha-linolenic acid in the serum of CXCL14 −/− defatting the sections, unstained slides were stained with Nissl Differentiation.Finally, slides were dehydrated by sinking into ddH 2 O, 70% ethyl alcohol (twice), 95% ethyl alcohol (twice), 100% ethyl alcohol (twice) and transparentized in xylene (twice) and mounted with neutral balsam.The morphological alterations were observed under a light microscope.

Quantitative RT-PCR
Major organs, including the brain, heart, kidney, liver, lung, small intestine, spleen, stomach, and skin, were collected from 8-week-old male C57 mice.The brain tissue analyzed was from the cerebral cortex.The total RNA of tissues and cell lines was extracted using the RNeasy Plus Universal Tissue Kit (Sangon Biotech Co., Shanghai, China) based on the manufacturer's instructions.The mRNA was purified as described in Hieff NGS ® mRNA Isolation Master Kit mRNA Purification Kit.(Yeasen Biotechnology, Shanghai, China).

Western Blot
From embryonic day 10.5 (E10.5, E12.5, E14.5, E16.5, E18.5), mouse parietal cortex tissues were collected.The mice were killed by cervical dislocation, the brain was rapidly removed from the skull and the cerebral hemispheres were frozen in isopentane within liquid nitrogen.Samples were stored at −80 • C for subsequent lysis.

Gene Set Enrichment Analysis
GEPIA2 (http://gepia2.cancer-pku.cn/#index,accessed on 3 December 2023) is an updated version of GEPIA.It can be used to analyze the RNA expression sequencing data of 9736 tumor samples and 8587 normal samples in TCGA and GTEx.The "similar gene detection" module of the GEPIA2 database was used to obtain a gene set that was similar to the CXCL14 expression pattern and ranked in the top 1000.Similar genes detection of function identifies a list of genes with similar expression patterns with CXCL14 and cerebral cortex (for given sets of GTEx expression data).The list of genes that have similar expression patterns ranked by Pearson correlation coefficient (PCC).A standard language with three levels of structure, including biological process (BP), cell composition (CC), and molecular function (MF), has been developed to provide consistent functional descriptions of gene products in various databases.We performed functional enrichment analysis of CXCL14-related genes by using the cluster profile package in R software (topGO) and visualized the results.Through the "correlation analysis" module in the GEPIA2 database, this function performs pair-wise gene expression correlation analysis for given sets of GTEx expression data, using methods including Pearson.One gene can be normalized by other genes.4.6.Membrane-Anchored Ligand and Receptor Yeast Two-Hybrid System 4.6.1.Cell Lines and Plasmids The E. coli strain DH5α (Code No. 9057) served as the host for plasmid construction and amplification.Yeast strains GoldY2H (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4∆, gal80∆, LYS2::GAL1 UAS -Gal1 TATA -His3, GAL2 UAS -Gal2 TATA -Ade2 URA3::MEL1 UAS -Mel1 TATA -LacZ, MEL1) and Y187 yeast cells (MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4∆, met-, gal80∆, URA3::GAL1 UAS -GAL1 TATA -lacZ), were obtained from Clonetech, USA.The plasmids pGAD-T7 and pGBK-T7 (Code No. 630442, 630443) were also acquired from Clonetech, USA.Our laboratory maintained the 293T cell line.The pcDNA3.4 plasmid (Code No. A14697) was purchased from Life Technology, USA.We performed mycoplasma contamination tests on the cells prior to their use.

Construction of Bait Plasmids
The plasmid was synthesized based on previous studies.The pGAD-T7 plasmid was utilized to express the bait, in which all elements between the ADH1 promoter and ADH1 terminator were deleted, with specific target sequences cloned using a one-step clone kit from Vazyme, Co. Nanjing China.The bait protein consisted of a signal peptide, a ligand protein, a transmembrane peptide, the C-terminal region of yeast ubiquitin (Cub) and reporter genes.In different experiments, a signal peptide derived from either SP CXCL14 (residues 1-28), SP WBP1 (WBP1, residues 1-31) was fused at the N-terminus of ligand The WBP1 transmembrane peptide and flanking sequences (residues 350-430) were used as the transmembrane peptide and linkers and fused between CXCL14 and the reporter genes.To examine subcellular localization, EGFP served as the reporter gene.For detecting PPIs (protein-protein interactions), the Cub-GAL4 cassette functioned as the reporter module.

