Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens

Meng Liu; Guixian Bu; Yiping Wan; Jiannan Zhang; Chunheng Mo; Juan Li; Yajun Wang

doi:10.1210/endocr/bqac073

. 2022 May 18;163(7):bqac073. doi: 10.1210/endocr/bqac073

Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens

Meng Liu ^1,^#, Guixian Bu ^2,^3,^#, Yiping Wan ⁴, Jiannan Zhang ⁵, Chunheng Mo ^6,⁷, Juan Li ⁸, Yajun Wang ^9,^✉

PMCID: PMC9170129 PMID: 35583189

Abstract

In vertebrates, adrenocorticotropin (ACTH), released by the pituitary gland, is a critical part of the stress axis and stress response. Generally, the biosynthesis and secretion of ACTH are controlled by both hypothalamic stimulatory factors and inhibitory factors [eg, ACTH-releasing inhibitory factor (CRIF)], but the identity of this CRIF remains unrevealed. We characterized the neuropeptide B (NPB)/neuropeptide W (NPW) system in chickens and found that NPW could directly target the pituitary to inhibit growth hormone (GH) and prolactin (PRL) secretion via neuropeptide B/W receptor 2 (NPBWR2), which is completely different from the mechanism in mammals. The present study first carried out a series of assays to investigate the possibility that NPW acts as a physiological CRIF in chickens. The results showed that (1) NPW could inhibit ACTH synthesis and secretion by inhibiting the 3′,5′-cyclic adenosine 5′-monophosphate/protein kinase A signaling cascade in vitro and in vivo; (2) NPBWR2 was expressed abundantly in corticotrophs (ACTH-producing cells), which are located mainly in cephalic lobe of chicken pituitary, as demonstrated by single-cell RNA-sequencing, immunofluorescent staining, and fluorescence in situ hybridization; (3) dexamethasone could stimulate pituitary NPBWR2 and hypothalamic NPW expression in chicks, which was accompanied by the decease of POMC messenger RNA levels, as revealed by in vitro and subcutaneous injection assays; and (4) the temporal expression profiles of NPW-NPBWR2 pair in hypothalamus-pituitary axis and POMC in pituitary were almost unanimous in chicken. Collectively, these findings provide comprehensive evidence for the first time that NPW is a potent physiological CRIF in chickens that plays a core role in suppressing the activity of the stress axis.

Keywords: NPW, NPBWR2, pituitary, ACTH, CRIF

In the neuroendocrine system, the hypothalamus-pituitary-adrenal gland (HPA) axis is involved in regulating the stress response and many physical activities (1-4). Neurons in the paraventricular nucleus of the hypothalamus synthesize and secrete corticotropin-releasing hormone (CRH), which can act on the anterior pituitary to stimulate adrenocorticotropin hormone [ACTH, encoded by the POMC gene (proopiomelanocortin)] release, which then promotes glucocorticoid (GC) secretion, principally corticosteroids. The corticosteroids exert a variety of physiological roles (5), and it could feedback on the hypothalamus and pituitary to inhibit CRH and ACTH synthesis/secretion to maintain homeostasis of the HPA axis. Additionally, it was reported that a purified bovine hypothalamus extract could inhibit basal ACTH secretion in rat anterior pituitary cells, strongly suggesting the existence of ACTH-releasing inhibitory factor (CRIF) in the hypothalamus (6, 7). Some peptides, such as somatostatin (SST) and atrial natriuretic peptide, and neurotransmitters, such as dopamine, were also found to attenuate ACTH release in tumor cell lines or in in vivo models but could not do so in normal anterior pituitary cells and are thus unlikely to be a physiological CRIF (8).

