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A single-cell transcriptome atlas during Cashmere goat hair 1
follicle morphogenesis 2
Wei Ge a, Wei-Dong Zhang a, Yue-Lang Zhang a, Yu-Jie Zhenga, Fang Li a, Shan-He Wang a, 3
Jin-Wang Liu c, Shao-Jing Tan b, Zi-Hui Yan b, Lu Wang b, Wei Shen b, Lei Qu c, Xin Wang a 4
5
a Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province,6
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 7
712100, China; 8
b College of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China; 9
c Life Science Research Center, Yulin University, Yulin, Shaanxi 719000, China 10
11
12
¶ Correspondence and reprint requests to: 13
Prof. Xin Wang; E-mail: wxwza@126.com 14
15
16
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted January 31, 2020. . https://doi.org/10.1101/2020.01.30.926287doi: bioRxiv preprint
https://doi.org/10.1101/2020.01.30.926287http://creativecommons.org/licenses/by-nc-nd/4.0/
Abstract: 17
Cashmere, also known as soft gold, is produced from the secondary hair follicles in 18
Cashmere goats and it’s therefore of significance to investigate the molecular profiles 19
during Cashmere goat hair follicle development. However, our current understanding of the 20
machinery underlying Cashmere goat hair follicle remains largely unexplored and 21
researches regarding hair follicle development mainly used the mouse as a research model. 22
To provides comprehensively understanding on the cellular heterogeneity and cell lineage 23
cell fate decisions, we performed single-cell RNA sequencing on 19,705 single cells from 24
induction (embryonic day 60), organogenesis (embryonic day 90) and cytodifferentiation 25
(embryonic day 120) stages of fetus Cashmere goat dorsal skin. Unsupervised clustering 26
analysis identified 16 cell clusters and their corresponding cell types were also 27
unprecedentedly characterized. Based on the lineage inference, we revealed detailed 28
molecular landscape along the dermal and epidermal cell lineage developmental pathways. 29
Notably, by cross-species comparasion of single cell data with murine model, we revelaed 30
conserved programs during dermal condensate fate commitment and the heterochrony 31
development of hair follicle development between mouse and Cashmere goat were also 32
discussed here. Our work here delineate unparalleled molecular profiles of different cell 33
populations during Cashmere goat hair follicle morphogenesis and provide a valuable 34
resource for identifying biomarkers during Cashmere goat hair follicle development. 35
36
Key Words: Single-cell transcriptome; Developmental trajectories; Hair follicle 37
morphogenesis 38
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted January 31, 2020. . https://doi.org/10.1101/2020.01.30.926287doi: bioRxiv preprint
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Introduction 39
Every year, more than 20,000 tons of Cashmere were generated in China and Cashmere 40
goat has become an important source of income for the people lived in north China (Scott 41
Waldron et al., 2014). Cashmere is the secondary hair follicles in Cashmere goats and forms 42
as early as the fetus stage and due to the commercial value of Cashmere (Ansari-Renani et 43
al., 2011, Geng et al., 2013), it’s therefore of great significance to reveal molecular 44
pathways during early hair follicle development in Cashmere goats. Besides, by using 45
mouse as a research model, research has demonstrated that molecular pathways during 46
early hair follicle morphogenesis play important roles in regulating the hair characteristics, 47
including hair fiber length, fineness and curvature (Duverger & Morasso, 2009), therefore, 48
further enhancing the significance of revealing the molecular pathways driving hair follicle 49
morphogenesis in Cashmere. However, due to the long-time duration of pregnancy (about 50
145~159 days) in Cashmere goats (AJ Ritar et al., 1989), researches focused on hair follicle 51
morphogenesis mainly used the mouse as a research model, while our current 52
understanding on hair follicle morphogenesis in Cashmere goat remains largely unknown. 53
Similar to murine hair follicle development, Cashmere goat hair follicle in uterus 54
development can also be divided into three main stages: induction stage (about embryonic 55
day 55 - 65), organogenesis (about embryonic day 85 - 95) and cytodifferentiation stages 56
(around embryonic day 115) (Zhang Y et al., 2006). In mice, the molecular underpinnings 57
underlying the induction stage has recently been comprehensively investigated by virtue of 58
the development of single-cell RNA sequencing technology, while the late two stages 59
remain not well-known (Saxena et al., 2019). According to what is known in mice, the 60
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formation of placodes and dermal condensates (DC) are two marking events in the 61
induction stage and requires a conserved crosstalk between dermal and epidermal cell 62
populations, including Wnt/β-catenin signaling, Edar signaling and Fgf signaling (Chen et 63
al., 2012, Huh et al., 2013, Zhang et al., 2009). More recently, Mok et al., demonstrated that 64
murine DC formation can be further divided into three sub-stages using single-cell RNA 65
sequencing (scRNA seq) technology and characterized detailed transcriptome signature 66
genes during each substage, they also found that ECM/Adhesion signaling was vital for the 67
early DC fate commitment (Mok et al., 2019). The organogenesis stage is characterized by 68
the formation of dermal papilla (DP) cells, hair shaft and inner root sheath (IRS) and the 69
molecular pathways involved includes PDGFα signaling and Shh signaling (Karlsson et al., 70
1999, Ouspenskaia et al., 2016). For the cytodifferentiation stage, the differentiation of IRS, 71
hair shaft and keratinocyte become obvious and Eda signaling is demonstrated to play a 72
role (Duverger & Morasso, 2009, Millar, 2002). Noteworthy, the asynchronous 73
development of different hair follicles, including guard hair follicles (starts from E13.5), 74
awl, auchene hair follicles (starts from E15.5), and zigzag hair follicle (starts from E17.5) 75
in mice, is also a marking event during the cytodifferentiation stage (Schlake, 2007). 76
However, the molecular machinery underlying the asynchronous development of different 77
hair follicles remains not well-known (Chi et al., 2013, Driskell et al., 2009). 78
To preliminarily reveal molecular pathways involved during Cashmere goat hair 79
follicle morphogenesis, several groups have collected skin samples from fetus goat and 80
performed transcriptome sequencing analysis to reveal gene expression dynamics between 81
different time points (Gao et al., 2016, Ren et al., 2016). However, due to the lack of 82
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted January 31, 2020. . https://doi.org/10.1101/2020.01.30.926287doi: bioRxiv preprint
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conserved markers to label particular cell types within the hair follicles, most studies used 83
skin tissues to perform transcriptome sequencing analysis and generated the “equalized” 84
expression matrices, which is sometimes, hard to reveal the real scenario. The paucity of 85
