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Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis
Journal Pre-proof Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis Yingfeng Tao, Xiaoliu Zhou, Zhiwei Liu, Xiaokang Zhang, Yangfan Nie, Xinting Zheng, Shaomei Li, Xuewen Hu, Ge Yang, Qianqian Zhao, Chunyan Mou PII:
S1567-133X(19)30063-8
DOI:
https://doi.org/10.1016/j.gep.2019.119078
Reference:
MODGEP 119078
To appear in:
Gene Expression Patterns
Received Date: 18 April 2019 Revised Date:
11 November 2019
Accepted Date: 15 November 2019
Please cite this article as: Tao, Y., Zhou, X., Liu, Z., Zhang, X., Nie, Y., Zheng, X., Li, S., Hu, X., Yang, G., Zhao, Q., Mou, C., Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis, Gene Expression Patterns (2019), doi: https:// doi.org/10.1016/j.gep.2019.119078. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Expression patterns of three JAK–STAT pathway
2
genes in feather follicle development during chicken
3
embryogenesis
4 5 6 7 8 9 10 11 12 13 14 15 16
Yingfeng Tao1, Xiaoliu Zhou1, Zhiwei Liu1, Xiaokang Zhang1, Yangfan Nie1, Xinting Zheng1, Shaomei Li1, Xuewen Hu1, Ge Yang1, Qianqian Zhao1, Chunyan Mou1* 1
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, HuaZhong Agricultural University, Wuhan, China *Correspondence: Chunyan Mou [email protected]
1
17
ABSTRACT
18
The Janus kinase (JAK)–signal transducer and activator of transcription (STAT)
19
(JAK–STAT) pathway is shown to restrain the hair follicles in catagen and telogen
20
and prevent anagen reentry in murine hair follicle cycling. The early roles of
21
JAK-STAT pathway genes in skin development remain uncharacterized in mouse and
22
chicken models. Here, we revealed the expression patterns of three JAK-STAT
23
pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6–E10 stages
24
which are key to feather follicle morphogenesis. Multiple sequence alignment of the
25
three genes from chicken and other species all showed a closely related homology
26
with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed
27
weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and
28
followed with the focally restricted signals in the feather follicles of neck and body
29
skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three
30
genes displayed stronger expression in feather follicles of neck skin than that of body
31
skin. The expression levels of JAK1 and TYK2 were much stronger than those of
32
JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased
33
expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the
34
much stronger expression in neck and body skin at later stages (E8-E10) than earlier
35
stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and
36
TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange
37
the proximal–distal axis of feather follicles, respectively, during the morphogenesis of
38
feather follicles in embryonic chicken skin.
39 40
Keywords: JAK-STAT, JAK1, JAK2, TYK2, Skin, Feather follicle, Chicken,
41
Morphogenesis
42
2
43
1. Introduction
44 45
Feathers are elaborate skin appendages with hierarchical branches that are
46
developed from the barb ridges (Cheng et al., 2018), and represented as flight feathers,
47
contour feathers, and downy feathers that serve to assist communication,
48
thermoregulation, and flight (Yu et al., 2004). The feather development is comprised
49
of morphogenesis in early stages and cycling growth post-hatching, including five
50
phases, termed macro-patterning, micro-patterning, intra-bud morphogenesis, follicle
51
morphogenesis, and regenerative feather cycling (Lin et al., 2006a). Although a
52
number of signaling pathways related to feather development have been investigated,
53
many potential regulators of feather follicle development are still waiting to be
54
characterized.
55
The investigations of skin appendages including feather follicles in birds and hair
56
follicles, mammary gland and tooth in mouse models confirm the partially conserved
57
genes and signaling pathways involved in the development of different appendages.
58
Of those, the Janus kinase–signal transducer and activator of transcription (JAK–
59
STAT) pathway has emerged as an important regulator of hair follicle development.
60
The binding of growth factors or cytokines to the receptors drives a conformational
61
change and assembles Janus kinases (JAK1, JAK2, TYK2) specifically with
62
intracellular domains of cytokine-receptor signaling chains, building STAT docking
63
sites
64
phosphorylation
65
Tyrosine-phosphorylated STAT proteins then activate tyrosine residues, transfer to the
66
nucleus, and bind with specific DNA elements to regulate the expression of target
67
genes (Aaronson and Horvath, 2002; Gurzov et al., 2016; Stark and Darnell, 2012).
68
Previous reports show that three JAK–STAT pathway genes, namely JAK1, JAK2, and
69
TYK2 are involved to regulate the growth and development of hair follicles in mice
70
(Udy et al., 1997; Xing et al., 2014). During the normal hair cycle, JAK-STAT
71
pathway genes display cyclic expression patterns with up-regulation (e.g., Jak1 and
through
catalyzing of
their
intracellular
own
ligand-induced
tyrosine
3
residues
phosphorylation in
the
and
receptor.
72
Jak3) in catagen and telogen, and repressed expression in early anagen. The inhibition
73
of JAK–STAT signaling with antagonists results in rapid reentry of hair follicles from
74
telogen to anagen of the hair cycle in wild type mice (Harel et al., 2015). This process
75
activates the key signaling pathways such as Wnt and Shh, and stimulates the hair
76
follicle progenitor cells in hair germ (Harel et al., 2015). Moreover, the STAT5 is
77
expressed in the dermal papilla (DP) of hair follicles during the hair cycle, with the
78
peaked expression in the early anagen phase (Legrand et al., 2016a; Wang et al.,
79
2016a). These observations demonstrate that JAK-STAT signaling is devoted to
80
maintain the hair follicles in the catagen and telogen phases, and prevent the transition
81
into anagen (Harel et al., 2015). Most of the studies are focused on interpreting the
82
functional roles of JAK-STAT signaling during the hair cycle postnatally, not the early
83
development prenatally. It would be interesting to conduct the related studies and
84
illustrate the potential roles of JAK-STAT during the early morphogenesis of hair
85
follicle in murine skin and feather follicles in chicken skin.
86
The present study aimed to examine the expression patterns of JAK–STAT
87
pathway genes (JAK1, JAK2, and TYK2) in chicken skin, particularly in the neck and
88
body regions located dorsally at different embryonic stages by using whole mount in
89
situ hybridization (WISH) and quantitative real-time PCR (qRT-PCR) validation. The
90
outcome of this study would add extra information to understand the regulatory roles
91
of JAK-START pathway functioned in the development of skin appendages.
92 93
2. Results
94 95
2.1. Characterization and phylogenetic analysis of chicken JAK–STAT pathway genes
96
(JAK1, JAK2, and TYK2)
97 98
Currently, the roles of JAK–STAT pathway genes in feather follicle and skin
99
development in chicken are not clear. Chicken JAK1, JAK2 and TYK2 are localized on
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chromosome 8, chromosome Z, and chromosome 30, respectively. Multiple alignment 4
101
of the protein sequences of JAK1, JAK2, and TYK2 showed a high degree of
102
homology among different species with conserved regions (Supplemental Fig. 1-3). A
103
phylogenetic tree was built to determine the relationship of JAK1, JAK2, TYK2
104
homologs. As is evident from the tree, the three genes in chicken are closely related to
105
orthologs in other species, particularly birds, namely Nipponia nippon, Coturnix
106
japonica (quail) and Anser cygnoides demisticus (goose) (Fig. 1).
107 108
2.2. Expression analysis of JAK1
109 110
The gene expression of JAK1, JAK2, and TYK2 influences hair growth in murine
111
skin (Harel et al., 2015; Xing et al., 2014). To determine the spatial distribution of
112
JAK1 in feather follicles, chicken embryos at different developmental stages were
113
fixed and examined by WISH using DIG-labeled antisense probes of the JAK1
114
transcripts. The hybridization signals of JAK1 were barely detectable in the dorsal
115
view of neck and body skin of chicken embryos at E6 and E7 (Fig. 2A, B, F, G). The
116
positive JAK1 expression signals were detected in the feather follicle primordia of
117
embryonic neck and body skin at E8 (Fig. 2C, C′, H, H′), E9 (Fig. 2D, D′, I, I′) and
118
E10 (Fig. 2E, E′, J, J′) stages.
119
It is interesting that the localization of JAK1 is slightly different between neck
120
and body skin regions. The feather follicles in body regions are more developed with
121
the main expression of JAK1 at the distal parts of feather follicles compared with
122
those of nearly homogeneous expression in feather follicles of neck skin (Fig. 2D, D′,
123
I, I′). Moreover, the expression of JAK1 in feather follicles showed stronger levels in
124
neck skin than that of body skin (Fig. 2D, D′, I, I′). Additionally, the weak JAK1
125
signals were also detected in the ring-like inter-follicular area outside of the feather
126
follicles at E10 stage. The sense probe of JAK1 was used as negative control showing
127
no hybridization signals during all the detected developmental stages (Supplemental
128
Fig. 4).
