Single-cell analysis of ecto-mesenchymal cells derived from the neural crest reveals the regulatory role of P75NTR in odontogenic and osteogenic differentiation

Abstract

Background

The aim to this study was to explore the effect of p75NTR on the mineralization and development of maxillofacial processes. Moreover, we tried to elaborate the potential mechanism of p75NTR’s effect on the odontogenic or osteogenic differentiation ability of neural crest cells (s).

Methods

We used gene p75NTR knockout (p75NTR^−/−) mice and wildtype (p75NTR^+/+) mice embryos as a model. And We isolated p75NTR^−/− and p75NTR^+/+ ectodermal mesenchymal stem cells (EMSCs) from the embryonic maxillofacial process of the same pregnant p75NTR^+/− mice. The potential mechanism of p75NTR’s effect on the odontogenic or osteogenic differentiation ability of EMSCs was analyzed by single cell RNA sequence(scRNA-seq). The results were further verified by mineralize induction and wound-healing Assay.

Results

We found the deletion of p75NTR could impact the communications between different cell types, influence differentiation and suppress odontogenesis via regulation networks of cytokines that associating with migration, proliferation and differentiation. The volcano plot characterized the distribution of differentially expressed genes (DEGs) in the selection at threshold level. p75NTR deletion suppressed EMSCs migration, proliferation and odonto/osteogenic differentiation in vitro.

Conclusions

This study provides a comprehensive analysis of the cellular fate of EMSCs in the maxillofacial process. p75NTR plays a crucial role in initiating hard tissue development and regulates the odontogenic and osteogenic differentiation of EMSCs during embryonic development.

Background

Tooth loss, a common issue in human life, affects both physical and mental health. We believe that the reconstruction and regeneration of tooth defect should be based on a thorough understanding of its developmental biology and molecular regulatory mechanisms. Therefore, understanding the mechanism of tooth development is essential for advancing tooth regeneration. In the early phase of embryogenesis, neural crest cells (NCCs) migrate to the maxillofacial processes and interact with the dental epithelium, differentiate, mature, and contribute to maxillofacial tissue formation. Upon migrating to the first arch, they are also referred to as cranial neural crest derived cells (CNCs) or ectodermal mesenchymal stem cells (EMSCs). Due to the unique characteristics of tooth development, research on NCCs in regenerative medicine is highly valuable [1]. However, since NCCs are transient during tooth development, studying their biological characteristics is challenging. Thus, clarifying the molecular mechanism underlying tooth development in NCCs is crucial and requires further exploration.

The p75 neurotrophic factor receptor (p75NTR), is a reliable cell surface marker to sort pure EMSCs [2, 3]. Previous studies have thoroughly elucidated that p75NTR mediates various cellular functions including cell adhesion, differentiation, signal transduction, metastasis and apoptosis [4,5,6,7]. Among numerous previous studies, p75NTR has primarily been used as a tracer marker for neural crest to observe their migration and differentiation, especially as a specific marker for neural crest derived stem cells in cell identification. Recent reports have increasingly shown that p75NTR functions as a key protein involved in tooth morphogenesis and development. In terms of odonto/osteogenic differentiation, p75NTR has been shown to play a role in the mineralization of EMSCs in rats [8]. Wang et al. reported that the deletion of p75NTR reduces the mineralization capacity of mouse femurs [9]. In our study, we used p75NTR−/− mice as a model to investigate its effects on tooth mineralization and development. Our previous studies on this model have yielded similar conclusions: P75NTR knockout resulted in a smaller remnant compared to the normal, and whole-body skeletal analysis further revealed some degree of bone loss. Additionally, further studies on this strain demonstrated that P75NTR expression was correlated with clock genes expression. Moreover, in p75NTR−/− mice, the arrangement, morphology, and even boundary in pre-odontoblast/pre-ameloblast layers were disordered [10].