information regarding the cell heterogeneity within the hair follicles has obviously become 86
the main obstacle in dissecting the hair follicle morphogenesis. scRNA seq has recently 87
became robust tool in dissecting cell heterogeneity and several groups have also 88
successfully used scRNA seq technology to reveal the molecular machinery underlying 89
murine hair follicle development (Ge et al., 2019, Gupta et al., 2019, Mok et al., 2019), 90
further emphasized its application prospect in hair follicle development-related researches. 91
Tackling the paucity of information regarding the cellular heterogeneity and molecular 92
pathway underlying key cell fate decisions during Cashmere hair follicle development. 93
Here, we reported a single-cell transcriptome landscape during Cashmere goat hair follicle 94
morphogenesis based on 19,705 single-cell transcriptional profiles. We successfully 95
identified different cell types during Cashmere goat hair follicle development and 96
delineated their cell type-specific gene expression profiles, which provides valuable 97
information for the identification of biomarkers and dissecting cellular heterogeneity during 98
Cashmere goat hair follicle development. Besides, cell lineage inference analysis provides a 99
comprehensively understanding of the molecular pathways underlying major cell lineage 100
fate decisions. Our study here provides a valuable resource for understanding Cashmere 101
goat hair follicle development, and will also have implications for future Cashmere goat 102
breeding. 103
104
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Results 105
Single-cell sequencing and characterization of cellular heterogeneity during Cashmere 106
goat hair follicle morphogenesis 107
To provide in-depth insight into molecular profiles during Cashmere goat hair follicle 108
development and main cell fates transitions, we collected skin samples from E60, E90 and 109
E120 stage fetus Cashmere goat skin (Supplemental. Fig. S1a), which correspond to hair 110
follicle induction, organogenesis and cytodifferentiation stage and performed single-cell 111
RNA sequencing (Fig. 1a). We totally captured 7,000 single cells for each sample, and for 112
each sample we detected at least 16,000 genes and the genome mapping rate was higher 113
than 90% for all the samples (Supplemental. Fig. S1b). For quality control, we filtered cells 114
according to the number of genes detected (Supplemental. Fig. S1c) and retained 115
high-quality cells for downstream analysis. After quality control, we totally analyzed 116
19,705 single-cell transcriptome expression profiles from E60 (6,825 single cells), E90 117
(6,873 single cells) and E120 (6,007 single cells) stage fetus Cashmere goat back skin. 118
To dissect cellular heterogeneity, we next performed t-distributed stochastic neighbor 119
embedding (t-SNE) analysis and we totally identified 16 different cell clusters across three 120
developmental times (Fig. 1b,c and Supplemental. Table 1). By analyzing cluster-specific 121
expressed gene expression, we successfully identified the different cell types according to 122
their marker gene expression (Supplemental. Fig. S1d). Briefly, we found that cluster 1, 4, 6, 123
7 and 13 expressed high level of dermal cell lineage markers LUM and COL1A1 (Gupta et 124
al., 2019), while cluster 0, 2, 3, 5, 8, 9 and 12 expressed high level of epithelial lineage 125
markers KRT14 and KRT17 (Gu & Coulombe, 2007, Joost et al., 2016). Besides, we also 126
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identified hair shaft clusters (LHX2 and MSX1, cluster 2, 5) (Yang et al., 2017), endothelial 127
cluster (KDR and PECAM1, cluster 11) (Detmar et al., 1998), DP cluster (SOX2 and SOX18, 128
cluster 13) (Driskell et al., 2009), pericyte cluster (ACTA2 and TPM2, cluster 15) 129
(Paquet-Fifield et al., 2009), muscle cell cluster (CNMD and ARS1, cluster 17) and 130
macrophage cell cluster (ALF1 and RGS1, cluster 16) (Lee et al., 2016). More importantly, 131
we further delineated transcriptional characteristics for each cell types and identified a 132
series of cell type-specific marker genes during Cashmere goat hair follicle development 133
(Fig. 1d), and it was also worth noting that many cell type-specific expressed marker genes 134
were also consistent with a murine scenario, such as dermal cell markers POSTN, DCN, 135
APOE, epithelial cell markers KRT14, KRT15, and DP cell markers SOX2, SOX18. 136
137
Defining dermal cell lineage and epidermal cell lineage developmental trajectory 138
along pseudoptime 139
After the characterization of different cell clusters, we then want to investigate major cell 140
fate transitions during hair follicle development. We, therefore, performed pseudotime 141
trajectory construction analysis on dermal and epidermal cell clusters (Fig. 2). Since we 142
have successfully characterized all cell clusters, we then selected dermal cell lineage cell 143
clusters (Fig. 2a, cluster 1, 4, 6, 7 and 12) and epidermal cell lineage cell clusters (Fig. 2b, 144
cluster 0, 2, 3, 5, 8, 9 and 11) to infer cell lineage developmental trajectory. For the dermal 145
cell lineage, pseudotime trajectory displayed 2 branch points (Fig. 2c), while the epidermal 146
cell lineage showed 3 branch points (Fig. 2d). Noteworthy, when the cells were color-coded 147
with their corresponding developmental time, they also showed a time-ordered pattern 148
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along the pseudotime. As for the branch point in the dermal and epidermal cell populations, 149
based on the prior knowledge on cell lineage dynamics, during early hair follicle 150
development, the dermal cell fate involves DC fate commitment and DP fate commitment 151
(Fig. 2e) (Saxena et al., 2019), while epidermal cell fate involves matrix cell fate 152
commitment, hair shaft/IRS fate commitment and keratinocyte fate commitment (Fig. 2f) 153
(Forni et al., 2012, Millar, 2002, Schmidt-Ullrich & Paus, 2005), it’s therefore different 154
branch points may represent the process of cell fate decisions. 155
156
Delineating developmental pathway during DC fate commitment 157
After dermal cell lineage trajectory inference, we firstly focused on the first branch point on 158
the dermal cell pseudotime trajectory to reveal the first dermal cell fate decision. By 159
analyzing gene expression dynamics along pseudotime, we observed 2,679 differentially 160
expressed genes at the end of cell fate commitment (Supplemental. Table 2) and gene 161
functional enrichment analysis revealed that these genes enriched GO terms of “tissue 162
morphogenesis, response to growth factor and cell morphogenesis involved in 163
differentiation” (Fig. 3a). A comparison of overlapped GO terms between each gene set 164
showed that they shared substantial GO terms (Supplemental. Fig. 2). Of particular notice, 165
we observed a series of canonical murine dermal condensate cell markers such as APOD, 166
LUM and APOE (Mok et al., 2019) in those differentially expressed genes, and the 167
pseudotime expression pattern of DC markers APOD, SOX18, CTNNB1 and SOX2 was 168
increased along pseudotime (Fig. 3b), therefore, we termed the first branch point as DC fate 169