129 5
130 131
2.3. Expression analysis of JAK2
132
We further explored the expression patterns of JAK2 in the feather follicles of
133
chicken embryos. The WISH results indicated that JAK2 was barely expressed in the
134
neck and body dorsal skin of E6 and E7 embryos (Fig. 3A, B, F, G). At E8, JAK2 was
135
weakly expressed in the feather follicles of neck and body skin (Fig. 3C, C′, H, H′). At
136
E9 and E10 stages, the expression of JAK2 was gradually increased in the feather
137
follicles in the body and neck skin of chicken embryos, with the strongest expression
138
at E10 stage (Fig. 3D, D′, I, I′ and E, E′, J, J′). At this stage, JAK2 was expressed with
139
higher levels in the feather follicles of neck skin than those of the body skin (Fig. 3E,
140
E′, J, J′). Interestingly, at E9 and E10 stages, hybridization signals in the feather
141
follicles of neck skin were detectable in whole feather follicle primordia, whereas in
142
body skin it was mainly expressed in the distal axis of the feather follicles (Fig. 3D,
143
D′, I, I′ and E, E′, J, J′). The WISH of JAK2 sense probes showed no positive signals
144
in chicken embryonic skin at listed developmental stages (Supplemental Fig. 4).
145 146
2.4. Expression analysis of TYK2
147
The expression patterns of TYK2 in feather follicles were detected in chicken
148
embryos. TYK2 hybridization signals were nearly undetectable at E6 and E7, whereas
149
the positive signals were faint in both neck and body skin regions at E8 (Fig. 4A, B, C,
150
C′, F, G, H, H′). At E9 and E10 stages, the expression of TYK2 was strongly enhanced
151
and focally detected in the neck and body feather follicles, with much stronger
152
expression in neck skin than those of body skin at both E9 and E10 stages (Fig. 4D,
153
D′, I, I′ and Fig. 4E, E′, J, J′). Interestingly, the expression became more apparent in
154
the neck and body feather follicles at E10 (Fig. 4E, E′, J, J′). The expression of TYK2
155
and JAK2 was both restricted in the feather follicles in the feather bud stage as
156
detected in skin of E9 and E10 embryonic skin except that TYK2 showed stronger
157
expression than JAK2. The sense probes of TYK2 was detected as negative control
158
with no hybridization signals during all the listed developmental stages of chicken
159
embryos (Supplemental Fig. 4). 6
160 161
2.5. Validation of the expression levels of JAKI, JAK2, and TYK2 in chicken
162
embryonic skin by quantitative real-time PCR
163 164
Quantitative real-time PCR (qRT-PCR) was performed to validate the expression
165
levels of JAK1, JAK2, and TYK2 in chicken skin during different embryonic stages
166
(E6–E10). JAK1 displayed upregulation from E7 to E10 in both neck and body skin,
167
with more prominent elevation in neck and body skin at E8, whereas it showed no
168
clear difference from E6 to E7 (Fig. 5A, D). JAK2 exhibited continuously increased
169
expression from E6 to E10 both in neck and body skin (Fig. 5B, E). TYK2 showed the
170
similar expression tendency as JAK2 with gradually upregulation in both neck and
171
body skin, particularly with strong increase from E8 to E9 in body skin (Fig. 5C, F).
172
The qRT-PCR results of TYK2 were consistent with WISH analyses showing no
173
obvious signals at E6 and E7, weak signals at E8 and strong signals at E9 and E10.
174
Taken together, the qRT-PCR results were in line with the WISH analysis, further
175
clarifying the expression levels of the three genes during embryonic development.
176 177
3. Discussion
178 179
Feather development starts with the morphogenesis of feather follicles,
180
characterized as the induction of weak and fuzzy presumptive feather follicle
181
primordia in chicken skin at E6 and E7 (macro-patterning phase) to develop the
182
feather tract area and later feather follicles at E7.5 and E8.5 (Gong et al., 2018).
183
During this period, the epidermal placodes are associated with the underlying
184
condensed dermal cells, specifying the location of the feather follicles (Davidson,
185
1983; Linsenmayer, 1972). At E9 and E10 stages, the intra-bud morphogenesis occurs
186
to develop the anterior–posterior (A–P) and proximal–distal (P–D) axes of the
187
elongated feather buds (Gong et al., 2018; Lin et al., 2006b). The morphogenesis of
188
feather follicles is regulated by a series of signaling pathways including 7
189
wingless-related integration site (WNT) (Chang et al., 2004; Chodankar et al., 2003),
190
bone morphogenetic proteins (BMP) (Noramly and Morgan, 1998; Scaal et al., 2002),
191
and fibroblast growth factor (FGF) (Song et al., 2004) signaling pathways. There are
192
many other pathways and genes that are involved in this process like JAK-STAT
193
signaling pathway. Previous studies have demonstrated that the JAK–STAT pathway
194
genes are probably gate-keepers to restrain the hair follicles in catagen and telogen
195
and prevent the re-entering anagen during the hair cycling growth (Harel et al., 2015;
196
Legrand et al., 2016b; Wang et al., 2016b), whereas the roles in the morphogenesis of
197
early developmental phases were largely unclear both in chicken and mouse models.
198
The complex expression patterns of JAK–STAT family genes (JAK1, JAK2, and
199
TYK2) in embryonic chicken feather follicles were reported here using WISH and
200
qRT-PCR. Multiple sequence alignment of the JAK1, JAK2, and TYK2 proteins
201
showed a close homology with other species, particularly with birds like goose and
202
quail. Overall, the three genes exhibited similar expression patterns with delicate
203
differences in the neck and body skin regions of comparable developmental stage. For
204
example, the expression levels of all three genes in E6 and E7 embryonic skin were
205
very weak with nearly undetectable expression with WISH and much lower than other
206
stages (E8 to E10). During the transition from E6 to E7, chicken embryonic skin is
207
developing to form presumptive feather follicle primordia consisting of dermal
208
condensate and overlying feather placode, combined with the thickening of the
209
epidermis and dermis (Gong et al., 2018; Olivera-Martinez et al., 2000). The poor
210
expression of JAK1, JAK2, and TYK2 genes at E6 and 7 indicates that all three genes
211
were dispensable of the very early phase of feather follicle initiation.
212
The three genes displayed interesting expression patterns with focal
213
hybridization signals in the feather follicles at E8 for JAK1, E10 for JAK2 and E9 for
214
TYK2 gene. Three genes were sequentially stimulated to regulate the feather follicle
215
development with the order of JAK1, TYK2 and then JAK2. Moreover, the expression
216
levels of JAK1 and TYK2 were much stronger that those of JAK2. The developmental
217
stage from E7 to E8 is a key time point to reinforce the initiation or induction of the 8
218
de novo formed epidermal placodes and associated condensation of dermal cells that
219
specify the location of the feather follicle primordia in skin (Gong et al., 2018). These
220
results show that JAK1 might be involved in the restriction of presumptive feather
221
follicle primordia, not in the early induction stage, whereas JAK2 and TYK2 were not
222
important during the induction of feather follicles.
223
The polarity of the feather follicle starts to emerge as the anterior–posterior (A–P)
224
and proximal–distal (P–D) axes in E9 and E10 stages. Notch signaling is involved in
225
setting up the asymmetric A–P axis (Lin et al., 2006b), whereas FGF signaling is
226
shown to regulate the formation of P–D axis (Cheng et al., 2018). With both WISH
227
and qRT-PCR, we observed fairly specific JAK1, JAK2, and TYK2 expression signals
228
at E9 and E10 stages, particularly stronger expression of JAK1 and TYK2 than that of
229
JAK2. The developmental period from E9 to E10 is considered as the phase of
230
intra-bud morphogenesis. The short buds can be apparently distinguishable in the
231
feather tracts at E9 stage, and with elongation at E10 stage (Gong et al., 2018). All
232
three of our tested genes were expressed in the feather follicle primordia of the neck
233
and body dorsal skin. The hybridization signals of JAK1, JAK2 and TYK2 were more
234
apparent in feather follicles of neck skin, particularly in the distal axis than in other
235
parts of feather follicles. All the data suggest that JAK1 and TYK2, not JAK2 are
236
important to specify the feather follicle primordia, and to regulate the P–D axis
237
formation, respectively during the development of feather follicles in embryonic
238
chicken skin.
