Proteomic analysis of fetal skin by iTRAQ reveals molecular signals underlying Inner Mongolia Cashmere goat hair follicle initiation

Figures & data

Figure 1. Morphological observation of hair follicles across three developmental stages in cashmere goat. (A–C) Skin at embryonic day 45 (E45), 55 (E55), 65 (E65), respectively. (A) Forty-five days of embryonic period: dermal signal initiation; The induction of hair follicles is initiated around E45 and characterized by crowded epidermal keratinocytes, hair placode, and dermal condensate. (B) Fifty-five days of embryonic period: primary hair follicle genesis; the number of primary follicles increases significantly. (C) Sixty-five days of embryonic period: secondary hair follicle genesis, secondary follicles (SFs) start to form.

Figure 2. (A) Distribution of proteins containing different numbers of identified peptides, (B) distribution of the identified proteins among the different molecular weight classes, (C) coverage of proteins by the identified peptides, (D) distribution of protein abundance ratio.

Figure 3. Hierarchical cluster analysis of protein expression profiles from nine skin samples with 159 proteins.

Table 1. Selected proteins constantly increased through the whole embryonic developmental stages of placode formation in cashmere goat skin.

Accession	Symbol	Description
Hair keratin family
O77727	KRT15	Keratin, type I cytoskeletal 15 OS = Ovis aries GN = KRT15 PE = 2 SV = 1 – [K1C15_SHEEP]
W5Q6E8	KRT75	Uncharacterized protein OS = Ovis aries GN = KRT75 PE = 3 SV = 1 – [W5Q6E8_SHEEP]
W5Q665	KRT13	Uncharacterized protein OS = Ovis aries GN = KRT13 PE = 3 SV = 1 – [W5Q665_SHEEP]
W5Q5U5	KRT4	Uncharacterized protein OS = Ovis aries GN = KRT4 PE = 3 SV = 1 – [W5Q5U5_SHEEP]
W5Q611	KRT1	Uncharacterized protein (Fragment) OS = Ovis aries GN = KRT1 PE = 3 SV = 1 – [W5Q611_SHEEP]
W5Q687	KRT5	Uncharacterized protein OS = Ovis aries GN = KRT5 PE = 3 SV = 1 – [W5Q687_SHEEP]
W5Q160	KRT10	Uncharacterized protein OS = Ovis aries GN = KRT10 PE = 3 SV = 1 – [W5Q160_SHEEP]
U3MXA9	KRT17	Keratin 17 OS = Ovis aries GN = KRT17 PE = 2 SV = 1 – [U3MXA9_SHEEP]
Annexins
W5PSZ5	ANXA1	Annexin OS = Ovis aries GN = ANXA1 PE = 3 SV = 1 – [W5PSZ5_SHEEP]
A2SW69	ANXA2	Annexin A2 OS = Ovis aries GN = ANXA2 PE = 1 SV = 1 – [ANXA2_SHEEP]
W5PEC3	ANXA6	Annexin OS = Ovis aries GN = ANXA6 PE = 3 SV = 1 – [W5PEC3_SHEEP]
Extracellular matrix (ECM) components
W5Q5H8	FGA	Fibrinogen alpha chain OS = Ovis aries GN = FGA PE = 4 SV = 1 – [W5Q5H8_SHEEP]
W5Q4M3	COL5A2	Uncharacterized protein OS = Ovis aries GN = COL5A2 PE = 4 SV = 1 – [W5Q4M3_SHEEP]
Q9MZW2	COL6A1	Type VI collagen alpha 3 chain (Fragment) OS = Ovis aries GN = COL6A1 PE = 2 SV = 1 – [Q9MZW2_SHEEP]
W5PTN6	COL6A2	Uncharacterized protein OS = Ovis aries GN = COL6A2 PE = 4 SV = 1 – [W5PTN6_SHEEP]
W5QCP9	COL6A3	Uncharacterized protein OS = Ovis aries GN = COL6A3 PE = 4 SV = 1 – [W5QCP9_SHEEP]
W5Q4S0	COL3A1	Uncharacterized protein OS = Ovis aries GN = COL3A1 PE = 4 SV = 1 – [W5Q4S0_SHEEP]
Growth factor and associated molecules
W5Q4S0	COL3A1	Uncharacterized protein OS = Ovis aries GN = COL3A1 PE = 4 SV = 1 – [W5Q4S0_SHEEP]
W5Q0F3	TGFBI	Uncharacterized protein (Fragment) OS = Ovis aries GN = TGFBI PE = 4 SV = 1 – [W5Q0F3_SHEEP]
Q9TTE2	DCN	Decorin OS = Ovis aries GN = DCN PE = 2 SV = 1 – [PGS2_SHEEP]