Construction of Prey Plasmids
Recombinant prey genes were inserted into the pGBK-T7 vector, which involved replacing the original ORF between the ADH1 promoter and terminator with mouse CXC motif receptors and reporter genes.To investigate subcellular localization, EGFP was utilized as a reporter gene, while for PPI detection, N ub G served as a reporter module.

The Yeast Plasmid Transformation
The mating process followed the LiAc method described in the commercial kit manual.Specifically, prey plasmids were transformed into the Y187 yeast strain, whereas bait plasmids were transformed into the GoldY2H cells strain.Transformants were selected using synthetic dropout nutrient medium/plate (SD medium) lacking leucine or tryptophan (SD/Leu − or SD/Trp − ).Mating occurred by suspending 2-3 clones of each cell in 0.5 mL YPD medium and incubating overnight with shaking.The resulting cells were collected through centrifugation and spread onto SD/Leu − Trp − media plates to facilitate interaction strength detection via growth assay.

Interaction Strength Detection
Growth assay involved dropping five microliters of 10-fold diluted cell suspension (OD 600 = 1, 0.1 and 0.01 series) onto SD/Leu − Trp − His − Ade − medium plates, followed by photographing the plates after three days of incubation to record cell growth levels accurately.

β-Galactosidase Activity Assay
Hybrid cells (with an OD 600 ranging from 0.6 to 0.8) were subjected to lysis using Z buffer (composed of 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , and adjusted to pH = 7).The lysis process involved three cycles of freezing in liquid nitrogen and thawing in a water bath set at 37 • C. Subsequently, the resulting cell lysates were resuspended in Z buffer containing 0.27% (v/v) 2-mercaptoethanol and 0.4% 2-nitrophenylβ-D-galactopyranoside at a temperature of 30 • C, allowing for chromogenic reaction.The reaction was brought to a halt after 40 min by adding 400 µL of 1 M Na 2 CO 3 .The absorbance of the supernatant was then measured at a wavelength of 420 nm.It is important to note that the OD 600 value corresponds to the absorbance of the yeast culture before lysis.Each sample was subjected to three repetitions of the procedure.The activity of β-Gal was determined using the subsequent equation: β-galactosidase units = 1000 × OD 420 t(min) × OD 600 (1) The test was used as the statistical method.These serum samples were thawed slowly at 4 • C and then 100 µL of each sample was added to 400 µL of precooled methanol/acetonitrile solution (1:1, v/v), vortexed and mixed, left at −20 • C for 60 min, centrifuged at 14,000× g for 20 min at 4 • C and the supernatant was taken and freeze-dried under vacuum.For mass spectrometry, 100 µL of aqueous acetonitrile solution (acetonitrile: water = 1:1, v/v) was added to redissolve, vortexed, centrifuged at 14,000× g at 4 • C for 15 min, and the supernatant was injected into the sample for analysis B at 40%.From 18 to 18.1 min, B changed linearly from 40% to 95%; B was maintained at 95% from 18.1 to 20 min.QC samples were inserted into the sample cohort to monitor and evaluate the stability of the system and the reliability of the experimental data.

LC-MS Method
Each sample was detected by electrospray ionization (ESI) in positive and negative ion modes, respectively.The samples were separated by UPLC and analyzed by mass spectrometry using a Triple-TOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA).The ESI Source conditions are as follows: Ion Source Gas1 (Gas1): 60, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600 • C, IonSapary Voltage Floating (ISVF) +5500 V (positive and negative modes); TOF MS scan m/z range: 60-1200 Da, product ion scan m/z range: 25-1200 Da, Time of Flight Mass Spectrometer (TOF) MS scan accumulation time 0.15 s/spectra, product ion scan accumulation time 0.03 s/spectra; Secondary mass spectra were obtained with information-dependent acquisition (IDA) and in high sensitivity mode with Declustering potential (DP): +60 V (positive and negative modes), Collision Energy: 30 eV, IDA Settings as follows Exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 6.

Data Analysis
The Content Management System (CMS) program was used for peak alignment, retention time correction and peak area extraction.The structure of metabolites was identified by accurate mass number matching and secondary spectrogram matching to search the laboratory database.For data extracted using LC/MS and GC/MS Data Analysis (XCMS 3.4) software, missing values within groups were eliminated with a specific target of 50% of ion peaks.Application software SIMCA-P 14.1 (Umetrics, Umea, Sweden) pattern recognition was conducted, and the data were preprocessed by Pareto-scaling.After that, multidimensional statistical analysis was performed, including unsupervised principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA).One-dimensional statistics the analysis included Student's t-test and variogram analysis and R software (ggplot2) mapping the volcano.