However, conflicting views exist regarding the role of p75NTR in regulating stem cell differentiation. Some researchers speculated that p75NTR may influence EMSCs differentiation through the Smad4, Wnt/β-catenin and PI3K/Akt pathway [9, 11, 12]. Additionally, other studies suggest that the role of p75NTR in tooth development may be associated with melanoma-associated antigen-D1 and sclerostin [12,13,14]. However, a point of controversy remains while some researchers have suggested that P75NTR inhibits the differentiation of deciduous dental pulp stem cells (DDPSC) others propose that it promotes the differentiation of permanent dental pulp stem cells [8]. Overall, these findings highlight the diverse biological functions of p75NTR; However, due to the lack of comprehensive bioinformatics analyses, simple phenotypic analysis of cells is insufficient to delineate the complex molecular regulatory network of EMSCs. Moreover, contradictions and inconsistencies in the findings further contribute to the ambiguity, leaving the detailed mechanisms unclear.

Single-cell RNA sequence(scRNA-seq) has been used to elaborate the potential mechanisms underlying p75NTR’s effect on the osteogenic differentiation capacity of EMSCs. Our findings suggest that the deletion of p75NTR disrupts communication between different cell types, influences differentiation and suppresses odontogenesis by modulating cytokine regulatory networks associated with migration, proliferation, and differentiation. This study sheds light on the molecular mechanisms of tooth development and provides an experimental foundation for future clinical research.

Materials and methods

Animals

Wild type (p75NTR+/+) mice and p75NTR−/− mice were bought from the Jackson Laboratory (Bar Harbor, ME, USA). These mutant mice, which exhibit a targeted deletion of exon III in the p75NTR locus, do not express a functional full-length p75NTR. The mice were raised and bred at the Animal Center of the School of Stomatology, Wenzhou Medical University. This study was approved by the Ethics Committee of School & Hospital of Stomatology, Wenzhou Medical University (No. 2018012).

Cell isolation and cultivation

p75+/− male and female mice were mated, and embryos at embryonic day 16.5 (E16.5) were obtained through abdominal surgery. Primary mesenchymal cells were then isolated from the embryonic maxillofacial processes. Briefly, pregnant mice at embryonic day 16.5 were euthanized, and homozygous embryos were obtained through abdominal surgery. The maxillofacial process tissue was then dissected under a stereo microscope. The collected tissue was washed three times with high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher Scientific, MA, USA) and subsequently digested with Trypsin-EDTA (Gibco/Thermo Fisher Scientific, MA, USA) at 37℃ for 10 min. The digestion was followed by centrifugation at 1000 rpm for 5 min. The resulting cell suspension was resuspended in growth medium consisting of DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific, MA, USA), and 1% antibiotics (100 ug/mL penicillin and 100 µg/mL streptomycin). The cells were maintained in a 5% CO2 humidified incubator at 37℃, and the culture medium was changed every 3 days. The genotype of the mice was confirmed using a one-step mouse genotyping kit (Vazyme, Nanjing, China). Since it was not possible to accurately distinguish the sex of the fetal mice in this experiment, potential gender-related effects on the results were not considered. EMSCs at the third passage were used for all experiments. The osteogenic induction medium consisted of DMEM/F12 supplemented with 10% FBS, 1% Penicillin-streptomycin, 10 mmol/L β-glycerophosphate (Sigma-Aldrich/Merck, Germany.) 100 nmol/L dexamethasone (Sigma-Aldrich/Merck, Germany.) and 50 µg/mL ascorbic acid (Sigma-Aldrich/Merck, Germany.).

Single-cell library preparation and single-cell RNA-sequence data processing

Single-cell cDNA, library preparation and 3′-end single-cell RNA-sequencing were performed by LC-Biotechnology (Hangzhou, China). All data processing was based on the R platform (version 3.6.2, https://www.R-project.org/). Seurat (version4.1.0, http://satijalab.org/seurat/install.html) was employed for cell clustering. Normalize data using the log-normalization method. Cluster Profiler demonstrated pathway descriptions such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis, and Gene Set Enrichment Analysis (GSEA) [15]. The P values were calculated using Pearson’s correlation. Values of P < 0.05 were considered significant. Cell-cell communication was analyzed by cellphoneDB (version 3.1.0, https://www.cellphonedb.org). See the details in supporting information.