commitment. 170
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To our knowledge, no reports yet have delineated cellular heterogeneity and 171
transcriptional landscape during Cashmere goat hair follicle morphogenesis, and most 172
researches regarding hair follicle development have been performed on the murine model. 173
To gain in-depth insight into machinery driving DC fate commitment, we then compared 174
DC fate signature genes with recently reported murine DC fate commitment signature 175
genes (Fig. 3c) and observed 729 (about 14.5% of all) overlapped genes between Cashmere 176
goat and murine DC signature genes (Supplemental. Table 3). Noteworthy, based on 177
single-cell RNA sequencing on E15.0 dorsal skin, Mok et al. recently demonstrated that 178
murine DC fate commitment can be divided into pre-DC, DC1 and DC2 stage at a more 179
detailed level (Mok et al., 2019). By analyzing murine DC marker genes at different stages, 180
we similarly found that DC signature genes of Cashmere goat also showed chronological 181
expression patterns along pseudotime (Fig. 3d). Briefly, murine pre-DC markers DKK1 and 182
LEF1 were elevated prior to DC1 and DC2 marker expressions, such as PRDM1, TRPS1, 183
INHBA and RSPO3, it’s therefore plausible that DC fate commitment in Cashmere goat 184
may also involve different stages. 185
186
Delineating DP cell heterogeneity along pseudotime 187
After defining DC fate commitment, we then focused on the next branch point. We firstly 188
compared differentially expressed gene expression between the two branches and observed 189
that cell fate 1 elevated canonical DP marker genes, such as APOD, SOX18, and enriched 190
GO terms of “tissue morphogenesis and epidermis development”, while cell fate 2 elevated 191
genes such as CENPW, TOP2A and enriched GO terms of “mitotic cell cycle process and 192
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DNA-dependent DNA replication” (Fig. 4a,b and Supplemental. Fig. 3a,b). To gain an 193
in-depth understanding of the differences between the two branches, we further identified 194
differentially expressed genes between the two branches using another differential analysis 195
method (Wilcox, Wilcoxon Rank Sum test). Consistent with Monocle analysis, cell fate 1 196
also showed higher expression of APOD, SOX18 and IGF1, while cell fate 2 showed 197
elevated expression of CENPW, TOP2A and DCN (Fig. 4c). GO enrichment analysis 198
similarly revealed that differentially expressed genes in cell fate 1 enriched GO terms of 199
“tissue morphogenesis and epithelial cell differentiation”, while cell fate 2 enriched GO 200
mainly related to the regulation of cell cycle (Supplemental. Fig. 3c). 201
To dissect the heterogeneity within DP cells, we further performed 202
immunofluorescence analysis on E120 Cashmere goat back skin tissues and observed 203
different staining patterns between primary hair follicles (PF) and secondary hair follicles 204
(SF) (Fig. 4d). For the BMP2 staining, we found that BMP2 specifically expressed in the 205
matrix cells surrounding DP cells, while in the secondary hair follicle, BMP2 positive cells 206
could be found in both DP cells and surrounding matrix cells. Besides, we found that 207
PCNA, a cell cycle-related marker, was expressed mainly in surrounding matrix cells in the 208
primary hair follicle, while in the secondary hair follicles, we observed high expression of 209
PCNA both in DP cells and surrounding matrix cells. Taken together, our analysis here 210
demonstrated that different hair follicles showed different gene expression patterns in 211
Cashmere goat, which may also plausible for the asynchronous development of different 212
hair follicles. 213
214
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Delineating developmental pathway during the first epidermal cell fate decision 215
After revealing dermal cell fate decisions, we then focused on the epidermal cell clusters. 216
Monocle pseudotime trajectory inference analysis revealed that epidermal cells showed 217
three different branch points. We then firstly focused on the first branch point and analyzed 218
differentially expressed gene dynamics along pseudotime. Based on k-means clustering, we 219
observed four distinct gene clusters. As expected, we observed a series matrix cell markers 220
at the end of pseodotime, including HOXC13, KRT25 and KRT71, thus deciphering a 221
matrix cell fate commitment (Fig. 5a). Gene functional enrichment analysis showed that 222
matrix cell expressed signature genes enriched GO terms of “keratinocyte differentiation, 223
epidermis development, and skin epidermis development” (Fig. 5b), while comparison of 224
GO terms between different gene set reveals that gene set 1,2 differs to that of gene set 3,4 225
(Supplemental. Fig. 4b). 226
To gain in-depth insight into molecular profiles during matrix cell fate commitment, we 227
further analyzed the signature gene expression pattern along pseudotime. For the gene set 4, 228
namely matrix cell fate, they enriched a series of keratin family genes, such as KRT25, 229
KRT27, KRT84, and their expression was increased along pseudotime (Fig. 5c). For the 230
gene set 3 and 2, we found that they transiently elevated expression of LEF1, SBSN, SOX18, 231
SOX9, and enriched GO terms of “cardiac epithelial to mesenchymal transition, 232
mesenchymal cell differentiation and skin development, morphogenesis of an epithelium, 233
respectively. For the gene set 1, they enriched genes such as VIM, LUM and COL1A1 and 234
both showed decreased expression along pseudotime. Immunohistochemistry staining 235
analysis also confirmed that LEF1 and CTNNB1 were expressed in the upper epidermis, 236
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which was also consistent with a murine scenario (Polakis, 2001, Tsai et al., 2014). 237
238
Delineating cell fate decisions during hair shaft and IRS cell fate commitment 239
After deciphering matrix cell fate commitment in the first epidermal trajectory point, we 240
next focused on the next branch point. By analyzing differentially expressed genes along 241
pseudotime, we found that cell fate 1 enriched canonical hair shaft markers, including SHH, 242
VDR, and HOXC13, while cell fate 2 enriched canonical IRS markers, such as SOX9, 243
KRT14, SBSN and LEF1 (Fig. 6a) (Yang et al., 2017). Our immunofluorescence results also 244
confirmed their expression in the Cashmere hair follicles (Supplemental. Fig. 5a). For the 245
pre-branch, we observed genes such as LUM, COL1A, OGN, SOX18 and they all showed 246
decreased expression along pseudotime (Fig. 6b). For the hair shaft fate (cell fate 1), we 247
also observed elevated expression of DCN, TOP2A, CENPW and H2AFZ and enriched GO 248
terms of “mitotic cell cycle process and DNA replication” (Supplemental. Fig. 5 a,b). For 249
the IRS cell fate (cell fate 2), we observed elevated expression of PRDM1, KRT1, SOX9, 250
KRT14 and enriched GO terms of “supramolecular fiber organization and tissues 251
morphogenesis”. 252
Besides, we also compared our identified hair shaft and IRS signature genes with our 253
recently identified murine hair shaft and IRS signature genes (Ge et al., 2019). Interestingly, 254