239
In conclusion, we report the systematic analysis of spatial and temporal
240
expressions of three JAK family genes during the initiation and development of
241
feather follicles in chicken models. The outcome of this study will considerably
242
facilitate the understanding of feather follicle and skin development in birds.
243 244
4. Experimental procedures
245 246
4.1. Experimental animals 9
247 248
White Leghorn chickens were raised in a local chicken farm. The fertilized eggs
249
were collected and incubated following the standard procedures. Chicken embryos
250
were harvested from fertilized eggs and determine the desired stages of embryonic
251
development according to the Hamburger–Hamilton stages (Hamburger and Hamilton,
252
1951). Subsequently, a number of chicken embryos from E6 to E10 stages were
253
collected in ice-cold phosphate-buffered saline (PBS), and stored in 4%
254
paraformaldehyde in PBS at 4 °C for in situ hybridization. To minimize variation,
255
embryos with clear individual differences in each developmental stage were removed.
256
Skin tissue samples were harvested from the body and neck skin located dorsally of
257
the embryos. The samples were immediately stored in TRIzol Reagent (Invitrogen,
258
USA) and kept frozen at −80 °C in a refrigerator for qPCR validation. All the
259
experiments on animals were approved by the Standing Committee of Hubei People’s
260
Congress and the ethics committee of Huazhong Agricultural University.
261 262
4.2. Phylogenetic analysis
263 264
The amino acid sequences of JAK1, JAK2, and TYK2 were retrieved from NCBI
265
(https://www.ncbi.nlm.nih.gov/protein) and were aligned using MAFFT (Nakamura et
266
al., 2018). The maximum-likelihood (ML) phylogenetic trees were drawn by
267
IQ-TREE (Chernomor et al., 2016). The best-fitting nucleotide substitution model
268
was generated by the program using 1000 bootstrap replicates.
269 270
4.3. RNA and probe preparation for WISH
271 272
Total RNAs were extracted from the samples using TRIzol Reagent (Invitrogen,
273
USA). Reverse transcription was performed to generate the first-strand cDNA using
274
the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). The transcript
275
sequences of JAK1, JAK2, and TYK2 genes for primer design were obtained from 10
276
public
277
(http://asia.ensembl.org/Chicken/Search/Results?q=;site=ensembl;facet_species=Chic
278
ken) to generate the cDNA templates for preparation of in situ hybridization probes.
279
The primer pairs for cDNA cloning were listed in Table 1. The primers were designed
280
to amplify the conserved regions of all splicing isoforms of each gene. The reaction
281
system of the PCR amplification was 10 µL including 0.5 µL first-strand cDNA, 1 µL
282
of upstream and downstream primers, 3.5 µL dd H2O, and 5 µL Premix Taq. The PCR
283
amplification conditions were as follows: pre-denaturation for 2 min at 98 °C,
284
followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 62 °C for 5 s,
285
and final extension at 72 °C for 10 min. Purified DNA fragments were linked using
286
the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, USA) and transformed into
287
Escherichia coli DH5α-competent cells for cloning. Clones were sequenced by
288
Sangon Biotech (Shanghai, China). The correct cloning sequences were used for
289
WISH plasmid preparation. Homology analysis was then performed on the sequences
290
using the BLAST suite module from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
databases
291 292
4.4. Whole mount in situ hybridization
293 294
Plasmid DNA was first linearized with restriction enzymes to prepare the
295
templates for probes. The templates for both sense and antisense probes were
296
generated and labeled with digoxigenin (DIG) (Roche, United States) through in vitro
297
transcription. Probes were applied to chicken embryos collected at the E6–E10 stages
298
for hybridization. Detailed probe preparation methods were described previously
299
(Arede and Tavares, 2008). WISH was performed following published protocols
300
(Hanaoka et al., 2006; Kawahara et al., 2009; Mou et al., 2011). More information is
301
listed in the Key Resource Table.
302 303
4.5. Quantitative real-time PCR (qRT-PCR) validation
304 11
305
The total RNAs were applied for reverse transcription for qRT-PCR identification.
306
The qRT-PCR primers were listed in Table 2. Amplification was performed in a Roche
307
LightCyclerR96 using iTaqTM Universal SYBR Green Supermix (Bio-Rad, United
308
States). The reaction mixture consisted of 4.5 µL cDNA, 5 µL SYBR Green Supmix,
309
and 0.5 µL primers. The amplification protocol was as follows: denaturation for 5 min
310
at 95 °C, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH was
311
used as the internal normalization control (Li et al., 2018; Nie et al., 2018). Relative
312
gene expression was calculated with the 2-
313
calculated using the Student’s t-test on data from six independent samples per
314
embryonic stage (n = 6).
Ct
method. The statistical analyses were
315 316
Acknowledgments
317 318
This work was supported by the National Natural Science Foundation of China
319
(No. 31972548) and National Key R&D Program of China (2018YFD0501301). We
320
would like to thank all the members involved in this work and Editage
321
[www.editage.cn] for English language editing.
322 323
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399
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401
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402
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403
Wang, E., Harel, S., Christiano, A.M., 2016b. JAK△STAT Signaling Jump Starts the Hair Cycle.
404
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405
Xing, L., Dai, Z., Jabbari, A., Cerise, J.E., Higgins, C.A., Gong, W., de Jong, A., Harel, S.,
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Clynes, R., 2014. Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK
408
inhibition. Nature medicine 20, 1043△1049.
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Yu, M., Yue, Z., Wu, P., Wu, D.Y., Mayer, J.A., Medina, M., Widelitz, R.B., Jiang, T.X., Chuong,
410
C.M., 2004. The developmental biology of feather follicles. International Journal of
411
Developmental Biology 48, 181.
412 413
16
414 415
Figure legends
416
Fig. 1. Phylogenetic tree of the JAK–STAT family members. The amino acid sequences
417
were aligned using MAFFT. The maximum-likelihood phylogenetic tree was built using
418
IQ-TREE. The best-fitting nucleotide substitution model was built by the program following
419
1000 bootstrap replicates to check the repeatability of the result. GenBank accession numbers:
420
Homo sapiens (JAK1) (NP_002218.2); Rattus norvegicus (Jak1) (NP_445918.1); Mus
421
musculus (Jak1) (NP_666257.2); Danio rerio (jak1) (NP_571148.1); Anser cygnoides
422
domesticus (JAK1) (XP_013048557.1); Oryctolagus cuniculus (JAK1) (XP_017201714.1);
423
Coturnix japonica (JAK1) (XP_015726229.1); Nipponia nippon (JAK1) (XP_009459479.1);
424
Gallus gallus (JAK1) (NP_990201.1); Homo sapiens (JAK2) (NP_004963.1); Rattus
425
norvegicus (Jak2) (NP_113702.1); Mus musculus (Jak2) (NP_001041642.1); Danio rerio
426
(jak2) (NP_571162.1); Anser cygnoides domesticus (JAK2) (XP_013052840.1); Oryctolagus
427
cuniculus (JAK2) (XP_002708048.1); Coturnix japonica (JAK2) (XP_015704401.1);
428
Nipponia nippon (JAK2) (XP_009473266.1); Gallus gallus (JAK2) (NP_001025709.2);
429
Homo sapiens (TYK2) (AAH14243.1); Rattus norvegicus (Tyk2) (NP_001244276.1); Mus
430
musculus (Tyk2) (AAH94240.1); Danio rerio (tyk2) (XP_003198201.2); Anser cygnoides
431
domesticus (TYK2) (XP_013057650.1); Oryctolagus cuniculus (TYK2) (XP_017194260.1);
432
Coturnix japonica (TYK2) (XP_015706188.1); Nipponia nippon (TYK2) (XP_009461880.1);
433
Gallus gallus (TYK2) (XP_025001507.1).
434 435
Fig. 2. Spatial expression pattern of JAK1 during early embryogenesis in chicken skin by
436
whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
437
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a neck
438
feather follicle and magnified in (C′) with a red arrowhead and dashed line indicating a neck
439
feather follicle. (D) The stained neck skin at E9 with red arrowhead indicating a neck feather
440
follicle and magnified in (D′) with a red arrowhead and dashed line indicating a neck feather
441
follicle boundary. (E) The stained neck skin at E10 with red arrowhead indicating a neck
442
feather follicle and magnified in (E′) with a red arrowhead and dashed line indicating a neck
443
feather follicle. (F) The stained body skin at E6. (G) The stained body skin at E7. (H) The 17
444
stained body skin at E8 with red arrowhead indicating a body feather follicle and magnified in
445
(H′) with a red arrowhead and dashed line indicating a body feather follicle. (I) The stained
446
body skin at E9 with red arrowhead indicating a body feather follicle and magnified in (I′)
447
with a red arrowhead and dashed line indicating a body feather follicle boundary. (J) The
448
stained body skin at E10 with red arrowhead indicating a body feather follicle and magnified
449
in (E′) with a red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1
450
mm for A–J; 200 µm for C′–E′ and I′–J′; 500 µm for H′.