Alkaline phosphatase (ALP) staining, Alizarin red staining (ARS) and quantification

We performed the ALP staining assay by using an ALP kit (Beyotime, China) to assess alkaline phosphatase expression in cells. On Day 7 of osteogenic induction, cells were fixed with paraformaldehyde (Servicebio, China) fixative solution for 30 min and then stained in the dark for an additional 30 min. The ARS assay was conducted with an ARS kit (Beyotime, China) to evaluate the deposition of mineralized substances in cells. On day 14 of osteogenic induction, cells were fixed and stained with 1% ARS at room temperature for 20 min. histochemical detection of ALP and ARS staining was observed and imaged using Nikon microscope (Nikon, Japan).

Wound-Healing assay

We performed wound healing migration test as described. In short, when cells reached 70% confluence in a 24-well plate (Biofil, China), scrapped off the holes with a sterile pipette tip (200 µl). Washed the cells twice to remove separated cells. Images of the wound closure were captured under a microscope at 24 hours. The wound area was quantified by measuring the pixel count using Adobe Photoshop.”

CCK-8 proliferation

Cell Counting Kit-8 (CCK-8; Dojindo Kagaku Co., Japan) was used to investigate proliferation rate of E16.5d p75NTR+/+ and p75NTR−/− EMSCs. EMSCs were seeded at a density of 2 × 103 cells per well in a 96-well plate (Biofil, China). After 1 to 7 days of in vitro culture, 10 µl CCK-8 solution was added into each well and further culture in the dark at 37℃ for 2 h. Absorbance at 450 nm was measured daily for 7 consecutive days using a microplate reader (BioTek Instruments, VT, USA).

Results

ScRNA-seq of the maxillofacial process tissue and identification of cell types

To study whether p75NTR regulates the odontogenic or osteogenic differentiation of NCCs during embryonic development, the maxillary process was isolated from E16.5d p75NTR+/+ and p75NTR−/−mouse embryos. The droplet-based scRNA-seq was performed on cells with p75NTR+/+ and p75NTR−/−. Using t-distributed stochastic neighbor embedding (t-SNE) for unsupervised clustering of a total of 25,464 cells (12665 cells for p75NTR−/− sample and 12799 cells for p75NTR+/+sample), 20 clusters with different gene expression characteristics were identified (Fig. 1a). We divided these clusters into discrete subgroups based on the expression of specific markers, including muscle cells, mesenchymal cells, macrophages, endothelial cells, hair cells, epithelial cells, T cells, and ependymal cells (Fig. 1b). Clusters 0, 1, 2, 3, 4, 7, 16, and 17 expressing Eln are defined as “mesenchymal cells” (Fig. 1b, Fig. S1). Clusters 5 and 8 expressing Tnnt1 are defined as’ muscle cells’ (Fig. 1b, Fig. S1). Clusters 6, 9, and 12 expressing Krt14 are defined as “epithelial cells” (Fig. 1b, Fig. S1). Cluster 10 expressing Egfl7 is defined as “endothelial cells” (Fig. 1b, Fig. S1). Clusters 13 and 18 expressing Pf4 are defined as “macrophages” (Fig. 1b, Fig. S1). Cluster 14 expressing Cma1 is defined as “T cells” (Fig. 1b, Fig. S1). Cluster 15 expressing Fabp7 is defined as “glial cells” (Fig. 1b, Fig. S1). Cluster 19 expressing Ccdc153 is defined as “ependymal cells” (Fig. 1b, Figure S1). Specific marker genes exhibit high cell type specificity in different subgroups (Fig. 1c).

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ScRNA-seq analysis of p75NTR−/− and p75NTR+/+ mesenchymal cells subpopulation