we only observed about ~8.8% overlapped IRS signature genes between mouse and goat 255
(Fig. 6c and Supplemental. Table 4), and these overlapped genes mainly consisted of 256
Keratin family genes, such as KRT14, KRT17, KRT79. As for overlapped hair shaft 257
signature genes, we only observed ~4.6% genes (Fig. 6d), including MSX1, HOXC13, 258
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APOD, LHX2, SHH, and VDR. To further validate our analysis, we compared the 259
expression of PCNA and VDR between E90 Cashmere goat skin and E16.5 mouse skin 260
tissues, and the immunohistochemistry showed that they showed similar expression 261
patterns during hair follicle development (Fig. 6e). These data together demonstrate that 262
hair shaft and IRS specification may require a conserved program during cell fate decisions. 263
264
Revealing the developmental pathway during keratinocyte cell fate commitment 265
After delineating the first two epidermal cell lineage cell fate decisions, we then focused on 266
the last branch point. Pseudotime trajectory analysis also revealed two different branches, 267
and we then analyzed pseudotime gene expression dynamics between the two branches (Fig. 268
7a). Analyzing differentially expressed genes along psusotime, we found that cell fate 1 269
enriched genes such as VDR, BMP4, STAR, KRT85 and KRT14, while cell fate 2 enriched 270
genes such as BMP2, SHH, CUX1 and ETV5. Noteworthy, KRT14 has been identified as a 271
marker for keratinocyte both in humans and mice (Green et al., 2003, Joost et al., 2016), we, 272
therefore, termed this fate as keratinocytes. To gain in-depth insight into their 273
corresponding cell type, we performed gene functional enrichment analysis for each gene 274
set and analyzed pseudotime GO enrichment dynamics (Fig. 7b and Supplemental. Fig. 6a). 275
The result showed that the pre-branch mainly enriched genes related to “regulation of 276
response process”, while for the cell fate 1, the signature genes mainly enriched GO terms 277
of “development differentiation of epidermal”. Interestingly, for cell fate 2, these 278
differentially expressed genes mainly enriched in GO terms of “regulation of cell cycle” 279
and showed lower expression of KRT14 (Fig. 7c), it’s therefore plausible that keratinocyte 280
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differentiation at this stage is not synchronized. To confirm such hypothesis, we performed 281
immunofluorescence analysis of VDR, PCNA, BMP2, CTNNB1 and LEF1, and the results 282
showed that the expression of VDR and BMP2 in the outer layer epidermis was not 283
homogeneous, with some cell clusters showed higher expression while some cell 284
populations showed lower expression (Fig. 7d). For the pre-branch enriched CTNNB1, it 285
was uniformly expressed in the interfollicular epidermis. Besides, the expression pattern of 286
PCNA and LEF1 was even more significant and they were partially expressed in the 287
epidermis. Taken together, these results together emphasize that keratinocyte differentiation 288
in Cashmere goat is asynchronous and requires different gene expression profiles. 289
290
Discussion 291
The development of scRNA seq in recent years has been demonstrated as a robust tool for 292
the developmental biologist to investigate organogenesis and has provided us with 293
unparalleled insight into mammalian development. In the past decades, the number of 294
papers using scRNA seq based technology has increased exponentially and scRNA has also 295
been awarded Science's 2018 Breakthrough of the Year (Angerer et al., 2017, Pennisi, 296
2018). Here, we successfully constructed single-cell atlas during Cashmere goat hair 297
follicle development. Based on the downstream analysis, we provided unparalleled insight 298
into the cellular heterogeneity and major cell fate decisions during the Cashmere hair 299
follicle in uterus morphogenesis. As far as we have known, this is the first study to 300
comprehensively delineating molecular profiles of various cell types and revealing major 301
cell fate decisions during Cashmere hair follicle development. More importantly, by 302
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analyzing cluster-specific gene expression profiles, our data here provides a valuable 303
resource for identification of markers for future studies in Cashmere skin tissues and also 304
provides an in-depth understanding of the Cashmere hair follicle development. 305
In the current study, we totally analyzed 19,705 single-cell transcriptional profiles from 306
three different points, which can represent major cell types during hair follicle development. 307
To dissect cellular heterogeneity during Cashmere goat hair follicle development after tSNE 308
analysis, we analyzed cluster-specific expressed signature genes for each cluster to infer 309
their corresponding cell types. It’s worth noting that all the cell markers used in the current 310
study were referenced from the murine model due to the paucity of information regarding 311
marker gene expression during Cashmere goat hair follicle development. However, our 312
study here showed that substantial murine cell type-specific biomarkers are identical to the 313
Cashmere goat. Besides, by comparison of cell type-specific signature genes between 314
Cashmere goat and mouse (Fig. 3c and Fig. 6c,d), we found that the transcriptome 315
similarity decreased along pseudotime (revealed by overlapped signature genes in this 316
study). Briefly, about 729 DC signature genes overlapped between Cashmere goat and 317
mouse, and murine DC signature genes at different stages (pre-DC, DC1 and DC2) showed 318
similar expression pattern along pseudotime in Cashmere goat (Mok et al., 2019). While for 319
the late-stage IRS and hair shaft cell population in the late stage of hair follicle 320
development, the percentage of overlapped genes decreased (126 and 93 overlapped genes, 321
respectively). Similarly, by comparing gene expression across 7 different species from brain, 322
heart, ovary, kidney, testis and liver, researches have demonstrated that organ-specific 323
molecular profiles are more similar in early development and become more distinct during 324
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development (Cardoso-Moreira et al., 2019), it’s therefore plausible that hair follicle 325
development in Cashmere goat and mouse may also consistent with such theory. 326
Similar to the murine model, we also observed differential gene expression profiles in 327
different DP cell populations from different hair follicles (primary hair follicles vs 328
secondary hair follicles), which was also consistent with our recent findings on mice (Ge et 329
al., 2019). Besides, an in-depth comparison of differentially expressed genes revealed that 330
different DP cell populations require different transcriptional profiles, and it’s, therefore, 331
plausible that the different induction signals may be involved during the asynchronous 332
development of hair follicles. Consistent with such hypothesis, researches found that SOX2 333
was specifically expressed in guard hair follicles, but not zigzag hair follicles, while SOX18 334
was demonstrated to regulate zigzag hair follicle morphogenesis (Driskell et al., 2009, 335
