Identification method for pig hair follicle placode precursor cell, and use of method

Abstract

The present invention relates to an identification method for a pig hair follicle placode precursor cell, and a use of the method. In the present invention, OGN and UCHL1 are used as marker genes of a pig hair follicle placode precursor cell, the gene number of OGN in the NCBI database is 106509723, and the gene number of UCHL1 in the NCBI database is 396637. The identification method comprises: subjecting an epidermal cell of a pig embryo to immunofluorescence staining, wherein a cell in which both OGN and UCHL1 can be detected is a pig hair follicle placode precursor cell, and the pig embryo is an embryo on or before the 37-th day of the pig embryo stage. The identification method for a pig hair follicle placode precursor cell provided by the present invention is used in the research of hair follicle development. It is disclosed in the present invention that OGN and UCHL1 are more suitable as marker genes of pig hair follicle placode precursor cells than BMP7 and TGFβ2.

Description

Identification method of pig hair follicle placode precursor cells and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an identification method for pig hair follicle placode precursor cells and an application thereof. Background Art

[0002] Hair follicle development is a complex morphogenetic process, divided into embryonic morphological development and postnatal cyclical development. Embryonic hair follicle development involves a complex interplay between the epithelium and mesenchyme, involving numerous molecular and signaling pathways. Induced by signals from the dermis, the epithelium begins to thicken and form the follicular placode, marking the initial stage of hair follicle development and a key morphological hallmark of its onset. The follicular placode can generate nearly all cell types of the adult hair follicle. Its formation determines the proper formation and development of the hair follicle, and the number of follicular placodes also determines the number of hairs. The events following follicular placode formation have been extensively studied due to its morphological ease and stable molecular markers. However, the lack of prospective molecular markers and the indistinguishable morphology have made it difficult to investigate the cellular origins and molecular mechanisms underlying the early formation of follicular placode cells. Further investigation of this process will contribute to our understanding of hair follicle development and provide insights into human genetic hair disorders. Technical issues

[0003] In the field of hair follicle development research, mice are often used as a model due to their short generation interval, synchronous hair development, ease of manipulation, and low cost. However, there are still certain differences in the similarity of their embryonic development with humans. The difficulty of obtaining human embryos and the resulting ethical issues have restricted further research on embryonic hair follicle development. As a species with a very high homology to humans, the pig is highly similar to humans in anatomy and physiology. Its embryonic development process and hair type and morphology are highly consistent with humans, making it a suitable model animal for hair follicle development. However, there is little research on the molecular events in the early formation of the hair follicle placode and the markers of early hair follicle placode cells in pigs. The Chinese patent 202310153985.0 discloses a method for identifying pig embryonic hair follicle placode precursor cells and its application, which is the research result of this team. It provides key gene marker genes BMP7 and TGFβ2 for identifying pig embryonic hair follicle placode precursor cells. However, in the subsequent practice of this team, it was found that when BMP7 and TGFβ2 were used as marker genes to screen hair follicle placode precursor cells, missed screening occurred. Some hair follicle placode precursor cells were missed during screening because they did not express BMP7 and TGFβ2. Since early pig embryos were used to screen hair follicle placode precursor cells, the sample source is relatively rare. If there are more efficient marker genes for hair follicle placode precursor cells, the efficiency of using early pig embryos will be greatly improved, and more hair follicle placode precursor cells will be obtained. Technical Solutions

[0004] To solve the above problems, the present invention proposes a method for identifying pig hair follicle placode precursor cells and its application, aiming to provide an effective and stable marker for calibrating pig hair follicle placode precursor cells, which can be used to track the development process of early hair follicles and explore the molecular mechanism of hair follicle placode formation.