In order to understand the regulations associated with the odontogenic or osteogenic differentiation of NCCs during embryonic development, we used the likelihood-ratio test (bimod) for single-cell gene expression to assign differentially expressed genes at a cutoff of adjusted P value < 0.01 and|log2 fold change| ≥ 0.26. We compared p75NTR−/− mesenchymal cells with p75NTR+/+ mesenchymal cells, the volcano plot characterized the distribution of differentially expressed genes (DEGs) in the selection at threshold level, the annotated genes were the top10 genes with the largest differential upregulation/downregulation (Fig. 2a). The Ebf1 was significantly upregulated in the p75NTR−/− mesenchymal cells compared with the p75NTR+/+ mesenchymal cells (Fig. 2a). The Ebf1 promotes the early differentiation of osteoblasts, plays a key switching role in commitment determination between adipogenic and osteogenic lineages, and regulates the bone homeostasis [16,17,18]. The Apoe was highly expressed after p75NTR deletion (Fig. 2a). The Apoe promotes osteocytocytosis through ERK1/2 pathway and inhibits osteocytic differentiation through c-Fos, NFATc1, and NF-κB pathways, playing a vital role in maintaining bone mass [19, 20]. The Aspn expression was suppressed by p75NTR deletion (Fig. 2a). The Aspn promotes cell migration by regulating TGF-β/Smad2/3 signaling pathway, limits the differentiation of the mesenchymal cells, and suppresses bone formation [20,21,22,23]. The Col1a1 was significantly downregulated after p75NTR deletion (Fig. 2a). The Col1a1 allele was completely absent in osteogenesis imperfecta, and the modification of Col1a1 in autologous adipose tissue-derived progenitor cells rescued bone phenotype in mice with osteogenesis imperfecta [24, 25].

DEGs between p75NTR−/− and p75NTR+/+ EMSCs were enriched in GO terms associated with osteoblast differentiation, ossification, and skeletal system development (Figs. 2b and 3b). Cellular component of DEGs revealed GO term enrichment in extracellular space, collagen-containing extracellular matrix, endoplasmic reticulum and intracellular anatomical structure (Fig. 2b). Molecular function of DEGs revealed GO term enrichment in DNA binding, metal ion binding, and protein binding (Fig. 2b). The pathway analysis of KEGG demonstrated that focal adhesion, regulation of action cytoskeleton, and signaling pathways regulating pluripotency of stem cells might involve in p75NTR regulating the odontogenic or osteogenic differentiation processing (Figs. 2c and 3a). The regulation signaling pathway, such as PI3K-Akt, ECM-receptor interaction, TNF and Apelin, might associate with mesenchymal differentiation processing regulated by p75NTR (Figs. 2c and 3a). We found that gene sets related to N-glycan biosynthesis, focal adhesion and ECM receptor interaction by GSEA. These results suggested that p75NTR is essential for mesenchymal cells migration and osteogenic differentiation during embryonic development (Fig. 3c-e). These results suggested that deletion of p75NTR affects the odontogenic and osteogenic differentiation of mesenchymal cells through cytokine regulatory networks associated with migration, proliferation, and differentiation.

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Deletion of p75NTR influenced the fate of the EMSCs fate of differentiation

The cell population in cluster 0 and 1 decreased following p75NTR deletion, whereas the populations in cluster 2 and 3 increased (Fig. 4a). additionally, the mesenchymal cell population was larger in p75NTR−/− compared to p75NTR+/+(Fig. 4b). However, the endothelial cell population significantly decreased after p75NTR deletion (Fig. 4b). We investigated the gene expression pattern of cluster 2. The top 10 marker genes of cluster 2 were showed in Fig. S2a, b. We compared cluster 2 of p75NTR−/− with p75NTR+/+, the volcano plot characterized the distribution of DEGs in the selection at threshold level (Fig. S3a). These DEGs were enriched in GO terms associated with positive regulation of endothelial cell migration, extracellular matrix and multicellular organism development (Fig. S3b, c). The osteoclast differentiation and relation signaling pathway were among the pathway analysis of KEGG organismal systems terms (Fig. S4a, b). The cell-cell communication networks showed that the major interactions were concentrated between mesenchymal cells with epithelial cells and endothelial cells in the p75NTR+/+ sample (Fig. 4c, Fig. S5a, c). However, the interactions intensity between mesenchymal cells with epithelial cells and endothelial cells decreased in the p75NTR−/− sample (Fig. 4d, Fig. S5b, d). To investigate the interaction information between cell types, we collected CellphoneDB results on the specific protein interaction relationships exist between different cell types. The dot plots showed the top 20 with higher average values average expression level and significance of protein interaction relationship in cell types of p75NTR+/+ or p75NTR−/− sample (Fig. S6a, b). It was discovered that mesenchymal cells could interact with the epithelial cells via NOTCH1/DLK1, PLXNB2/PTN ligand–receptor pairs (Fig. S6a, b), while the interactions intensity weaken in the p75NTR−/− sample (Fig. S6b). These data implied that the deletion of p75NTR of EMSCs during embryonic development could impact the communications between different cell types, finally influence the EMSCs fate of differentiation.