James et al., 2003, Pennisi et al., 2000). Although our study here also demonstrates the 336
asynchronous development of different hair follicles in Cashmere goat, the detailed 337
machinery underlying such phenomenon was not investigated here and future studies may 338
focus on such topics, which will definitely provide us new insight into the hair follicle 339
biology and hair follicle regeneration. 340
Based on single-cell pseudotime trajectory inference, our study here also highlighted 341
the underappreciated cell fate decisions during hair follicle development. Different from 342
our previous studies performed in mice, our pseudotime lineage trajectory of epidermal cell 343
lineage showed three different branch points while murine epidermal cell lineage trajectory 344
showed two branch points. by analyzing the gene expression profiles of the first two branch 345
points, they actually showed similar cell fate decisions, while the additional branch point in 346
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Cashmere goat mainly comes from keratinocyte differentiation. Such differences in 347
pseudotime trajectory may be partially explained by the differences in the development of 348
hair follicles across species, for example, E120 stage Cashmere goat showed obvious hair 349
fiber on the surface of the skin, while it was not until 6-7 postnatal day that the hair follicles 350
in the mouse skin surface become visible. The difference in the pseudotime trajectory 351
reveals that hair follicle development was heterochrony between Cashmere goat and mouse, 352
which was frequent when comparing the development of specific organs across species. 353
Finally, our dataset here provides an important resource for understanding the cellular 354
heterogeneity and major cell fate decisions during Cashmere goat hair follicle development. 355
For the first time, the detailed transcriptional landscape of different cell populations was 356
delineated here at single-cell resolution, which provided valuable resources for the 357
identification of biomarkers. Besides, our trajectory inference analysis here successfully 358
recapitulated major cell fate decisions during Cashmere goat hair follicle development, 359
which enabled us to comprehensively study the detailed developmental pathways involved 360
during Cashmere goat hair follicle morphogenesis, and will also have implications for the 361
Cashmere goat breeding work in animal husbandry. 362
363
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Materials and methods 364
Experimental Animals 365
All the experimental Shaanbei White Cashmere goats involved in this study were obtained 366
from the Shaanbei Cashmere Goat Engineering Technology Research Center of Shaanxi 367
Province and were fed with Cashmere goat feeding standard (DB61/T583-2013) of Shaanxi 368
Province. All pregnant goats were prepared using artificial insemination and all the 369
experimental procedures involved goats in this study were approved by the Experimental 370
Animal Manage Committee of Northwest A&F University. 371
Single-cell suspension preparation 372
The goat fetus at desired dates was isolated using cesarean operation when the pregnant 373
goats were anesthetic with the compound ketamine. The skin tissues (0.5 cm × 0.5 cm) 374
were isolated from the fetus back skin and were immediately transferred to the ice-cold 375
DMEM/F12 media (Gibco, Beijing, China) with 50 U/ml penicillin and 50 mg/ml 376
streptomycin (HyClone, Beijing, China). After washing three times with DMEM/F12 to 377
remove contaminative blood cells, the skin tissues were then dissociated into single cells 378
prior to sequencing. For E60 and E90 skin tissues, the obtained skin tissues were firstly 379
incubated with 2 mg/ml collagenase IV (Sigma, St Louis, MO, USA) for 30 min at 37°C, 380
and then the skin tissues were mechanically dissociated into single-cell suspensions with a 381
1ml pipette tip. For E120 skin tissues, the skin tissues were firstly cut into ~3 mm skin 382
pieces and then incubated with 2 mg/ml collagenase IV for 30 min. After incubation, the 383
hair follicles within the skin tissues were isolated with a pair of precise forceps and the 384
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pooled hair follicles were further dissociated into single cells with TypLE Express (Gibco, 385
Grand Island, NY, USA) for 30 min at 37°C. The obtained single-cell suspensions were 386
then washed three times with PBS supplemented with 0.04% BSA (Sigma, St Louis, MO, 387
USA) and were filtered with a 40 μm cell strainer (BD Falcon, BD Biosciences, San Jose, 388
CA, USA) to remove debris and cell aggregations. For each stage, the samples were 389
obtained from at least 2 different goat fetus and for each fetus goat, the single-cell 390
suspension was prepared separately until finally pooled together prior to single-cell 391
barcoding. 392
Single-cell library construction and sequencing 393
Single-cell library was constructed using 10x Genomics’ Chromium Single Cell 3’ V3 Gel 394
Beads Kit (10x Genomics, Pleasanton, CA, USA) according to the manufacture’s 395
instructions. After cell counting, the single-cell suspension was adjusted to 1000 cells/μl 396
and about 7,000 cells were obtained for each stage. The single-cell barcoding procedure 397
was performed using a 10x Genomics Chromium barcoding system (10x Genomics, 398
Pleasanton, CA, USA) according to the manufacturer's guide. After single-cell library 399
construction, the Illumina HiSeq X Ten sequencer (Illumina, San Diego, CA, USA) was 400
used to sequence and 150 bp pair ended reads were generated. 401
10x Genomics single-cell RNA sequencing data processing 402
The obtained raw sequencing files were processed with standard CellRanger (v2.2.0) 403
pipeline according to the 10x Genomics official guide (https://www.10xgenomics.com/cn/). 404
The produced raw base call files were firstly transformed into fastq files by using 405
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Cellranger mkfastq function. The goat ARS1 reference genome downloaded from ensemble 406
was used as a reference genome (https://asia.ensembl.org/Capra_hircus/Info/Index). 407
Cellranger count function was used to perform mapping, filtering low-quality cells, 408
barcoding counting and UMI counting. 409
After the standard Cellranger pipeline, the generated gene expression matrice files were 410
then analyzed with Seurat (V2.3.4) package according to the official user guide 411
(https://satijalab.org/seurat/vignettes.html). Quality control was performed using FilterCells 412
function and cells with detected genes less than 200 and genes expressed less than 3 cells 413
were filtered. After normalization and data scaling, the different datasets were integrated by 414
using RunMultiCCA function. tSNE was used to perform dimension reduction analysis and 415
different cell clusters were identified by using FindClusters function. The cluster 416
specifically expressed genes were analyzed with FindAllMarkers function and with the 417
parameter “min.pct = 0.25, thresh.use = 0.25”. 418
Single-cell pseudotime lineage trajectory reconstruction 419