Table 2. Specific information of these eight proteins in the form of tables.

	Protein expression
Description	45–1	45–2	45–3	55–1	55–2	55–3	65–1	65–2	65–3	ANOVA p-value
KRT15	0.8453	0.6505	0.7181	1.3406	0.9549	0.8693	1.2997	1.2695	1.0076	.0235
KRT75	0.9311	0.9189	0.8163	0.9755	0.6965	0.8107	1.7067	1.4699	1.5065	.0019
KRT13	0.2857	0.4753	0.4084	0.9288	0.6141	0.9441	1.6172	2.2305	2.0617	.0053
KRT4	0.5475	0.6275	0.6620	0.9492	0.7447	0.9118	1.4046	1.9674	1.7361	.0093
KRT1	0.7870	0.8393	0.7176	0.8253	0.8229	0.9061	2.0776	1.9124	1.6516	.0026
KRT5	0.5297	0.5369	0.5632	0.8161	0.5326	0.8755	2.4429	2.1586	1.9363	.0019
KRT10	0.7557	0.6805	0.6544	0.7807	0.7984	0.7595	1.7205	1.8384	1.5921	.0003
KRT17	0.6063	0.7183	0.6827	0.8900	0.7321	0.8426	1.6998	1.9234	1.8364	.0003

Figure 4. Venn diagram depicting the overlap between proteins differentially expressed between E55 vs. E45, E65 vs. E55, and E65 vs. E45.

Figure 5. GO Analysis of differentially expressed Proteins in three developmental stages of hair follicles (P < .05).

Table 3. KEGG pathway analysis.

Pathway	Count	Proteins
Metabolic pathways	11	ALDH9A1, ANPEP, BCAT1, CKB, CKMT2, CS, GFPT1, ISYNA1, PHGDH, PTGES3, PTGIS
ECM–receptor interaction	7	COL6A1, COL6A2, COL6A3, FN1, HSPG2, THBS1, TNC
Focal adhesion	7	CDC42, COL6A1, COL6A3, FLNB, FN1, THBS1, TNC
PI3K-Akt signaling pathway	6	COL6A1, COL6A3, FN1, HSP90AB1, THBS1, TNC
MAPK signaling pathway	3	CDC42, FLNB, NFATC1
TGF-beta signaling pathway	2	DCN, THBS1
VEGF signaling pathway	1	CDC42
Wnt signaling pathway	1	NFATC1
p53 signaling pathway	1	THBS1

Figure 6. (A) The protein–protein interaction network for the differentially expressed proteins. (B) The differentially expressed proteins are highlighted in yellow.

Table 4. KEGG pathway analysis.

Pathway	Count	Proteins
Metabolic pathways	11	ALDH9A1, ANPEP, BCAT1, CKB, CKMT2, CS, GFPT1, ISYNA1, PHGDH, PTGES3, PTGIS
ECM–receptor interaction	7	COL6A1, COL6A2, COL6A3, FN1, HSPG2, THBS1, TNC
Focal adhesion	7	CDC42, COL6A1, COL6A3, FLNB, FN1, THBS1, TNC
PI3K-Akt signaling pathway	6	COL6A1, COL6A3, FN1, HSP90AB1, THBS1, TNC
MAPK signaling pathway	3	CDC42, FLNB, NFATC1
TGF-beta signaling pathway	2	DCN, THBS1
VEGF signaling pathway	1	CDC42
Wnt signaling pathway	1	NFATC1
p53 signaling pathway	1	THBS1

Data availability statement

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository (Ma et al. 2019) with the dataset identifier PXD035366.