[0005] The present invention provides an identification method for pig hair follicle placode precursor cells, using OGN and UCHL1 as marker genes for pig hair follicle placode precursor cells, wherein the gene number of OGN in the NCBI database is 106509723, and the gene number of UCHL1 in the NCBI database is 396637. The identification method comprises the following steps: subjecting pig embryo epidermal cells to immunofluorescence staining, and cells in which OGN and UCHL1 can be simultaneously detected are pig hair follicle placode precursor cells, wherein the pig embryo refers to an embryo on or before the 37th day of the embryonic stage.

[0006] The gene sequences of OGN (Gene ID: 106509723) and UCHL1 (Gene ID: 396637) can be found in the Suscrofa11.1 reference genome (ftp: / / ftp.ensembl.org / pub / release-95 / fasta / sus_scrofa / ).

[0007] The identification method of pig hair follicle placode precursor cells provided by the present invention is applied in hair follicle development research. Beneficial effects

[0008] The beneficial effects of the present invention are as follows:

[0009] 1. The present invention found that compared with BMP7 and TGFβ2, OGN and UCHL1 are more highly expressed in a subpopulation of pig hair follicle placode precursor cells and less expressed in other subpopulations, indicating that OGN and UCHL1 are more suitable as marker genes for pig hair follicle placode precursor cells than BMP7 and TGFβ2. The present invention combines bioinformatics and molecular biology to analyze hair follicle development-related cells at different time points during the pig embryonic period to explore the process of early development of the hair follicle placode, screen out specific markers and test them, and find that OGN and UCHL1 are more suitable markers. The presence of pig hair follicle placode precursor cells can be determined by combined expression, which will help to explore the subsequent molecular mechanism of hair follicle placode development.

[0010] 2. The present invention provides two suitable markers, OGN and UCHL1, which can be used to isolate and culture pig hair follicle placode progenitor cells expressing the two markers in the early pig embryo using flow cytometry sorting technology. On this basis, a stable pig hair follicle placode progenitor cell line can be established, providing materials for the study of the molecular mechanism of hair follicle placode formation.

[0011] 3. In previous experiments, we used single-cell and spatial transcriptome sequencing to explore the process of hair follicle morphogenesis in pigs. We identified multiple cell subtypes involved in hair follicle development and analyzed the spatiotemporal landscape of gene expression during hair follicle morphogenesis. We also elucidated the progenitor cell origin, cell signaling, and transcriptional regulatory networks involved in hair follicle placode formation. Based on this analysis and combined with cell-based experiments, we proposed that the optimal time to identify pig hair follicle placode precursor cells using OGN and UCHL1 as marker genes is embryonic day 37 or earlier. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic diagram of phenotypic identification of pig embryo samples and single-cell sample preparation;

[0013] FIG2 is a schematic diagram of cell clustering in the porcine embryonic epidermis;

[0014] FIG3 is the expression detection of hair follicle placode precursor cell marker genes at E37 in Example 2;

[0015] FIG4 is the expression detection of the TC2 key gene described in Example 2 in E41. Modes for Carrying Out the Invention

[0016] The present invention will be further described below with reference to the examples.

[0017] Example 1: Single-cell transcriptome sequencing and screening of candidate genes in early hair follicle placode cells.

[0018] The process of collecting cells from hairy and hairless pig embryo samples for clustering and annotating cell types based on the expression of classic cell type markers in the cells is consistent with the content disclosed in Patent 202310153985.0.