The NPB/NPW system consists of neuropeptide B (NPB), neuropeptide W (NPW), and their cognate receptors (NPBWR1 and NPBWR2) (9). The NPB/NPW system has been reported to be widely and mainly expressed in the central nervous system (10-14). After binding their ligands, Gi protein-coupled receptor activation reduces intracellular 3′,5′-cyclic adenosine 5′-monophosphate (cAMP) accumulation, thus driving multiple pharmacological and physiological processes (15-17), including energy homeostasis (15, 18-21), obesity (22, 23), stress (24-26), pain (15, 21, 27-29), sleep (30), feeding (10, 19, 20, 31-33), emotion (15, 21), and social behavior (34, 35). Interestingly, previous intracerebroventricular (ICV) injection studies found that NPB and NPW also participated in regulating the plasma level of hormones, and the actions of NPB and NPW in modulating pituitary hormone secretion were dependent on neuroendocrine signals but did not directly act on the pituitary gland in mammals (24, 36).

Our previous work characterized the NPB/NPW system in chickens (37), and the NPB/NPW system is mainly mediated by NPW through the NPBWR2, which is dependent on reducing intracellular cAMP accumulation (20, 37, 38). Moreover, we found that NPW could serve as an inhibitory secretagogue for GH and PRL via NPBWR2 in the chicken pituitary, which clearly indicates that the working mechanism and physiological roles of the NPB/NPW system in chickens are quite different from those in mammals. In addition, the inhibitory potency of NPW/NPBWR2 on GH/PRL was much weaker than that of SST and cortistatin (37, 39). Considering the surprisingly high messenger RNA (mRNA) level of NPW-NPBWR2 in the chicken hypothalamus-pituitary axis (37), these results cast doubt on whether NPW could regulate the release of other chicken pituitary hormones, including ACTH, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone (40), and the newly identified cocaine- and amphetamine-regulated transcript (41-43) and gastrin-releasing peptide (44). In chickens, both cCRH and cAVT are ACTH secretion/synthesis stimulating factors (45-48). Our present study attempts to investigate whether NPW could act as a physiological CRIF in chickens. Undoubtedly, these findings will not only help us to better understand the roles of the NPB/NPW system in vertebrates and to provide a reference for the study of inhibitory factors of HPA axis activity in poultry but also provide a baseline for clarifying the complicated regulatory network of the HPA axis.

Materials and Methods

Animals and Tissues

Three-week-old, 6-week-old, 10-week-old, 15-week-old, and adult male chickens and male chicken embryos at embryonic days 12, 16, and 19 were purchased from a local commercial company. All animal experiments were performed according to the guidelines provided by the Animal Ethics Committee of Sichuan University.

Chemicals, Peptides, Antibodies, and Primers

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The pharmacological agent forskolin (an adenylate cyclase activator that can elevate intracellular cAMP levels and stimulate pituitary ACTH secretion in chickens) was supplied by Cayman Chemical Company. cNPW23 (the mature peptide form of chicken NPW that can functionally activate NPBWR2, WYKHVASPRYHTVGRASGLLMGV), cCRH (SEEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII) and cAVT (CYIQNCPRG) were synthesized by solid-phase Fmoc chemistry (GL Biochem Ltd, Shanghai, China). The purity of the synthesized chicken peptides was greater than 95% (analyzed by high-performance liquid chromatography), and their structures were verified by mass spectrometry. The chemically synthesized peptides were dissolved to 100 μM in Medium 199 (M199, Gibco) and stored at −80°C. A monoclonal antibody against β-actin was purchased from Cell Signaling Technology, Inc. (catalog no. 4970, RRID : AB_2223172), and a polyclonal antibody against ACTH was purchased from Abcam Company (catalog no. ab74976, RRID: AB_1280736). All primers used in this study (Table 1) were synthesized by the Beijing Genome Institute (BGI) Biological Company.

Table 1.