Figure 1 .Figure 1 .
Figure 1.CXCL14 knockout caused the reduction of the number of pyramidal cells in the external pyramidal layer and survival of mice.(A) The genotype proportion of offspring born by crossbreeding of 8-week-old male CXCL14 −/− and female CXCL14 +/− mice, they produced a total of 59 mice, 42 mice of CXCL14 +/− and 17 mice of CXCL14 −/− , and the birth rate of CXCL14 −/− was 28.8%, which was significantly different from expected percentage of 50% (n = 59, p < 0.01).(B) Cortical neuron staining of CXCL14 +/− and CXCL14 −/− 8-week-old mice by Nissl staining.(a,b) were slice images of CXCL14 +/− 8-week-old mice cortexes; (c,d) were slice image of CXCL14 −/− 8-week-old mice cortexes.(Layer I: molecular layer; II: external granular layer; III: external pyramidal layer; IV: internal granular layer; V: external pyramidal layer; VI: Multiform Layer.).(C) The density of neurons in each layer of cere-Figure 1. CXCL14 knockout caused the reduction of the number of pyramidal cells in the external pyramidal layer and survival of mice.(A) The genotype proportion of offspring born by crossbreeding Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 4 of 1

Figure 2 .
Figure 2. The CXCL14 expression of mice in spatial and temporal.(All data are expressed as mea ± SD, n = 3).(A) The expression of CXCL14 in major organs of adult male and female 8-week-ol mice by Q-PCR, which was higher in the skin and brain than in other organs.(B) The expression o CXCL14 by Q-PCR at embryonic days 10.5, 12.5, 14.5, 16.5, 18.5, 19.5 and postnatal days 1, 14, 5 (C) The expression of CXCL14 by Western Blot at embryonic days 10.5, 12.5, 14.5, 16.5 18.5 an postnatal days 0, 56.

Figure 2 .
Figure 2. The CXCL14 expression of mice in spatial and temporal.(All data are expressed as mean ± SD, n = 3).(A) The expression of CXCL14 in major organs of adult male and female 8-week-old mice by Q-PCR, which was higher in the skin and brain than in other organs.(B) The expression of CXCL14 by Q-PCR at embryonic days 10.5, 12.5, 14.5, 16.5, 18.5, 19.5 and postnatal days 1, 14, 56.(C) The expression of CXCL14 by Western Blot at embryonic days 10.5, 12.5, 14.5, 16.5 18.5 and postnatal days 0, 56.

Figure 2 .
Figure 2. The CXCL14 expression of mice in spatial and temporal.(All data are expressed as mea ± SD, n = 3).(A) The expression of CXCL14 in major organs of adult male and female 8-week-ol mice by Q-PCR, which was higher in the skin and brain than in other organs.(B) The expression o CXCL14 by Q-PCR at embryonic days 10.5, 12.5, 14.5, 16.5, 18.5, 19.5 and postnatal days 1, 14, 56 (C) The expression of CXCL14 by Western Blot at embryonic days 10.5, 12.5, 14.5, 16.5 18.5 an postnatal days 0, 56.

Figure 3 .
Figure 3. GO enrichment analysis was performed using GEPIA website to analyze the genes wit similar expression patterns in the cerebral cortex of CXCL14 in the GTEx database.(A) The top 2 Gene Ontology (GO) enrichment terms in Biological Processes (BP), Cellular Components (CC), an

Figure 3 .
Figure 3. GO enrichment analysis was performed using GEPIA website to analyze the genes with similar expression patterns in the cerebral cortex of CXCL14 in the GTEx database.(A) The top 20 Gene Ontology (GO) enrichment terms in Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF) in STAD (Stomach Adenocarcinoma) were identified.(B) The main genes in the neural projection pathway were positively correlated with CXCL14.