[0019] Specifically, the samples used in this invention primarily come from a genetic resource population of hairless pigs previously established by our research group. While normal Large White pigs have a certain amount of hair on their backs, our research group discovered a population of naturally hairless or sparsely haired Large White pigs within their natural population. Through artificial mating experiments, we determined that this hairlessness trait conforms to Mendelian inheritance and is an autosomal recessive trait, with normal hair as the dominant trait and hairlessness as the recessive trait. Heterozygotes exhibit normal hair. In their research on hair follicle placode precursor cells, our research group mated a sample of semen from a heterozygous normal boar with four homozygous hairless sows. Medical abortions were then performed on one of the sows on days 37, 41, 52, and 85 of gestation, resulting in over ten embryos from each sow (from the same litter). Our research group found that the epidermis of normal Large White pigs is uniformly distributed in a single layer at 37 days of embryonic period. At 41 days of embryonic period, the epidermis grows and sinks toward the dermis, forming the structure of the hair follicle basement, which will develop into hair follicles and produce hair in the future. However, the epidermis of hairless pigs does not form or forms very little hair follicle basement structure at 41 days of embryonic period. The epidermis of hairless pigs remains uniform from 41 days of embryonic period to adulthood, which is specifically manifested by the absence of hair follicle structure and the formation of no hair throughout life. Therefore, at 41 days of embryonic period and later, longitudinal sections of the skin can be prepared and HE staining can be performed to identify whether the pig embryo is normal or hairless. The standard for division is based on the number of hair follicles per 1 cm. 2 Embryos are categorized based on the number of hair follicles. Embryos with less than one hair follicle are classified as hairless (H), while those with more than four hair follicles are classified as normal (N). At embryonic day 37, hair follicle development has not yet begun in both normal and hairless pigs, and the pig's phenotype cannot be determined. These pigs are classified as unidentified (U).

[0020] Building on this research, our research team collected seven skin samples from the same litter (one sample at embryonic day 37) and one sample each from hairy and hairless pigs from the same litter at embryonic days 41, 52, and 85. Single-cell suspensions were prepared from these seven embryonic skin samples and subjected to single-cell transcriptome sequencing and spatial transcriptome sequencing (see Figure 1). Analysis of the sequencing results from these samples yielded approximately 51,871 cells and approximately 18,000 genes. Cells from both hairy and hairless embryonic samples were first clustered based on the top 2000 highly variable genes. Cells were annotated based on the expression of classic cell type markers, resulting in six cell types: epidermal cells, dermal cells, pericytes, endothelial cells, vascular smooth muscle cells, and Schwann cells. Because the hair follicle placode is derived exclusively from epidermal cells, after the coarse classification and annotation of epidermal-type cells, epidermal cells were sub-clustered and further divided into nine subpopulations. The significantly different genes and functions calculated for epidermal cell subpopulations were analyzed, and the subpopulations were annotated. Cell subpopulation 0 is interfollicular basal cells; cell subpopulation 2 is mitotically active cells; cell subpopulation 3 is hair follicle bulge cells; cell subpopulation 4 is considered to be hair follicle placode precursor cells; cell subpopulation 5 is keratinocytes; cell subpopulation 6 is progenitor cells (stromal / hair follicle stem cells); cell subpopulation 7 is interfollicular granular cells; cell subpopulation 8 is keratinocytes (sebaceous glands); cell subpopulation 1 has no hair follicle-related markers identified and is labeled epidermis 1 (see Figure 2).

[0021] Our research team discovered that OGN and UCHL1 may be more suitable marker genes for hair follicle placode progenitor cells than BMP7 and TGFβ2. Therefore, we used the "FindMarkers" function in Seurat software to compare the two sets of differentially expressed genes, identifying more specific marker genes. pct.1 represents the expression ratio of the gene in all cells of the corresponding cell subpopulation, while pct.2 represents the expression ratio of the gene in all cells of the other subpopulations. The larger the difference between pct.1 and pct.2, the more specific the gene is for that particular subpopulation. Compared to the previously discovered BMP7 and TGFβ2 genes disclosed in Patent 202310153985.0, this Example found that the proportion of cells expressing OGN and UCHL1 in cell subpopulation 4 (pct. 1) was significantly higher than the proportion of cells expressing BMP7 and TGFβ2. The proportion of cells expressing OGN and UCHL1 in all subpopulations except cell subpopulation 4 (pct. 2) was much lower than the proportion of cells expressing BMP7 and TGFβ2. At the same time, the difference between pct. 1 and pct. 2 for OGN and UCHL1 in cell subpopulation 4 was significantly greater than that for BMP7 and TGFβ2 (see Table 1). The results of this Example demonstrate that, compared to BMP7 and TGFβ2, OGN and UCHL1 are expressed at higher levels in cell subpopulation 4 and at lower levels in other subpopulations, indicating that the OGN and UCHL1 genes are more specific and more representative of the characteristics of cell subpopulation 4, indicating that OGN and UCHL1 are more suitable marker genes for porcine hair follicle placode precursor cells.