Single-cell lineage reconstruction analysis was performed using Monocle (V2) packages 420
according to the online tutorial (http://cole-trapnell-lab.github.io/monocle-release/docs/). 421
The monocle object was constructed from Seurat object with newCellDataSet function, and 422
Seurat determined variable genes were used as ordering genes to order cells in pseudotime 423
along a trajectory. Dimension reduction was performed using DDRTree methods. To 424
analyze differential gene expression between different cell branches, BEAM function was 425
used and differentially expressed genes were identified with q-val < 1e-4. Branch-specific 426
gene expression heatmap was plotted with plot_genes_branched_heatmap function and 427
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different gene set was calculated according to k-means clustering. 428
Immunohistochemistry staining analysis 429
The immunofluorescence or enzyme horseradish peroxidase (HRP)-based 430
immunohistochemistry analysis procedure was performed as we previously described (Ge 431
et al., 2017, Liu et al., 2017). For immunofluorescence analysis, the paraffin-embedded skin 432
tissues were firstly deparaffinized in xylene and further rehydrated in ethanol solutions. 433
Antigen retrieval was performed in 0.01 M sodium citrate buffer at 96°C. Following a 434
permeabilization procedure in 0.5 M Tris-HCI buffer supplemented with 0.5% TritonX-100 435
(Sorlabio, Beijing, China) for 10 min, the slides were then blocked with 3 % BSA and 10 % 436
donkey serum (Boster, Wuhan, China) in 0.5 M Tris-HCI buffer for 30 min. The primary 437
antibodies diluted in the blocking buffer were incubated with the slides at 4°C overnight. 438
The next morning, the corresponding secondary antibodies were then added in the slides 439
and incubated at 37°C for 1 h. DAPI was used to stain the nuclei and the pictures were 440
taken using Nikon AR1 confocal system (Nikon, Tokyo, Japan). For HRP based 441
immunohistochemistry analysis, following antigen retrieval, the samples were firstly 442
incubated with 3% H2O2 for 10 min at room temperature to remove endogenous peroxidase 443
activity. The primary antibodies were incubated 4 °C overnight, and the corresponding 444
HRP-labeled secondary antibodies were added in the next morning for 40 min at room 445
temperature. After that, peroxidase substrate DAB (Zsbio, Beijing, China) was used for 446
chromogenic reaction with hematoxylin used to stain nuclei. The slides were finally 447
mounted with neutral resins and pictures were captured with an Olympus BX51 microscope 448
imaging system (Olympus, Tokyo, Japan). All the primary and secondary antibodies used in 449
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this study were listed in Supplementary Table 5. 450
451
Data availability 452
The single-cell RNA sequencing data used in this research is deposited in NCBI GEO 453
databases under accession number: GSE144351. 454
455
Acknowledgments 456
This work was supported by the National Natural Science Foundation of China (31972556 457
and 31671554). 458
459
460
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554 555 556
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Figure Legends 557
Fig. 1 Dissecting cellular heterogeneity during Cashmere goat hair follicle 558
development. (a) Overall experimental design. (b) tSNE plot of all single cells labeled with 559
developmental time. Cells from the different developmental points were color-coded with 560
different colors. (c) tSNE plot of all single cells labeled with cell types according to their 561
marker gene expression. Different colors represent different cell clusters and the cell 562
number for each cluster was listed in the bracket. (d) Dot plot of representative marker 563
genes for different cell clusters. The color intensity represents its expression level, and the 564
dot size represents the positive cell percentage. 565
566
Fig. 2 Delineating dermal and epidermal cell lineage pseudotime developmental 567
trajectory. (a) Dermal cell lineage highlighted in the tSNE plot. (b) Developmental 568
trajectory of dermal cell lineage along pseudotime. Cells were color-coded with cell types 569
identified by Seurat (left panel) and developmental time (right panel), respectively. (c) 570
Epidermal cell lineage highlighted in the tSNE plot. (d) Developmental trajectory of 571
epidermal cell lineage along pseudotime. Cells were also color-coded with cell types (left 572
panel) and developmental time (right panel), respectively. (e) Main dermal cell lineage 573
decisions during hair follicle morphogenesis. (f) Main epidermal cell lineage fate decisions 574
during hair follicle morphogenesis. 575
576
Fig. 3 Revealing molecular profiles during DC fate commitment. (a) Pseudotime 577
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expression heatmap during DC fate commitment. The four gene sets were determined by 578
k-means clustering according to their expression pattern and GO terms for each gene set 579
were listed in the right panel. (b) Immunofluorescence analysis of BMP2, CTNNB1, LEF1 580
and K15 in E60 Cashmere goat skin tissues. Scale bars = 50 μm. (c) Veen diagram 581
illustrating overlapped signature genes between E60 Cashmere goat DC cells and murine 582
E13.5 DC signature genes. (d) Visualizing murine DC signature genes of different stages 583
along pseudotime in Cashmere goat. The murine signature DC markers were listed in the 584
top panel and their corresponding expression pattern in Cashmere goat was listed in the 585
lower panel. Cells were color-coded according to their cluster identify. 586
587
Fig 4. DP cells from primary hair follicles and secondary hair follicles in Cashmere 588
goat showed distinct gene expression profiles. (a) Heatmap illustrating dynamic gene 589
expression profiles during DP cell fate commitment. The gene expression pattern for each 590
gene set was listed in the middle panel, and the top 5 enriched GO terms for each gene set 591
were listed in the right panel. (b) Pseudotime expression of cell fate 1 enriched genes 592
APOD, SOX18 and cell fate 2 enriched genes CENPW, TOP2A. Cells were colored-coded 593
with their corresponding cluster identity. (c) Volcano plot illustrating cell fate 1 and cell fate 594
significantly enriched differentially expressed genes. (d) Immunofluorescence analysis of 595
BMP2, K15, VDR and PCNA in the primary hair follicles and secondary hair follicles from 596
E120 Cashmere skin sections. Scale bars = 50 μm. 597
598
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Fig. 5 Delineating matrix cell fate decision along pseudotime. (a) Heatmap 599
demonstrating dynamic gene expression patterns during matrix cell fate commitment. The 600
differentially expressed genes were divided into 4 different gene sets based on k-means 601
clustering. (b) Gene functional enrichment analysis of signature genes in the 4 different 602
gene sets identified in Fig. 5a. (c) The pseudotime expression pattern of representative 603
signature genes from each gene set. Cells were color-coded according to their cluster 604
identity. (d) Immunofluorescence analysis of LEF1 and CTNNB1 in E60 Cashmere skin 605
tissues. Scale bars = 50 μm. 606
607
Fig. 6 Revealing pseudotime gene expression dynamics during IRS and hair shaft cell 608