The primers used in the present study

Gene	Primer sequence, 5’- to -3’	Size, bp
cNPW
Sense	CGCCTCCTGCAGCGCCTGCT	133
Antisense	GCCGTCAGCGCAGCAGAGCGAT
cNPBWR2
Sense	GCTCCTACCTGGACAGCGAT	152
Antisense	GGGAGCCTTGAGGATCACAT
cPOMC
Sense	AGAACAGCAAGTGCCAGGACCT	179
Antisense	TGCTGTTGCGACGGCCGAACTT
β-actin
Sense	CCCAGACATCAGGGTGTGATG	123
Antisense	GTTGGTGACAATACCGTGTTCAAT

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Total RNA Extraction for RNA-sequencing and Assays on Gene Expression

Different tissues including the hypothalamus, pituitary glands, adrenal glands, and various brain regions of 3-week-old chicks, adult chickens, or chickens at other developmental stages were collected. Total RNA was extracted by using RNAzol (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions, dissolved in diethyl pyrocarbonate-treated H₂O, and either sent to the BGI for transcriptome sequencing or reverse transcribed by using M-MLV reverse transcriptase (Takara, Japan) to prepare samples to examine the mRNA levels of target genes. Quantitative real-time PCR assay (qPCR) was conducted with a CFX96 Real-time PCR Detection System (Bio-Rad), as described in our previous study (49).

Effect of cNPW23 on ACTH Secretion and of Dexamethasone on NPBWR2 Expression in Cultured Chick Pituitary Cells

As previously established (39, 50), pituitary cells were cultured at a density of 5 × 10⁵ cells/well in Corning CELLBIND 48-well plates with M199 supplemented with 15% fetal bovine serum at 37°C with 5% CO₂. After 18 hours of culture, the culture medium was removed, and the cells were treated with 200 μL M199 containing the desired dosages of peptide (cNPW23, 1-100 nM) in the presence of cCRH (5 nM), cAVT (10 nM), or forskolin (2 μM). 24 hours later, the cells were collected for RNA extraction and subsequent gene expression detection with β-actin as a reference gene. To prepare samples to examine ACTH secretion, cells were treated for 4 hours. Briefly, the culture medium was collected to measure ACTH secretion, and pituitary cells were lysed in 1 × passive lysis buffer (Promega) to examine the expression levels of ACTH with β-actin as an internal control. In this study, Western blotting was employed to detect protein levels by using antibodies against ACTH (1:2000) or β-actin (1:3000) as previously described (49, 50). For the dexamethasone (DEX) treatment group, cells were treated with 200 μL of M199 containing DEX (0.1-100 nM). After 4, 6, 12, 24, or 48 hours of treatment, cells were collected to isolate total RNA for subsequent transcriptome sequencing or qPCR assays.

Subcutaneous Injection Experiments

Three-week-old chicks were bred in an animal facility equipped with an automatic 12-hour light and dark cycle, and all experiments were conducted between 2 and 4 pm. A drug-free syringe needle was plunged subcutaneously into the abdomen for 7 consecutive days for adaptive training. Thereafter, chicks weighing approximately 300 g were selected randomly and injected subcutaneously with drugs in a volume of 0.5 mL per chick. To investigate the role of NPW in modulating pituitary ACTH secretion/synthesis, chicks were randomly assigned to 9 groups (n ≥ 6) as follows: sham group, control group (saline), cCRH group (5 nM cCRH/100 g), cAVT group (10 nM cAVT/100 g), cNPW23 group (10 nM cNPW23/100 g), cCRH + cNPW23 group (5 nM cCRH and 10 nM cNPW23/100 g), cAVT + cNPW23 group (10 nM cAVT and 10 nM cNPW23/100 g), cAVT + cCRH group (10 nM cAVT and 5 nM cCRH/100 g), and cAVT + cCRH + cNPW23 group (10 nM cAVT, 5 nM cCRH and 10 nM cNPW23/100 g). At 1 hour after injection, the chicks were sacrificed, and pituitary gland samples were collected to prepare for RNA extraction. To detect the effect of GCs on NPW-NPBWR2 expression in the chicken hypothalamus-pituitary axis, the chicks were subcutaneously injected with DEX (0.2 mg/100 g) once a day for 7 continual days as the long-term treatment group (n ≥ 6). For short-term treatment (n ≥ 6), chicks were injected with DEX (0.4 mg/100 g) once and sacrificed 3 hours after injection, and the samples (pituitary and hypothalamus) were collected to prepare RNA samples. All of the plasma samples were collected to measure the concentrations of ACTH (catalog no. RXJ600484CH, RRID: AB_2910566) and corticosterone (catalog no. RXJ600484CH, RRID: AB_2910567) by Quanzhou Ruixin Biotechnology company.