[0022]

[0023] Meanwhile, although some literatures mentioned that both OGN and UCHL1 are related to the morphological development of hair follicles, this example found for the first time that OGN and UCHL1 are more suitable marker genes for pig hair follicle placode precursor cells than BMP7 and TGFβ2.

[0024] Example 2: Functional verification of candidate genes

[0025] OGN and UCHL1 were identified as marker genes of hair follicle placode progenitor cells at the spatial transcriptome level and tissue protein level.

[0026] In the analysis at the spatial transcriptome level, the sample preparation, raw sequencing data processing, and analysis methods are consistent with those disclosed in Patent 202310153985.0, with the difference that the marker genes for the mapped cells are OGN and UCHL1. Specifically:

[0027] S1. Perform Visium spatial transcription on the E37 and E41 embryos obtained in Example 1;

[0028] S2, library preparation;

[0029] S3, processing raw sequencing data to generate a UMI count matrix;

[0030] S4. Spot Identification and Annotation: After the aforementioned quality control, the spatial transcriptome data were processed and subsequently analyzed using Seurat. The spots captured by ST were classified into distinct cell types covering the corresponding tissue regions. The spatial layout of these spots revealed a thin epidermis, with the dermis underlying and relatively thicker than the epidermis, consistent with their true anatomical locations and morphologies. This resulted in a spatial map of epidermal and dermal cell subtypes in E37 (Figure 3A) and E41N (Figure 4A) / E41 (Figure 4B).

[0031] O / U cells were first detected in the E37 ST sample (Figure 3B), overlapping with epidermal cells and accurately localized. TC1 cells showed no significant difference between normal embryonic (E41N) and hairless embryonic (E41H) samples from E41 (Figure 4C). However, PC (TC2) cells were detected in the epidermis of the normal pig sample E41N, whereas PC (TC2) cells were significantly reduced in the epidermis of the hairless pig sample E41H (Figure 4C). This suggests that in normal pigs, O / U cells differentiate into hair follicle placode PC (TC2) cells. The accumulation of sufficient PC cells in the epidermis underlies the formation of the hair follicle placode, which is the initial structural feature observable during hair follicle morphogenesis and marks the onset of hair follicle formation. In hairless pigs, however, O / U cells generate few or no TC2 cells, indicating that the development of hair follicle placode precursor cells into normal hair follicles is blocked in hairless pig samples. The overall results demonstrated the accuracy of spatial positioning of the two marker genes, OGN and UCHL1, and were highly consistent with the single-cell transcriptome results, indicating that OGN and UCHL1 are suitable markers that can well reflect the characteristics of hair follicle placode precursor cells.

[0032] In the verification of tissue protein levels, except for the selection of primary antibodies, the remaining steps are consistent with the content disclosed in patent 202310153985.0. Specifically:

[0033] (1) Paraffin sections were performed on the E37 and E41 embryos obtained in Example 1, and the sections were placed in an incubator at 60°C for 1 h;

[0034] (2) Place in 100% xylene in sequence (Ⅰ→Ⅱ→Ⅲ), 10 minutes each time, for a total of 30 minutes;

[0035] (3) Place in 100%, 95%, and 80% ethanol in sequence (I → II → III), 10 min / time, for a total of 30 min;

[0036] (4) Rinse with running water for 5 minutes;

[0037] (5) Wash once with PBS (10-15 min);