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p75NTR deletion suppressed EMSCs migration, proliferation and odonto/osteogenic differentiation

The expression of p75NTR was suppressed significantly in mesenchymal cells (Fig. S7). In order to analysis the expression pattern in mesenchymal cells on a higher resolution level, the mesenchymal cells were selected for reclustering. Mesenchymal cells were reclustered into 23 clusters (Fig. 5b). The heatmap showing the top 10 marker genes in the 23 clusters (Fig. 5a). We found that cluster 12 was totally eliminated in p75NTR−/− mesenchymal cells that p75NTR+/+ mesenchymal cells (Fig. 5b, c). The marker genes highly expressed in the cluster 12 of p75NTR+/+ mesenchymal cells were Itm2a, Aspn, which were associated with chondrogenic differentiation of NCCs (Fig. S8a, b) [26]. The Mfap4 was associated with cartilage development pathways, epithelial to mesenchymal transition [27], which was highly expressed in cluster 12 (Fig. 5d, e). Aspartic acid regulated chondrogenesis by inhibiting the expression of cartilage genes induced by transforming growth factor beta 1 [28], and inducing collagen mineralization [29], which was highly expressed in cluster 12 (Fig. 5d, e). The Dpt, Vim, tkt, which is responsible for differentiation of mesenchymal stem cells into osteoblasts [30,31,32], were highly expressed in cluster 12 (Fig. 5d, e). Cluster 12 was also highly expressing Pdlim4, which associated with bone mineral density regulation (Fig. 5d, e) [33]. LAMA2, Runx1 regulated the fate commitment of mesenchymal stem cells and osteogenesis for bone homeostasis (Fig. 5d, e) [34, 35].

KEGG analysis demonstrated that osteoclast differentiation, apoptosis, and the TGF-β, Wnt, PI3k-Akt signaling pathway associated with osteoclast differentiation, were enriched in cluster 12 of p75NTR+/+ mesenchymal cells (Fig. 6a, b). GO revealed that collagen-containing extracellular matrix, extracellular matrix organization, cell adhesion, angiogenesis and multicellular organism development were enriched in cluster 12 of p75NTR+/+ mesenchymal cells (Fig. 6c). Subsequently, ALP staining assay and alizarin red staining were performed to measure odonto/osteogenic differentiation of p75NTR+/+ and p75NTR−/− NCCs that were dissected from the first branching sheets of mouse embryos. The results showed that ALP activity of p75NTR−/− NCCs was deeply suppressed compared to p75NTR+/+ NCCs (Fig. 6d). Alizarin red staining showed that calcified nodes of p75NTR+/+ NCCs were significantly higher than p75NTR−/− NCCs (Fig. 6e). Scratch and CCK8 assays were used to compare proliferation and migration of p75NTR+/+ and p75NTR−/− NCCs. In the scratch assay, the migration capability of p75NTR−/−NCCs compared to p75NTR+/+ NCCs was significantly suppressed (Fig. 6f). The CCK8 assay showed that the proliferation was also inhibited by p75NTR deletion in NCCs (Fig. 6g). These results indicated that p75NTR was required for NCCs migration, proliferation, and odonto/osteogenic differentiation, and the deletion of p75NTR suppressed these cell biological processes.