fate commitment. (a) Heatmap illustrating pseudotime gene expression pattern of 609
differentially expressed genes during IRS and hair shaft cell development. Cell fate 1 610
depicts IRS fate, while cell fate 2 depicts hair shaft fates. (b) Pseudotime expression pattern 611
of representative marker genes during IRS and hair shaft cell fate commitment. Cells were 612
color-coded with their corresponding cluster identity and the solid line depicts cell fate 1, 613
while the dashed line depicts cell fate 2. (c) Veen diagram demonstrating overlapped 614
murine and Cashmere goat IRS signature genes. The representative overlapped genes were 615
listed in the corresponding boxes. (d) Comparison of murine hair shaft signature genes with 616
Cashmere goat hair shaft signature genes. The representative overlapped genes were listed 617
in the rectangular boxes. (e) Comparison of PCNA and VDR expression in E90 Cashmere 618
goat and E16.5 mouse skin tissues. Scale bars = 50 μm. 619
620
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Fig. 7 Dissecting the asynchronous development of keratinocyte. (a) Heatmap 621
illustrating gene expression dynamics during keratinocyte differentiation. (b) GO terms 622
corresponding to the gene set in Figure 7a. The arrow indicates the elongation of 623
pseudotime. (c) Expression of cell fate 1 signature genes KRT14, KRT17 and cell fate 2 624
signature genes TOP2A, PCNA along the pseudotime trajectory. (d) Immunofluorescent 625
staining analysis of VDR, PCNA, CTNNB1, BMP2, LEF1 and K15 in E90 Cashmere goat 626
skin tissues. Scale bars = 50 μm. 627
628
Supplementary Figure Legends 629
Supplementary Fig. 1 Single-cell dataset quality control and representative marker 630
expression across all single cells. (a) Morphology of fetus Cashmere goat at E60, E90 and 631
E120 and its corresponding skin structure revealed by HE staining. (b) Quality matrices 632
revealed by CellRanger of all datasets used in this study. (c) Comparison of the number of 633
genes detected and the number of UMI detected in each cell. (d) Evaluating key cell type 634
markers across all single cells in the tSNE plot. 635
636
Supplementary Fig. 2 Gene function enrichment of differentially expressed genes 637
during DC fate commitment. (a) The network of enriched GO terms for different gene 638
sets during DC fate commitment. Each dot represents one GO terms and different colors 639
represent different gene sets. (b) Circos plot demonstrating the number of overlapped GO 640
terms between different gene sets. 641
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Supplementary Fig. 3 Gene function enrichment of differentially expressed genes 642
during DP cell fate commitment. (a) The network of enriched GO terms for different gene 643
sets during DP fate commitment. Each dot represents one GO terms and different colors 644
represent different gene sets. (b) Circos plot demonstrating the number of overlapped GO 645
terms between different gene sets. (c) Comparison of enriched GO terms between two 646
different cell fate. 647
648
Supplementary Fig. 4 Comparison of enriched GO terms during matrix cell fate 649
commitment. (a) Heatmap illustrating gene set specific enriched GO terms. (b) Circos plot 650
demonstrating the number of overlapped GO terms between different gene sets. 651
652
Supplementary Fig. 5 Validation of hair shaft/IRS marker expression and comparison 653
of enriched GO terms. (a) Immunohistochemistry analysis of hair shaft marker HOXC13 654
and IRS marker SOX9 expression in Cashmere goat skin. Scale bars = 50 μm. (b) The 655
network of enriched GO terms for different gene sets during hair shaft and IRS fate 656
commitment. Each dot represents one GO terms and different colors represent different 657
gene sets. (c) Heatmap illustrating gene set specific enriched GO terms. 658
659
Supplementary Fig. 6 Gene functional enrichment and representative gene expression 660
along pseudtotime. (a) The network of enriched GO terms for different gene sets during 661
keratinocyte differentiation. Each dot represents one GO terms and different colors 662
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represent different gene sets. (b) Psedotime expression pattern of representative cell fate 1 663
and cell fate 2 signature genes. 664
665
Supplementary Tables 666
Supplementary table 1: All cluster specific differentially expressed genes. 667
Supplementary table 2: DC signature genes for different gene sets. 668
Supplementary table 3: DC signature genes comparasion between goat and mice. 669
Supplementary table 4: IRS and hair shaft signature genes comparasion between goat and 670
mice. 671
Supplementary table 5: All antibodies used in this study. 672
673
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E60E90E120
Figure 1
(a)
E60
E90
E120
Fetus Cashmere goat skin biospy Single cell barcoding
Beads Oil
Sequencing & Dissecting heterogeneity
(b) (c) Cell type (cells)
(d)
CD
H19
JSR
P1
ARSI
CN
MD
L1C
AM
−1 2
e
0 25 50 750
1
23
4
5
67
89
11
1
111
LUM
COL1
A1
POST
N
DCN
APO
E
KRT1
5
KRT1
7
SOX9
KRT1
4
KLF5
RSP
O2
APO
D
SOX1
8
SOX2
PCO
LCE2
PLVA
P
VWF
PEC
AM1
KDR
DEP
P1
KRT1
0
KRT1
SBSN
KRTD
AP
LRAT
TBX2
ACTA
2
TPM
2
EBF2
ABC
C9
LCP1
FCER
1A
RG
S1
RG
S10
LTC
4S
tSNE 1
tSN
E 2
Clu
ster
ID
dermal epithelial DP endothelial keratinocyte pericyte macrophagy muscle
Developmental time
tSNE 1
tSN
E 2
0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
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1 2
Component 1
Com
pone
nt 2
Figure 2
(a)1
4
1 2
Component 1
Com
pone
nt 2
Cell type (cells)Developmental trajectory Developmental time
( )
( )
( )
Component 1
Com
pone
nt 2
1
2
3
Cell type (cells)Developmental trajectory
( )
( )
( )
Dermal lineage
Epidermal lineage
tSNE 1
tSN
E 2
tSNE 1
tSN
E 2
Component 1
Com
pone
nt 2
1
2
3
Developmental time
(c)
(b) (d)
(e)
Undiff. DC
Fibro.
DP
(f)
Undiff.
Matrix Hair shaft Keratinocyte
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Figure 3
(a)
Pseudotime
FbAPODHES1LUMDCNVIMSOX18SOX9APOESOX2LHX2COL1A1HOXC13JUNPDGFADKK1TPX2NOTCH3
PTNLCKCORINCENPCCCDC66SFPQ
SYNRGBDKRB2AUTS2MRPL16ACADLHUNKWDR36CAB39LANGPT1PRXL2APURBKLHL24UBE2J2NDUFS5
0 10 20 30 40
0.0 2.5 5.0 7.5
0 2 4 6
0 2 4 6
tissue morphogenesisresponse to growth factor
cell morphogenesis involved in differentiationepidermis development
GO (BP) -Log(P)
negative regulation of cell cycle
positive regulation of organelle organization
negative regulation of protein modification
DNA-dependent DNA replication
Wnt signaling pathway
regulation of type I interferon production
viral translational termination-reinitiation
multicellular organismal homeostasis
proteasomal protein catabolic process
nucleobase-containing compound catabolic
intracellular receptor signaling pathway
anatomical structure homeostasis
(b)
1951(38.8%)
2345(46.7%)
729(14.5%)
Cashmere goat DC signature genes
E13.5 murine DCsignature genes
TRPS1
PRDM1
0 25 50 75 100
13
10
13
10
Pseudotime
Expr
essi
on
RSPO3
INHBA
0 25 50 75 100
13
1030
13
1030
Pseudotime
Expr
essi
on 1
13
467
(c)
LEF1
DKK1
0 25 50 75 100
13
10
0.51.0
3.05.0
Pseudotime
Expr
essi
on
Pre-DC DC1 DC2
Lef1, Dkk1, Twist, Fst Prdm1, Trps1, Sox18, Foxd1 Inhba, Rspo3, Foxp1, Hey1
Pseudotime
(d)
Dc
-3
3Exp.