451 452
Fig. 3. Spatial expression pattern of JAK2 during early embryogenesis in chicken skin by
453
whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
454
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a weak
455
hybridization signal of a neck feather follicle and magnified in (C′) with a red arrowhead and
456
dashed line indicating a neck feather follicle. (D) The stained neck skin at E9 with red
457
arrowhead indicating a neck feather follicle and magnified in (D′) with a red arrowhead and
458
dashed line indicating a neck feather follicle boundary. (E) The stained neck skin at E10 with
459
red arrowhead indicating a neck feather follicle and magnified in (E′) with a red arrowhead
460
and dashed line indicating a neck feather follicle. (F) The stained body skin at E6. (G) The
461
stained body skin at E7. (H) The stained body skin at E8 with red arrowhead indicating a
462
weaker hybridization signal of a body feather follicle and magnified in (H′) with a red
463
arrowhead and dashed line indicating a body feather follicle. (I) The stained body skin at E9
464
with red arrowhead indicating a body feather follicle and magnified in (I′) with a red
465
arrowhead and dashed line indicating a body feather follicle boundary. (J) The stained body
466
skin at E10 with red arrowhead indicating a body feather follicle and magnified in (E′) with a
467
red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1 mm for A–J;
468
200 µm for C′–E and H′–J′.
469 470
Fig. 4. Spatial expression pattern of TYK2 during early embryogenesis in chicken skin
471
by whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
472
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a weak 18
473
hybridization signal of a neck feather follicle and magnified in (C′) with a red arrowhead and
474
dashed line indicating neck feather follicle. (D) The stained neck skin at E9 with red
475
arrowhead indicating a neck feather follicle and magnified in (D′) with a red arrowhead and
476
dashed line indicating neck feather follicle boundary. (E) The stained neck skin at E10 with
477
red arrowhead indicating a neck feather follicle and magnified in (E′) with a red arrowhead
478
and dashed line indicating neck feather follicle. (F) The stained body skin at E6. (G) The
479
stained body skin at E7. (H) The stained body skin at E8 with red arrowhead indicating a
480
weaker hybridization signal of a body feather follicle and magnified in (H′) with a red
481
arrowhead and dashed line indicating a body feather follicle. (I) The stained body skin at E9
482
with red arrowhead indicating a body feather follicle and magnified in (I′) with a red
483
arrowhead and dashed line indicating a body feather follicle boundary. (J) The stained body
484
skin at E10 with red arrowhead indicating a body feather follicle and magnified in (E′) with a
485
red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1 mm for A–J;
486
200 µm for C′–E′ and H′–J′.
487 488
Fig. 5. Quantitative real-time PCR validation of gene expression levels. (A) Quantitative
489
expression of JAK1 in chicken neck skin at developmental stages E6–E10. (B) Quantitative
490
expression of JAK2 in chicken neck skin from stage E6 to E10. (C) Quantitative gene
491
expression of TYK2 in chicken neck skin from stage E6 to E10. (D) Quantitative expression
492
of JAK1 in chicken dorsal (body) skin from stage E6 to E10. (E) Quantitative expression of
493
JAK2 in chicken dorsal skin from stage E6 to E10. (F) Quantitative gene expression of TYK2
494
in chicken dorsal skin from stage E6 to E10. Result are shown as means ± SEM (n = 6). * p <
495
0.05 and ** p < 0.01 (Student’s t-test).
496 497
Supplemental Fig. 1. Multiple sequence alignment of chicken JAK1 with its orthologs from
498
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
499
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate that 100%,
500
75%, and 50% conservation, respectively.
501 19
502
Supplemental Fig. 2. Multiple sequence alignment of chicken JAK2 with its orthologs from
503
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
504
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate 100%, 75%,
505
and 50% conservation, respectively.
506 507
Supplemental Fig. 3. Multiple sequence alignment of chicken TYK2 with its orthologs from
508
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
509
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate 100%, 75%,
510
and 50% conservation, respectively.
511 512
Supplemental Fig. 4. Spatial expression pattern of sense probes of JAK1, JAK2 and
513
TYK2 during early embryogenesis in chicken skin. The five developmental stages of
514
chicken embryos from E6 to E10 hybridized with JAK1 sense probes showed no clear
515
positive signals both in neck as shown in figure (A1) E6, (B1) E7, (C1) E8, (D1) E9 and (E1)
516
E10, and body skin as shown in figure (F1) E6, (G1) E7, (H1) E8, (I1) E9 and (J1) E10. The
517
five developmental stages of chicken embryos from E6 to E10 hybridized with JAK2 sense
518
probes showed no clear positive signals both in neck as shown in figure (A2) E6, (B2) E7,
519
(C2) E8, (D2) E9 and (E2) E10, and body skin as shown in figure (F2) E6, (G2) E7, (H2) E8,
520
(I2) E9 and (J2) E10. The five developmental stages of chicken embryos from E6 to E10
521
hybridized with TYK2 sense probes showed no clear positive signals both in neck as shown in
522
figure (A3) E6, (B3) E7, (C3) E8, (D3) E9 and (E3) E10, and body skin as shown in figure
523
(F3) E6, (G3) E7, (H3) E8, (I3) E9 and (J3) E10. Scale bars=1 mm
524
20
525
Table 1. Primers used for cloning. Gene JAK1
Sequence (5′-3′)
Function
F: CGTTGAACAAGACCATCAGG
Cloning
R: GAGCCTCCACTGGATTCCAT JAK2
F: CAGAGGCACAATGTCAGCCAGA R: CACTCAGTGGTTTGTCTCCTCC
TYK2
F: GCACTTCTGTGACTTCCAAGAG R: GTTGAGGATCCGCTTCGCGTTG
534 535
Cloning
Table 2. Primers used for quantitative real-time PCR. Gene JAK1
Sequence (5′-3′)
Function
F: ATCCTTCGCACAGACAACATC
qPCR
R: GCATTCCTGAGCCTTCTTGG JAK2
F: GAGCGTGAGAATGCCACTGAC R: TGGAGGACAGCACTTGATGAAC
TYK2
F: TCTCCTTGGACGTCTCCAATG R: GAAATATCCGCGGTGGGAAAT
GAPDH
F: GAAGGCTGGGGCTCATCTG
qPCR qPCR qPCR
R: CAGTTGGTGGTGCACGATG
536
Cloning
Sequences for primer design span all isoforms from NCBI.
21
526 527 528 529 530 531 532 533
KEY RESOURCE TABLE REAGENT or RESOURCE Antibodies
SOURCE
IDENTIFIER
Anti-Digoxigenin, Fab fragments
Roche
Cat#11093274910
Local chicken farm
(Wuhan, China)
Takara Bio-Rad Invitrogen Invitrogen Roche
Cat#RR047A Cat#172-5124 Cat#1804946 Cat#15596026 Cat#11277073910
Ensemble database
http://asia.ensembl.o rg/Chicken/Search/R esults?q=;site=ense mbl;facet_species=C hicken
This paper This paper
N/A N/A
(Nakamura et al., 2018)
https://mafft.cbrc.jp/ alignment/software/ http://www.iqtree.or g/
Biological Samples
White Leghorn chicken embryos from E6 to E10 Critical Commercial Assays PrimeScript RT reagent kit with gDNA Eraser iTaqTM Universal SYBR Green Supermix Zero Blunt® TOPO® PCR Cloning Kit TRIzol™ Reagent Dig-RNA labeling mix Deposited Data The amino acids sequences of JAK1, JAK2, and TYK2
Primers Primers for cloning, see Table 1 Primer for real-time PCR, see Table 2 Software and Algorithms MAFFT IQ-TREE
Chernomor et al., 2016
Highlights: • • •
Assessment of JAK–STAT pathway genes in skin and feather follicle development. JAK1 contributes to reinforce the presumptive feather follicle primordium. TYK2 supports the arrangement of proximal-distal axis of feather follicles.
Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis Yingfeng Tao1, Xiaoliu Zhou1, Zhiwei Liu1, Xiaokang Zhang1, Yangfan Nie1, Xinting Zheng1, Shaomei Li1, Xuewen Hu1, Ge Yang1, Qianqian Zhao1, Chunyan Mou1*
1
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, HuaZhong Agricultural University, Wuhan, China
*Correspondence: Chunyan Mou [email protected]
Declaration of interest: The authors declare no competing interests.
S1567-133X(19)30063-8
DOI:
https://doi.org/10.1016/j.gep.2019.119078
Reference:
MODGEP 119078
To appear in:
Gene Expression Patterns
Received Date: 18 April 2019 Revised Date:
11 November 2019
Accepted Date: 15 November 2019
Please cite this article as: Tao, Y., Zhou, X., Liu, Z., Zhang, X., Nie, Y., Zheng, X., Li, S., Hu, X., Yang, G., Zhao, Q., Mou, C., Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis, Gene Expression Patterns (2019), doi: https:// doi.org/10.1016/j.gep.2019.119078. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Expression patterns of three JAK–STAT pathway
2
genes in feather follicle development during chicken
3
embryogenesis
4 5 6 7 8 9 10 11 12 13 14 15 16
Yingfeng Tao1, Xiaoliu Zhou1, Zhiwei Liu1, Xiaokang Zhang1, Yangfan Nie1, Xinting Zheng1, Shaomei Li1, Xuewen Hu1, Ge Yang1, Qianqian Zhao1, Chunyan Mou1* 1
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, HuaZhong Agricultural University, Wuhan, China *Correspondence: Chunyan Mou [email protected]
1
17
ABSTRACT
18
The Janus kinase (JAK)–signal transducer and activator of transcription (STAT)
19
(JAK–STAT) pathway is shown to restrain the hair follicles in catagen and telogen
20
and prevent anagen reentry in murine hair follicle cycling. The early roles of
21
JAK-STAT pathway genes in skin development remain uncharacterized in mouse and
22
chicken models. Here, we revealed the expression patterns of three JAK-STAT
23
pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6–E10 stages
24
which are key to feather follicle morphogenesis. Multiple sequence alignment of the
25
three genes from chicken and other species all showed a closely related homology
26
with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed
27
weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and
28
followed with the focally restricted signals in the feather follicles of neck and body
29
skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three
30
genes displayed stronger expression in feather follicles of neck skin than that of body
31
skin. The expression levels of JAK1 and TYK2 were much stronger than those of
32
JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased
33
expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the
34
much stronger expression in neck and body skin at later stages (E8-E10) than earlier
35
stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and
36
TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange
37
the proximal–distal axis of feather follicles, respectively, during the morphogenesis of
38
feather follicles in embryonic chicken skin.
39 40
Keywords: JAK-STAT, JAK1, JAK2, TYK2, Skin, Feather follicle, Chicken,
41
Morphogenesis
42
2
43
1. Introduction
44 45
Feathers are elaborate skin appendages with hierarchical branches that are
46
developed from the barb ridges (Cheng et al., 2018), and represented as flight feathers,
47
contour feathers, and downy feathers that serve to assist communication,
48
thermoregulation, and flight (Yu et al., 2004). The feather development is comprised
49
of morphogenesis in early stages and cycling growth post-hatching, including five
50
phases, termed macro-patterning, micro-patterning, intra-bud morphogenesis, follicle
51
morphogenesis, and regenerative feather cycling (Lin et al., 2006a). Although a
52
number of signaling pathways related to feather development have been investigated,
53
many potential regulators of feather follicle development are still waiting to be
54
characterized.
55
The investigations of skin appendages including feather follicles in birds and hair
56
follicles, mammary gland and tooth in mouse models confirm the partially conserved
57
genes and signaling pathways involved in the development of different appendages.
58
Of those, the Janus kinase–signal transducer and activator of transcription (JAK–
59
STAT) pathway has emerged as an important regulator of hair follicle development.
60
The binding of growth factors or cytokines to the receptors drives a conformational
61
change and assembles Janus kinases (JAK1, JAK2, TYK2) specifically with
62
intracellular domains of cytokine-receptor signaling chains, building STAT docking
63
sites
64
phosphorylation
65
Tyrosine-phosphorylated STAT proteins then activate tyrosine residues, transfer to the
66
nucleus, and bind with specific DNA elements to regulate the expression of target
67
genes (Aaronson and Horvath, 2002; Gurzov et al., 2016; Stark and Darnell, 2012).
68
Previous reports show that three JAK–STAT pathway genes, namely JAK1, JAK2, and
69
TYK2 are involved to regulate the growth and development of hair follicles in mice
70
(Udy et al., 1997; Xing et al., 2014). During the normal hair cycle, JAK-STAT
71
pathway genes display cyclic expression patterns with up-regulation (e.g., Jak1 and
through
catalyzing of
their
intracellular
own
ligand-induced
tyrosine
3
residues
phosphorylation in
the
and
receptor.
72
Jak3) in catagen and telogen, and repressed expression in early anagen. The inhibition
73
of JAK–STAT signaling with antagonists results in rapid reentry of hair follicles from
74
telogen to anagen of the hair cycle in wild type mice (Harel et al., 2015). This process
75
activates the key signaling pathways such as Wnt and Shh, and stimulates the hair
76
follicle progenitor cells in hair germ (Harel et al., 2015). Moreover, the STAT5 is
77
expressed in the dermal papilla (DP) of hair follicles during the hair cycle, with the
78
peaked expression in the early anagen phase (Legrand et al., 2016a; Wang et al.,
79
2016a). These observations demonstrate that JAK-STAT signaling is devoted to
80
maintain the hair follicles in the catagen and telogen phases, and prevent the transition
81
into anagen (Harel et al., 2015). Most of the studies are focused on interpreting the
82
functional roles of JAK-STAT signaling during the hair cycle postnatally, not the early
83
development prenatally. It would be interesting to conduct the related studies and
84
illustrate the potential roles of JAK-STAT during the early morphogenesis of hair
85
follicle in murine skin and feather follicles in chicken skin.
86
The present study aimed to examine the expression patterns of JAK–STAT
87
pathway genes (JAK1, JAK2, and TYK2) in chicken skin, particularly in the neck and
88
body regions located dorsally at different embryonic stages by using whole mount in
89
situ hybridization (WISH) and quantitative real-time PCR (qRT-PCR) validation. The
90
outcome of this study would add extra information to understand the regulatory roles
91
of JAK-START pathway functioned in the development of skin appendages.
92 93
2. Results
94 95
2.1. Characterization and phylogenetic analysis of chicken JAK–STAT pathway genes
96
(JAK1, JAK2, and TYK2)
97 98
Currently, the roles of JAK–STAT pathway genes in feather follicle and skin
99
development in chicken are not clear. Chicken JAK1, JAK2 and TYK2 are localized on
100
chromosome 8, chromosome Z, and chromosome 30, respectively. Multiple alignment 4
101
of the protein sequences of JAK1, JAK2, and TYK2 showed a high degree of
102
homology among different species with conserved regions (Supplemental Fig. 1-3). A
103
phylogenetic tree was built to determine the relationship of JAK1, JAK2, TYK2
104
homologs. As is evident from the tree, the three genes in chicken are closely related to
105
orthologs in other species, particularly birds, namely Nipponia nippon, Coturnix
106
japonica (quail) and Anser cygnoides demisticus (goose) (Fig. 1).
107 108
2.2. Expression analysis of JAK1
109 110
The gene expression of JAK1, JAK2, and TYK2 influences hair growth in murine
111
skin (Harel et al., 2015; Xing et al., 2014). To determine the spatial distribution of
112
JAK1 in feather follicles, chicken embryos at different developmental stages were
113
fixed and examined by WISH using DIG-labeled antisense probes of the JAK1
114
transcripts. The hybridization signals of JAK1 were barely detectable in the dorsal
115
view of neck and body skin of chicken embryos at E6 and E7 (Fig. 2A, B, F, G). The
116
positive JAK1 expression signals were detected in the feather follicle primordia of
117
embryonic neck and body skin at E8 (Fig. 2C, C′, H, H′), E9 (Fig. 2D, D′, I, I′) and
118
E10 (Fig. 2E, E′, J, J′) stages.
119
It is interesting that the localization of JAK1 is slightly different between neck
120
and body skin regions. The feather follicles in body regions are more developed with
121
the main expression of JAK1 at the distal parts of feather follicles compared with
122
those of nearly homogeneous expression in feather follicles of neck skin (Fig. 2D, D′,
123
I, I′). Moreover, the expression of JAK1 in feather follicles showed stronger levels in
124
neck skin than that of body skin (Fig. 2D, D′, I, I′). Additionally, the weak JAK1
125
signals were also detected in the ring-like inter-follicular area outside of the feather
126
follicles at E10 stage. The sense probe of JAK1 was used as negative control showing
127
no hybridization signals during all the detected developmental stages (Supplemental
128
Fig. 4).