Figure 4 .
Figure 4. Interaction test of the CXCL14 and CXCR family members.(A) Diagram of Membrane-Anchored Ligand and Receptor Yeast Two-Hybrid, MALAR-Y2H.(i) The interaction between CXCL14 and the CXCR series receptor protein was detected.CXCL14 was fused to the C-terminus of ubiquitin protein (Cub) through a transmembrane peptide (TMP) and conjugated to the transcription factor GAL4.The C-terminus of the receptor protein is coupled to the N-terminus of the ubiquitin protein (Nub).When CXCL14 interacts with the extracellular domain of the receptor, Cub and Nub recombine into active ubiquitin monomers that are hydrolyzed by ubiquitin-specific hydrolase (UBP) to release GAL4.(ii) Negative control, CXCL14 and yeast membrane protein OST1 were used as bait and prey to form a negative control group.(B) Yeast Two-hybrid.GoldY2H cells expressing the CXCL14-CXCL14-TMP-Cub-GAL4 plasmid were hybridized with Y187 cells expressing each OST1-CXCR-NubG fusion protein, and diploid cells were dropped onto SD/Leu − Trp − Ade − His − plates containing α-X-gal and 15 mM 3-AT.The growth of cells was observed after 72 h cultivation.CXCL14-TMP-Cub-GAL4 × OST1-NubG was used as a negative control and WBP1-Cub-GAL4 × OST1-NubG was used as a positive control.(C) β-Gal assay of diploid cells expressing SP-CXCL14-TMP-Cub-GAL4 and each of the SP-OST1-CXCR-NubG proteins.The experiment was repeated three times independently and every sample was tested twice in each experiment (n = 3).

Figure 4 .
Figure 4. Interaction test of the CXCL14 and CXCR family members.(A) Diagram of Membrane-Anchored Ligand and Receptor Yeast Two-Hybrid, MALAR-Y2H.(i) The interaction between CXCL14 and the CXCR series receptor protein was detected.CXCL14 was fused to the C-terminus of ubiquitin protein (C ub ) through a transmembrane peptide (TMP) and conjugated to the transcription factor GAL4.The C-terminus of the receptor protein is coupled to the N-terminus of the ubiquitin protein (N ub ).When CXCL14 interacts with the extracellular domain of the receptor, C ub and N ub recombine into active ubiquitin monomers that are hydrolyzed by ubiquitin-specific hydrolase (UBP) to release GAL4.(ii) Negative control, CXCL14 and yeast membrane protein OST1 were used as bait and prey to form a negative control group.(B) Yeast Two-hybrid.GoldY2H cells expressing the CXCL14-CXCL14-TMP-C ub -GAL4 plasmid were hybridized with Y187 cells expressing each OST1-CXCR-N ub G fusion protein, and diploid cells were dropped onto SD/Leu − Trp − Ade − His − plates containing α-X-gal and 15 mM 3-AT.The growth of cells was observed after 72 h cultivation.CXCL14-TMP-C ub -GAL4 × OST1-N ub G was used as a negative control and WBP1-C ub -GAL4 × OST1-N ub G was used as a positive control.(C) β-Gal assay of diploid cells expressing SP-CXCL14-TMP-C ub -GAL4 and each of the SP-OST1-CXCR-N ub G proteins.The experiment was repeated three times independently and every sample was tested twice in each experiment (n = 3).

Figure 5 .
Figure 5. Multivariate data analysis from UPLC-MS/MS.(A) PCA score plots of serum samples collected from different groups.(B) Score scatter plot of the OPLS-DA model for group CXCL14 +/− (W) and CXCL14 −/− (C).(C) Permutation test of the OPLS-DA model for group CXCL14 +/− (W) and CXCL14 −/− (C).The abscissa represented the permutation retention of the permutation test and the ordinate represents the value of R2Y or Q2; the green dot represents the value of R2Y obtained by the permutation test and the blue square dot represents the value of Q2 obtained by the permutation test; the two dashed lines represent the regression lines of R2Y and Q2.(D) Hierarchical clustering of metabolites with significant differences.

Figure 5 .
Figure 5. Multivariate data analysis from UPLC-MS/MS.(A) PCA score plots of serum samples collected from different groups.(B) Score scatter plot of the OPLS-DA model for group CXCL14 +/− (W) and CXCL14 −/− (C).(C) Permutation test of the OPLS-DA model for group CXCL14 +/− (W) and CXCL14 −/− (C).The abscissa represented the permutation retention of the permutation test and the ordinate represents the value of R2Y or Q2; the green dot represents the value of R2Y obtained by the permutation test and the blue square dot represents the value of Q2 obtained by the permutation test; the two dashed lines represent the regression lines of R2Y and Q2.(D) Hierarchical clustering of metabolites with significant differences.