[0038] (6) Fill the slice box with antigen retrieval solution, cover it, and place it in a microwave oven on high heat for 5 minutes, then turn to low heat for 20 minutes;

[0039] (7) Take out the box containing the slices and let it cool naturally for about an hour;

[0040] (8) Wash three times with PBS, 5 min each time;

[0041] (9) Place some PBS in the incubation box, wipe the slide dry (be careful not to wipe the tissue) and place it flat in a humidified chamber. Add 5% goat serum (the specific amount depends on the number of slides) and use a pipette to draw it onto the tissue. Each tissue requires about 30 μL of liquid. Make sure the goat serum completely covers the tissue and keep the tissue moist throughout the process. Then place the slide in a moisturizing box and leave it at room temperature for 30 minutes.

[0042] (10) Primary antibody: Take two E37U samples, shake off the sheep serum on the slide, wipe off the residual liquid around the tissue with clean paper, and perform OGN+KRT14 (epidermal marker) double labeling and UCHL1+KRT14 double labeling respectively (OGN antibody was purchased from Proteintech, 12755-1-AP, Rabbit polyclonal antibody to OGN, dilution ratio 1:100; UCHL1 antibody was purchased from Boster, BM4990, Rabbit polyclonal antibody to UCHL1, 1:100 dilution; KRT14 antibody: purchased from Santa, sc-53253, 1:200 dilution), dilute the primary antibody with PBS, add the diluted primary antibody and completely cover the tissue, place the slide with the primary antibody in a moisturizing box and leave it at 4°C overnight. The same method was used to select samples from E41N and E41H, and the expression of important TC2 genes (wnt5a, wnt10b) was verified (wnt5a was purchased from abmart, catalog number T56869; wnt10b was purchased from abmart, catalog number TD9038);

[0043] (11) Secondary antibody: Alexa Fluor 488 (green fluorescence) labeled goat anti-mouse IgG (H+L) and Alexa Fluor 647 (red fluorescence) labeled goat anti-rabbit IgG (H+L) were diluted at 1:500 and then dropped on the tissue. The slide was placed in a wet box and left at room temperature for 30 minutes. All operations after the secondary antibody application were carried out in a dark environment.

[0044] (12) Wash with PBS 3 times, 5 min each time;

[0045] (13) Wipe away the liquid around the tissue, add ready-to-use DAPI, and incubate for 2 minutes;

[0046] (14) Wash with PBS 3 times, 5 min each time;

[0047] (15) Sealing: Wipe the liquid around the tissue dry, drop a drop of anti-fluorescence attenuation mounting medium on the tissue, and cover with a coverslip. Since the coverslip is easy to slide, you can apply nail polish around the coverslip to fix it after covering it;

[0048] (16) Filming: Immediately after staining, films were taken and analyzed using a confocal microscope. As shown in Figure 3C, OGNs were detected in the E37 sample. + / UCHL1 + This indicates that the markers OGN and UCHL1 of cell subpopulation 4, identified through data analysis in Example 1, are indeed expressed in early precursor cells involved in hair follicle placode formation, consistent with the single-cell analysis results and spatial transcriptome results. Together, these two findings validate the accuracy of the hair follicle placode precursor cell markers OGN and UCHL1 identified by the present invention.

[0049] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0050] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for identifying pig hair follicle placode precursor cells, characterized in that: OGN and UCHL1 are used as marker genes for pig hair follicle placode precursor cells, the gene number of OGN in the NCBI database is 106509723, and the gene number of UCHL1 in the NCBI database is 396637; the identification method is: pig embryo epidermal cells are subjected to immunofluorescence staining, and cells in which OGN and UCHL1 can be simultaneously detected are pig hair follicle placode precursor cells, and the pig embryo is an embryo on or before the 37th day of the pig embryonic period.

2. Use of the method for identifying pig hair follicle placode precursor cells according to claim 1 in hair follicle development research.

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