129 5
130 131
2.3. Expression analysis of JAK2
132
We further explored the expression patterns of JAK2 in the feather follicles of
133
chicken embryos. The WISH results indicated that JAK2 was barely expressed in the
134
neck and body dorsal skin of E6 and E7 embryos (Fig. 3A, B, F, G). At E8, JAK2 was
135
weakly expressed in the feather follicles of neck and body skin (Fig. 3C, C′, H, H′). At
136
E9 and E10 stages, the expression of JAK2 was gradually increased in the feather
137
follicles in the body and neck skin of chicken embryos, with the strongest expression
138
at E10 stage (Fig. 3D, D′, I, I′ and E, E′, J, J′). At this stage, JAK2 was expressed with
139
higher levels in the feather follicles of neck skin than those of the body skin (Fig. 3E,
140
E′, J, J′). Interestingly, at E9 and E10 stages, hybridization signals in the feather
141
follicles of neck skin were detectable in whole feather follicle primordia, whereas in
142
body skin it was mainly expressed in the distal axis of the feather follicles (Fig. 3D,
143
D′, I, I′ and E, E′, J, J′). The WISH of JAK2 sense probes showed no positive signals
144
in chicken embryonic skin at listed developmental stages (Supplemental Fig. 4).
145 146
2.4. Expression analysis of TYK2
147
The expression patterns of TYK2 in feather follicles were detected in chicken
148
embryos. TYK2 hybridization signals were nearly undetectable at E6 and E7, whereas
149
the positive signals were faint in both neck and body skin regions at E8 (Fig. 4A, B, C,
150
C′, F, G, H, H′). At E9 and E10 stages, the expression of TYK2 was strongly enhanced
151
and focally detected in the neck and body feather follicles, with much stronger
152
expression in neck skin than those of body skin at both E9 and E10 stages (Fig. 4D,
153
D′, I, I′ and Fig. 4E, E′, J, J′). Interestingly, the expression became more apparent in
154
the neck and body feather follicles at E10 (Fig. 4E, E′, J, J′). The expression of TYK2
155
and JAK2 was both restricted in the feather follicles in the feather bud stage as
156
detected in skin of E9 and E10 embryonic skin except that TYK2 showed stronger
157
expression than JAK2. The sense probes of TYK2 was detected as negative control
158
with no hybridization signals during all the listed developmental stages of chicken
159
embryos (Supplemental Fig. 4). 6
160 161
2.5. Validation of the expression levels of JAKI, JAK2, and TYK2 in chicken
162
embryonic skin by quantitative real-time PCR
163 164
Quantitative real-time PCR (qRT-PCR) was performed to validate the expression
165
levels of JAK1, JAK2, and TYK2 in chicken skin during different embryonic stages
166
(E6–E10). JAK1 displayed upregulation from E7 to E10 in both neck and body skin,
167
with more prominent elevation in neck and body skin at E8, whereas it showed no
168
clear difference from E6 to E7 (Fig. 5A, D). JAK2 exhibited continuously increased
169
expression from E6 to E10 both in neck and body skin (Fig. 5B, E). TYK2 showed the
170
similar expression tendency as JAK2 with gradually upregulation in both neck and
171
body skin, particularly with strong increase from E8 to E9 in body skin (Fig. 5C, F).
172
The qRT-PCR results of TYK2 were consistent with WISH analyses showing no
173
obvious signals at E6 and E7, weak signals at E8 and strong signals at E9 and E10.
174
Taken together, the qRT-PCR results were in line with the WISH analysis, further
175
clarifying the expression levels of the three genes during embryonic development.
176 177
3. Discussion
178 179
Feather development starts with the morphogenesis of feather follicles,
180
characterized as the induction of weak and fuzzy presumptive feather follicle
181
primordia in chicken skin at E6 and E7 (macro-patterning phase) to develop the
182
feather tract area and later feather follicles at E7.5 and E8.5 (Gong et al., 2018).
183
During this period, the epidermal placodes are associated with the underlying
184
condensed dermal cells, specifying the location of the feather follicles (Davidson,
185
1983; Linsenmayer, 1972). At E9 and E10 stages, the intra-bud morphogenesis occurs
186
to develop the anterior–posterior (A–P) and proximal–distal (P–D) axes of the
187
elongated feather buds (Gong et al., 2018; Lin et al., 2006b). The morphogenesis of
188
feather follicles is regulated by a series of signaling pathways including 7
189
wingless-related integration site (WNT) (Chang et al., 2004; Chodankar et al., 2003),
190
bone morphogenetic proteins (BMP) (Noramly and Morgan, 1998; Scaal et al., 2002),
191
and fibroblast growth factor (FGF) (Song et al., 2004) signaling pathways. There are
192
many other pathways and genes that are involved in this process like JAK-STAT
193
signaling pathway. Previous studies have demonstrated that the JAK–STAT pathway
194
genes are probably gate-keepers to restrain the hair follicles in catagen and telogen
195
and prevent the re-entering anagen during the hair cycling growth (Harel et al., 2015;
196
Legrand et al., 2016b; Wang et al., 2016b), whereas the roles in the morphogenesis of
197
early developmental phases were largely unclear both in chicken and mouse models.
198
The complex expression patterns of JAK–STAT family genes (JAK1, JAK2, and
199
TYK2) in embryonic chicken feather follicles were reported here using WISH and
200
qRT-PCR. Multiple sequence alignment of the JAK1, JAK2, and TYK2 proteins
201
showed a close homology with other species, particularly with birds like goose and
202
quail. Overall, the three genes exhibited similar expression patterns with delicate
203
differences in the neck and body skin regions of comparable developmental stage. For
204
example, the expression levels of all three genes in E6 and E7 embryonic skin were
205
very weak with nearly undetectable expression with WISH and much lower than other
206
stages (E8 to E10). During the transition from E6 to E7, chicken embryonic skin is
207
developing to form presumptive feather follicle primordia consisting of dermal
208
condensate and overlying feather placode, combined with the thickening of the
209
epidermis and dermis (Gong et al., 2018; Olivera-Martinez et al., 2000). The poor
210
expression of JAK1, JAK2, and TYK2 genes at E6 and 7 indicates that all three genes
211
were dispensable of the very early phase of feather follicle initiation.
212
The three genes displayed interesting expression patterns with focal
213
hybridization signals in the feather follicles at E8 for JAK1, E10 for JAK2 and E9 for
214
TYK2 gene. Three genes were sequentially stimulated to regulate the feather follicle
215
development with the order of JAK1, TYK2 and then JAK2. Moreover, the expression
216
levels of JAK1 and TYK2 were much stronger that those of JAK2. The developmental
217
stage from E7 to E8 is a key time point to reinforce the initiation or induction of the 8
218
de novo formed epidermal placodes and associated condensation of dermal cells that
219
specify the location of the feather follicle primordia in skin (Gong et al., 2018). These
220
results show that JAK1 might be involved in the restriction of presumptive feather
221
follicle primordia, not in the early induction stage, whereas JAK2 and TYK2 were not
222
important during the induction of feather follicles.
223
The polarity of the feather follicle starts to emerge as the anterior–posterior (A–P)
224
and proximal–distal (P–D) axes in E9 and E10 stages. Notch signaling is involved in
225
setting up the asymmetric A–P axis (Lin et al., 2006b), whereas FGF signaling is
226
shown to regulate the formation of P–D axis (Cheng et al., 2018). With both WISH
227
and qRT-PCR, we observed fairly specific JAK1, JAK2, and TYK2 expression signals
228
at E9 and E10 stages, particularly stronger expression of JAK1 and TYK2 than that of
229
JAK2. The developmental period from E9 to E10 is considered as the phase of
230
intra-bud morphogenesis. The short buds can be apparently distinguishable in the
231
feather tracts at E9 stage, and with elongation at E10 stage (Gong et al., 2018). All
232
three of our tested genes were expressed in the feather follicle primordia of the neck
233
and body dorsal skin. The hybridization signals of JAK1, JAK2 and TYK2 were more
234
apparent in feather follicles of neck skin, particularly in the distal axis than in other
235
parts of feather follicles. All the data suggest that JAK1 and TYK2, not JAK2 are
236
important to specify the feather follicle primordia, and to regulate the P–D axis
237
formation, respectively during the development of feather follicles in embryonic
238
chicken skin.