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Discussion

Since Lee et al. developed the P75NTRExonIII knock out model, these mice have been widely used to study neurotrophin receptor and their associated signaling pathways [36]. Numerous studies have demonstrated that P75NTR plays a crucial important role in neurogenesis [37], neuronal development [38], nerve regeneration [39, 40], and axon growth [41]. Interestingly, studies investigating hippocampal function in p75NTR knockout mice have found that downregulation or blockade of p75NTR effectively enhances hippocampal function [42, 43]. This finding provides a novel and promising therapeutic approach for the treatment of Alzheimer’s disease. Additionally, a study by Mahdi Abbasian et al. found that p75NTR−/− mice exhibit severe abnormalities in walking, gait, balance, and strength [44]. As a receptor for neurotrophic factors, research on p75NTR has focused on its neurological functions. They suggested that these gait and balance abnormalities in mice may be related to impaired development of sensory neurons in the dorsal root ganglia, while the potential effects on tooth and bone have largely been overlooked. In our previous study, we demonstrated that p75NTR+/+ EMSCs exhibit greater odontogenic potential than p75NTR−/− EMSCs. Furthermore, we found that p75NTR can inhibit the expression of DSPP and DMP1 during the odontogenic differentiation of EMSCs via the NF-kB signaling [45]. In subsequent studies, Xing et al. reported that deletion of p75NTR leads to the downregulation of mineralization in EMSCs [11]. Furthermore, accumulating evidence has consistently demonstrated that p75NTR knockout mice exhibit some degree of bone loss [9, 13, 45,46,47]. However, whether in the nervous system or stem cell differentiation, in-depth studies on the role of p75NTR in regulating the ultimate fate of these cells remain limited.

ScRNA-seq offers distinct advantages in studying cellular heterogeneity across complex biological systems [48]. Also, scRNA-seq provides new opportunities to disclose the changes of gene expression during the development process of embryos. As ideal models to study the differential potential, human and mouse embryonic stem cells have been traced by scRNA-seq [49, 50].Therefore, we performed the scRNA-seq on p75NTR deficient embryos to investigate the underlying mechanism.

In our study, the scRNA-seq of p75NTR+/+ and p75NTR−/− embryos has been utilized to figure out the effects of p75NTR in tooth development. The unsupervised clustering uncovered and identified 20 clusters. We found that mesenchymal cells which marked by Eln occupied the majority, which confirming the feasibility of developing vitro stem cell as models to represent NCCs-derived cells in previous studies [2]. The odontogenic or osteogenic differentiation of EMSCs is controversial; however, most researchers agreed that during early stages of tooth development, p75NTR derived from NCCs interacts with factors from odontogenic epithelial cells to regulate tooth formation [51]. Our previous results concluded that p75NTR+/+ NCCs had higher tooth differentiation potential under an induction of odontogenic epithelial conditioned medium. In order to figure out it, we selected mesenchymal cells to identify differentially expressed genes in single-cell gene expression analysis. We observed a significant increase in Ebf1 and Apoe expressions, which related with bone homeostasis in p75NTR−/− mesenchymal cells [16, 19]. Our data also exhibited a suppression of Aspn and Col1a1, which correlated with osteogenesis by p75NTR deletion. The re-cluster analysis exposed an eliminated cluster in mesenchymal cells. It marked by Itm2a, Aspn. which were reported to have functions in cell differentiation during odontogenesis [52].The expression of Mfap4 and Dpt, Vim, Tkt were highly expressed in p75NTR+/+ samples, along with Pdlim4, LAMA2, Runx1, which regulated the fate commitment of mesenchymal stem cells [30,31,32, 34, 35]. These results provide new evidence for p75NTR influence the osteogenesis of NCCs cells.

Additional important signal pathways related with p75NTR may have been missed, and the relevant pathways should be explored further. To investigate the mechanism of p75NTR in the mesenchymal cells, we had made some wet lab work, further study should be performed in the sooner future.

Conclusions

p75NTR can not only serve as a marker for cell isolation and purification, but also involved in multiple biological effects of cells. In our study, we provided molecular evidence that p75NTR is involved in the initiation of dental development and the regulation of mineralization. Also, we preliminarily explored the potential signaling mechanisms of P75NTR regulation. However, the precise role and signaling pathway of p75NTR in tooth development and tissue regeneration still need to further investigation. With the deepening of research, a deeper understanding of p75NTR’s regulatory mechanisms in tooth development is expected, which may contribute to uncovering the molecular basis of odontogenesis and advancing tooth tissue engineering.

Data availability

Sequence data that support the findings of this study can be found in the NCBI SRA: 506PRJNA891206.

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