1
2
3
4
SOX18
APOD
0 25 50 75 100
13
1030
1
10
100
Pseudotime
Expr
essi
on
SOX2
CTNNB1
0 25 50 75 100
0.51.0
3.05.0
13
1030
Pseudotime
Expr
essi
on
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Cell Fate 1 vs Cell Fate 2
Figure 4
(a)
-3
3Exp. 0 10 20
0 20 40 60
0 5 10 15 20
0 1 2 3
tissue morphogenesisepidermis development
epithelial cell differentiationneuron projection morphogenesis
GO (BP)
mitotic cell cycle process
DNA-dependent DNA replication
cellular response to DNA damage stimulus
microtubule cytoskeleton organization
regulation of cell morphogenesis
citrulline metabolic process
protein-containing complex disassembly
protein localization to membrane
extracellular matrix organization
skeletal system development
blood vessel development
cellular response to growth factor stimulus
Pre-Branch Cell Fate 1 Cell Fate 2
Pseudotime
-Log(P)Signature gene expression
1,53
91.
077
559
201
BMP2
/K15
/DAP
I
Primary
BMP2
/K15
/DAP
I
Secondary
VDR
/PC
NA/
DAP
I
VDR
/PC
NA/
DAP
I
(b) (c) (d)
SOX18
APOD
0 25 50 75 100
13
1030
1
10
100
Pseudotime
Expr
essi
on
1
13
467
TOP2A
CENPW
0 25 50 75 100
13
1030
13
1030
Pseudotime
Expr
essi
on
SOX18 SOX2
FGF7KLF2
H2AFZ
CENPWLUM
KIF20B TOP2A
VIM
DLK1
0
50
100
150
200
−2 20
avg_logFC
log1
0(p_
val_
adj)
Significant None
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Figure 5
(a)KRT25KRT71KRT85KRT27KRT35KRT84AHCYFGF21HBP1HPRT1CLTBGANTIMP2KCNK7CTSVTXN
POSTNVIMCOL1A1LUMACTC1COL1A2OGNTOP2A
KRT28PADI3SBSNKRT14KRT19LRATKRT23KRT73BMP4LEF1KRT4KRT1KRT17KRT10PDGFASOX9HES1SOX18
keratinocyte differentiationepidermis development
skin epidermis developmentregulation of keratinocyte differentiation
GO (BP) -Log(P)
cardiac epithelial to mesenchymal transition
mesenchymal cell differentiation
epithelial to mesenchymal transition
BMP signaling pathway
skin development
morphogenesis of an epithelium
negative regulation of cell proliferation
keratinocyte proliferation
cell division
skeletal system development
ossification
regulation of endothelial cell proliferation
0.0 2.5 5.0 7.5
0 2 4
0 5 10 15
0 5 10 15
Pseudotime
Undiff. Matrix
KRT27
KRT25
0 25 50 75 100
1e+01
1e+03
1e+05
1e+021e+051e+08
Expr
essi
on
SBSN
LEF1
0 25 50 75 100
1
3
10
1
10
100Exp
ress
ion
POSTN
LUM
0 25 50 75 100
1
10
100
110
1001000
Exp
ress
ion
SOX9
SOX18
0 25 50 75 100
13
1030
13
1030
Exp
ress
ion
gene
set 1
gene
set 2
gene
set 3
gene
set 4
Pseudotime
Cell clusters:0 2 3 5 8 9 12
(b) (c)
-3
3Exp.
4 3 2 1
LEF1/DAPILEF1 DAPI
E60
skin
(d)
CTNNB1/DAPICTNNB1 DAPI
E60
skin
epidermal
dermal
epidermal
dermal
epidermal
dermal
epidermal
dermal
epidermal
dermal
epidermal
dermal
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Figure 6
(a)
Pseudotime
Cell fate 2
-3
3Exp.
ALCAMCOL1A2LUMOGNVIMCOL1A1SOX18VCANFSTAPODDKK1DLK1NR3C1CDH11MICU3ACADSLRCH3
H2AFZDCNTOP2APCNASHHVDREGFRLCKDCTETV5DCKSLF1MT3SNRPB
SBSNKRT4KRT1KRT14SOX9HES1PRDM1ELF1
MSX1KRT35ID3ID1AHCYHOXC13LEF1APOOUSP16RPS9MARS
Cell fate 1
OGN
LUM
0 25 50 75 100
1
10
100
13
1030
Expr
essi
on
TOP2A
DCN
0 25 50 75 100
1
10
100
13
1030
Expr
essi
on
SOX18
COL1A1
0 25 50 75 100
1
10
100
13
1030
Expr
essi
on
H2AFZ
CENPF
0 25 50 75 100
13
1030
13
1030
Expr
essi
on
Cell clusters:0 2 3 5 8 9 12
Pseudotime
Cell fates:fate 1fate 2
Pre-branch(b)
581 (40.3%)
306 (21.3%)
57 (4%)
427 (29.7%)
69 (4.8%)
Cashmere goat IRSsignature genes
Murine IRS-1signature genes
Murine IRS-2signature genes
Cell fate 1
KRT79, SBSN, KRT17,KRT7, KRT79, DUSP1
KRT14, KRT5, KRT1,KRT10, KRT77, KLF5,KLF4, KLF3 ...
(c)
Murine hair shaftsignature genes
491(23.9%)
389(18.9%)
1,085(52.7%)
26(1.3%)
67(3.3%)
Goat hair shaft HS-1
Goat hair shaftHS-2
MSX1,KRT35, HOXC13APOO, BMP4 ...
VDR, CUX1, SHH,LHX2, PCNA, DCN...
(d)
1
2
3
4
(e) E90 goat E16.5 mouse
PCN
A
VDR
E90 goat E16.5 mouse
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1
2
3
KRT14
Figure 7
(a) Cell fate 2
-3
3Exp.
KRT85KRT35KRT14KRT84KRT28VDRBMP4STAR
Cell fate 1 Pre-branch
BMP2SHHCUX1ETV5IGSF8NARSURI1DNLZ
SBSNKLK10KRT17SOX9FSTPARM1LAMA3
CTNNB1POSTNH2AFZTIMP2KIF23TUBB2ABGNDLK1APOEVCANVIMASPHFMR1
regulation of cell cycledevelopment differentiationepidermal cell population proliferation
intermediate filament organization
GO enrichment dynamics along pseudotime
endocytosis virus host
lipid metabolic process
process metabolic cellular
folding 'de novo'
start
cell fate 1 cell fate 2
regulation response process
1
2
3
PCNA
log10( + 0.1)
1
2
3
TOP2A
component 1
com
pone
nt 2
Cel
l fat
e 1
Cel
l fat
e 2
1
2
3
KRT17
log10( + 0.1)
BMP2/K15/DAPI LEF1/PCNA/DAPI
Hi Hi
low low
low low
Hi Hi
VDR/PCNA/DAPI CTNNB1/DAPI
(b)
(c) (d)
1
2
3
4
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