239
In conclusion, we report the systematic analysis of spatial and temporal
240
expressions of three JAK family genes during the initiation and development of
241
feather follicles in chicken models. The outcome of this study will considerably
242
facilitate the understanding of feather follicle and skin development in birds.
243 244
4. Experimental procedures
245 246
4.1. Experimental animals 9
247 248
White Leghorn chickens were raised in a local chicken farm. The fertilized eggs
249
were collected and incubated following the standard procedures. Chicken embryos
250
were harvested from fertilized eggs and determine the desired stages of embryonic
251
development according to the Hamburger–Hamilton stages (Hamburger and Hamilton,
252
1951). Subsequently, a number of chicken embryos from E6 to E10 stages were
253
collected in ice-cold phosphate-buffered saline (PBS), and stored in 4%
254
paraformaldehyde in PBS at 4 °C for in situ hybridization. To minimize variation,
255
embryos with clear individual differences in each developmental stage were removed.
256
Skin tissue samples were harvested from the body and neck skin located dorsally of
257
the embryos. The samples were immediately stored in TRIzol Reagent (Invitrogen,
258
USA) and kept frozen at −80 °C in a refrigerator for qPCR validation. All the
259
experiments on animals were approved by the Standing Committee of Hubei People’s
260
Congress and the ethics committee of Huazhong Agricultural University.
261 262
4.2. Phylogenetic analysis
263 264
The amino acid sequences of JAK1, JAK2, and TYK2 were retrieved from NCBI
265
(https://www.ncbi.nlm.nih.gov/protein) and were aligned using MAFFT (Nakamura et
266
al., 2018). The maximum-likelihood (ML) phylogenetic trees were drawn by
267
IQ-TREE (Chernomor et al., 2016). The best-fitting nucleotide substitution model
268
was generated by the program using 1000 bootstrap replicates.
269 270
4.3. RNA and probe preparation for WISH
271 272
Total RNAs were extracted from the samples using TRIzol Reagent (Invitrogen,
273
USA). Reverse transcription was performed to generate the first-strand cDNA using
274
the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). The transcript
275
sequences of JAK1, JAK2, and TYK2 genes for primer design were obtained from 10
276
public
277
(http://asia.ensembl.org/Chicken/Search/Results?q=;site=ensembl;facet_species=Chic
278
ken) to generate the cDNA templates for preparation of in situ hybridization probes.
279
The primer pairs for cDNA cloning were listed in Table 1. The primers were designed
280
to amplify the conserved regions of all splicing isoforms of each gene. The reaction
281
system of the PCR amplification was 10 µL including 0.5 µL first-strand cDNA, 1 µL
282
of upstream and downstream primers, 3.5 µL dd H2O, and 5 µL Premix Taq. The PCR
283
amplification conditions were as follows: pre-denaturation for 2 min at 98 °C,
284
followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 62 °C for 5 s,
285
and final extension at 72 °C for 10 min. Purified DNA fragments were linked using
286
the Zero Blunt TOPO PCR Cloning Kit (Invitrogen, USA) and transformed into
287
Escherichia coli DH5α-competent cells for cloning. Clones were sequenced by
288
Sangon Biotech (Shanghai, China). The correct cloning sequences were used for
289
WISH plasmid preparation. Homology analysis was then performed on the sequences
290
using the BLAST suite module from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
databases
291 292
4.4. Whole mount in situ hybridization
293 294
Plasmid DNA was first linearized with restriction enzymes to prepare the
295
templates for probes. The templates for both sense and antisense probes were
296
generated and labeled with digoxigenin (DIG) (Roche, United States) through in vitro
297
transcription. Probes were applied to chicken embryos collected at the E6–E10 stages
298
for hybridization. Detailed probe preparation methods were described previously
299
(Arede and Tavares, 2008). WISH was performed following published protocols
300
(Hanaoka et al., 2006; Kawahara et al., 2009; Mou et al., 2011). More information is
301
listed in the Key Resource Table.
302 303
4.5. Quantitative real-time PCR (qRT-PCR) validation
304 11
305
The total RNAs were applied for reverse transcription for qRT-PCR identification.
306
The qRT-PCR primers were listed in Table 2. Amplification was performed in a Roche
307
LightCyclerR96 using iTaqTM Universal SYBR Green Supermix (Bio-Rad, United
308
States). The reaction mixture consisted of 4.5 µL cDNA, 5 µL SYBR Green Supmix,
309
and 0.5 µL primers. The amplification protocol was as follows: denaturation for 5 min
310
at 95 °C, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. GAPDH was
311
used as the internal normalization control (Li et al., 2018; Nie et al., 2018). Relative
312
gene expression was calculated with the 2-
313
calculated using the Student’s t-test on data from six independent samples per
314
embryonic stage (n = 6).
Ct
method. The statistical analyses were
315 316
Acknowledgments
317 318
This work was supported by the National Natural Science Foundation of China
319
(No. 31972548) and National Key R&D Program of China (2018YFD0501301). We
320
would like to thank all the members involved in this work and Editage
321
[www.editage.cn] for English language editing.
322 323
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16
414 415
Figure legends
416
Fig. 1. Phylogenetic tree of the JAK–STAT family members. The amino acid sequences
417
were aligned using MAFFT. The maximum-likelihood phylogenetic tree was built using
418
IQ-TREE. The best-fitting nucleotide substitution model was built by the program following
419
1000 bootstrap replicates to check the repeatability of the result. GenBank accession numbers:
420
Homo sapiens (JAK1) (NP_002218.2); Rattus norvegicus (Jak1) (NP_445918.1); Mus
421
musculus (Jak1) (NP_666257.2); Danio rerio (jak1) (NP_571148.1); Anser cygnoides
422
domesticus (JAK1) (XP_013048557.1); Oryctolagus cuniculus (JAK1) (XP_017201714.1);
423
Coturnix japonica (JAK1) (XP_015726229.1); Nipponia nippon (JAK1) (XP_009459479.1);
424
Gallus gallus (JAK1) (NP_990201.1); Homo sapiens (JAK2) (NP_004963.1); Rattus
425
norvegicus (Jak2) (NP_113702.1); Mus musculus (Jak2) (NP_001041642.1); Danio rerio
426
(jak2) (NP_571162.1); Anser cygnoides domesticus (JAK2) (XP_013052840.1); Oryctolagus
427
cuniculus (JAK2) (XP_002708048.1); Coturnix japonica (JAK2) (XP_015704401.1);
428
Nipponia nippon (JAK2) (XP_009473266.1); Gallus gallus (JAK2) (NP_001025709.2);
429
Homo sapiens (TYK2) (AAH14243.1); Rattus norvegicus (Tyk2) (NP_001244276.1); Mus
430
musculus (Tyk2) (AAH94240.1); Danio rerio (tyk2) (XP_003198201.2); Anser cygnoides
431
domesticus (TYK2) (XP_013057650.1); Oryctolagus cuniculus (TYK2) (XP_017194260.1);
432
Coturnix japonica (TYK2) (XP_015706188.1); Nipponia nippon (TYK2) (XP_009461880.1);
433
Gallus gallus (TYK2) (XP_025001507.1).
434 435
Fig. 2. Spatial expression pattern of JAK1 during early embryogenesis in chicken skin by
436
whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
437
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a neck
438
feather follicle and magnified in (C′) with a red arrowhead and dashed line indicating a neck
439
feather follicle. (D) The stained neck skin at E9 with red arrowhead indicating a neck feather
440
follicle and magnified in (D′) with a red arrowhead and dashed line indicating a neck feather
441
follicle boundary. (E) The stained neck skin at E10 with red arrowhead indicating a neck
442
feather follicle and magnified in (E′) with a red arrowhead and dashed line indicating a neck
443
feather follicle. (F) The stained body skin at E6. (G) The stained body skin at E7. (H) The 17
444
stained body skin at E8 with red arrowhead indicating a body feather follicle and magnified in
445
(H′) with a red arrowhead and dashed line indicating a body feather follicle. (I) The stained
446
body skin at E9 with red arrowhead indicating a body feather follicle and magnified in (I′)
447
with a red arrowhead and dashed line indicating a body feather follicle boundary. (J) The
448
stained body skin at E10 with red arrowhead indicating a body feather follicle and magnified
449
in (E′) with a red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1
450
mm for A–J; 200 µm for C′–E′ and I′–J′; 500 µm for H′.
451 452
Fig. 3. Spatial expression pattern of JAK2 during early embryogenesis in chicken skin by
453
whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
454
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a weak
455
hybridization signal of a neck feather follicle and magnified in (C′) with a red arrowhead and
456
dashed line indicating a neck feather follicle. (D) The stained neck skin at E9 with red
457
arrowhead indicating a neck feather follicle and magnified in (D′) with a red arrowhead and
458
dashed line indicating a neck feather follicle boundary. (E) The stained neck skin at E10 with
459
red arrowhead indicating a neck feather follicle and magnified in (E′) with a red arrowhead
460
and dashed line indicating a neck feather follicle. (F) The stained body skin at E6. (G) The
461
stained body skin at E7. (H) The stained body skin at E8 with red arrowhead indicating a
462
weaker hybridization signal of a body feather follicle and magnified in (H′) with a red
463
arrowhead and dashed line indicating a body feather follicle. (I) The stained body skin at E9
464
with red arrowhead indicating a body feather follicle and magnified in (I′) with a red
465
arrowhead and dashed line indicating a body feather follicle boundary. (J) The stained body
466
skin at E10 with red arrowhead indicating a body feather follicle and magnified in (E′) with a
467
red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1 mm for A–J;
468
200 µm for C′–E and H′–J′.
469 470
Fig. 4. Spatial expression pattern of TYK2 during early embryogenesis in chicken skin
471
by whole mount in situ hybridization (WISH). (A) The stained neck skin at E6. (B) The
472
stained neck skin at E7. (C) The stained neck skin at E8 with red arrowhead indicating a weak 18
473
hybridization signal of a neck feather follicle and magnified in (C′) with a red arrowhead and
474
dashed line indicating neck feather follicle. (D) The stained neck skin at E9 with red
475
arrowhead indicating a neck feather follicle and magnified in (D′) with a red arrowhead and
476
dashed line indicating neck feather follicle boundary. (E) The stained neck skin at E10 with
477
red arrowhead indicating a neck feather follicle and magnified in (E′) with a red arrowhead
478
and dashed line indicating neck feather follicle. (F) The stained body skin at E6. (G) The
479
stained body skin at E7. (H) The stained body skin at E8 with red arrowhead indicating a
480
weaker hybridization signal of a body feather follicle and magnified in (H′) with a red
481
arrowhead and dashed line indicating a body feather follicle. (I) The stained body skin at E9
482
with red arrowhead indicating a body feather follicle and magnified in (I′) with a red
483
arrowhead and dashed line indicating a body feather follicle boundary. (J) The stained body
484
skin at E10 with red arrowhead indicating a body feather follicle and magnified in (E′) with a
485
red arrowhead and dashed line indicating a body feather follicle. Scale bars, 1 mm for A–J;
486
200 µm for C′–E′ and H′–J′.
487 488
Fig. 5. Quantitative real-time PCR validation of gene expression levels. (A) Quantitative
489
expression of JAK1 in chicken neck skin at developmental stages E6–E10. (B) Quantitative
490
expression of JAK2 in chicken neck skin from stage E6 to E10. (C) Quantitative gene
491
expression of TYK2 in chicken neck skin from stage E6 to E10. (D) Quantitative expression
492
of JAK1 in chicken dorsal (body) skin from stage E6 to E10. (E) Quantitative expression of
493
JAK2 in chicken dorsal skin from stage E6 to E10. (F) Quantitative gene expression of TYK2
494
in chicken dorsal skin from stage E6 to E10. Result are shown as means ± SEM (n = 6). * p <
495
0.05 and ** p < 0.01 (Student’s t-test).
496 497
Supplemental Fig. 1. Multiple sequence alignment of chicken JAK1 with its orthologs from
498
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
499
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate that 100%,
500
75%, and 50% conservation, respectively.
501 19
502
Supplemental Fig. 2. Multiple sequence alignment of chicken JAK2 with its orthologs from
503
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
504
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate 100%, 75%,
505
and 50% conservation, respectively.
506 507
Supplemental Fig. 3. Multiple sequence alignment of chicken TYK2 with its orthologs from
508
different species. Deduced amino acid sequences were aligned in CLUSTAL X (2.0). Amino
509
acid sequences are highly conserved. Asterisks, two dots, and one dot indicate 100%, 75%,
510
and 50% conservation, respectively.
511 512
Supplemental Fig. 4. Spatial expression pattern of sense probes of JAK1, JAK2 and
513
TYK2 during early embryogenesis in chicken skin. The five developmental stages of
514
chicken embryos from E6 to E10 hybridized with JAK1 sense probes showed no clear
515
positive signals both in neck as shown in figure (A1) E6, (B1) E7, (C1) E8, (D1) E9 and (E1)
516
E10, and body skin as shown in figure (F1) E6, (G1) E7, (H1) E8, (I1) E9 and (J1) E10. The
517
five developmental stages of chicken embryos from E6 to E10 hybridized with JAK2 sense
518
probes showed no clear positive signals both in neck as shown in figure (A2) E6, (B2) E7,
519
(C2) E8, (D2) E9 and (E2) E10, and body skin as shown in figure (F2) E6, (G2) E7, (H2) E8,
520
(I2) E9 and (J2) E10. The five developmental stages of chicken embryos from E6 to E10
521
hybridized with TYK2 sense probes showed no clear positive signals both in neck as shown in
522
figure (A3) E6, (B3) E7, (C3) E8, (D3) E9 and (E3) E10, and body skin as shown in figure
523
(F3) E6, (G3) E7, (H3) E8, (I3) E9 and (J3) E10. Scale bars=1 mm
524
20
525
Table 1. Primers used for cloning. Gene JAK1
Sequence (5′-3′)
Function
F: CGTTGAACAAGACCATCAGG
Cloning
R: GAGCCTCCACTGGATTCCAT JAK2
F: CAGAGGCACAATGTCAGCCAGA R: CACTCAGTGGTTTGTCTCCTCC
TYK2
F: GCACTTCTGTGACTTCCAAGAG R: GTTGAGGATCCGCTTCGCGTTG
534 535
Cloning
Table 2. Primers used for quantitative real-time PCR. Gene JAK1
Sequence (5′-3′)
Function
F: ATCCTTCGCACAGACAACATC
qPCR
R: GCATTCCTGAGCCTTCTTGG JAK2
F: GAGCGTGAGAATGCCACTGAC R: TGGAGGACAGCACTTGATGAAC
TYK2
F: TCTCCTTGGACGTCTCCAATG R: GAAATATCCGCGGTGGGAAAT
GAPDH
F: GAAGGCTGGGGCTCATCTG
qPCR qPCR qPCR
R: CAGTTGGTGGTGCACGATG
536
Cloning
Sequences for primer design span all isoforms from NCBI.
21
526 527 528 529 530 531 532 533
KEY RESOURCE TABLE REAGENT or RESOURCE Antibodies
SOURCE
IDENTIFIER
Anti-Digoxigenin, Fab fragments
Roche
Cat#11093274910
Local chicken farm
(Wuhan, China)
Takara Bio-Rad Invitrogen Invitrogen Roche
Cat#RR047A Cat#172-5124 Cat#1804946 Cat#15596026 Cat#11277073910
Ensemble database
http://asia.ensembl.o rg/Chicken/Search/R esults?q=;site=ense mbl;facet_species=C hicken
This paper This paper
N/A N/A
(Nakamura et al., 2018)
https://mafft.cbrc.jp/ alignment/software/ http://www.iqtree.or g/
Biological Samples
White Leghorn chicken embryos from E6 to E10 Critical Commercial Assays PrimeScript RT reagent kit with gDNA Eraser iTaqTM Universal SYBR Green Supermix Zero Blunt® TOPO® PCR Cloning Kit TRIzol™ Reagent Dig-RNA labeling mix Deposited Data The amino acids sequences of JAK1, JAK2, and TYK2
Primers Primers for cloning, see Table 1 Primer for real-time PCR, see Table 2 Software and Algorithms MAFFT IQ-TREE
Chernomor et al., 2016
Highlights: • • •
Assessment of JAK–STAT pathway genes in skin and feather follicle development. JAK1 contributes to reinforce the presumptive feather follicle primordium. TYK2 supports the arrangement of proximal-distal axis of feather follicles.
Expression patterns of three JAK–STAT pathway genes in feather follicle development during chicken embryogenesis Yingfeng Tao1, Xiaoliu Zhou1, Zhiwei Liu1, Xiaokang Zhang1, Yangfan Nie1, Xinting Zheng1, Shaomei Li1, Xuewen Hu1, Ge Yang1, Qianqian Zhao1, Chunyan Mou1*
1
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, HuaZhong Agricultural University, Wuhan, China
*Correspondence: Chunyan Mou [email protected]
Declaration of interest: The authors declare no competing interests.