Single-cell Sequencing and RNA Sequencing

Adult chickens were killed, and fresh pituitary tissues were quickly collected, submerged in tissue protection reagent, stored at low temperature, and sent to the BGI for single-cell isolation, preparation, and sequencing analysis as soon as possible. The complimentary DNA (cDNA) library was generated with a 10 × Genomics 3’ Gex V2 kit according to the protocol (Chromic Single Cell 3’ Reagents Kits V2), and the cDNA library was quantitatively analyzed on an Agilent Tapestation system (Agilent Technologies Inc, USA), sequenced and analyzed with a BGI500 (BGI). Before RNA sequencing, an Agilent 2100 Bioanalyzer was used to perform quality detection of the fragment size and concentration of the constructed cDNAs in the library. Quality control analysis of the raw data was carried out with SOAPnuke software to remove impurity data. To reduce the deviation of read values, using Salmon software, we matched and quantitated clean data with the reference transcript of chicken GRCg6a (Gallus_Gallus.GRCg6a.cdna.all.fa) in the ENSEMBL database, converted the read values into transcripts per kilobase per million mapped reads values, used the Tximport function for matching the data to the gene name, and exported the data with R studio. We used the DESeq2 software package to normalize the data for subsequent analysis of differential genes. According to the adjusted P-value (adjusted P-value) < 0.01, up- and downregulated genes were defined as multiple range > 1.4, <−1.4 (as log₂ fold-change > 0.5, <−0.5) as critical value. Finally, the Cluster Profiler R package (51) was used for enrichment analysis.

Immunohistochemical Staining, Immunofluorescent Staining, and Fluorescence In Situ Hybridization

Anterior pituitaries collected from 3-week-old chickens were fixed in 4% paraformaldehyde overnight and embedded in paraffin wax for immunohistochemistry (IHC), immunofluorescence (IF), and fluorescence in situ hybridization (FISH) as described in our previous study (50, 52). Briefly, IHC, IF, and FISH were performed in chicken pituitary sections of 5 μm thickness. A polyclonal antibody against ACTH (1:200) was used to probe the spatial distribution of ACTH protein in the pituitary. Sections incubated with rabbit serum were used as negative controls. The sections were incubated with horseradish peroxidase–conjugated goat anti-rabbit immunoglobin G secondary antibody (1:1000; Thermo Fisher Scientific, catalog no. 32460, RRID: AB_1185567) and treated with a DAB kit (Boster, Wuhan, China) according to the manufacturer’s instructions for IHC and Alexa Fluor-488 secondary antibody (1:1000, Thermo Fisher Scientific, catalog no. A-11008, RRID: AB_143165), followed by counterstaining with diaminopyrolylindole 4,6-diamino, 2-pyrolylindole (DAPI; 1:1000, Thermo Fisher Scientific, catalog no. D1306, RRID:AB_2629482) for IF.

To evaluate whether ACTH and NPBWR2 are coexpressed in cells in the chicken pituitary gland, FISH was used to locate NPBWR2 in the anterior pituitary with a standard in situ hybridization protocol (53). First, the synthetic NPBWR2 mRNA probe was successfully labeled with the digoxigenin tag, and then the probe was visualized by using a tyramide signal amplification plus fluorescence kit (TSA+, PerkinElmer, USA) according to the manufacturer’s instructions. The stained sections were photographed and then prepared for ACTH (1:200) IF with the previously discussed protocol. Finally, sections were viewed under a Leica fluorescence microscope, photographed and processed using DP Controller Manager software.

Data Analysis

The staining intensity of the bands from Western blotting was determined and analyzed using Quantity One software (Bio-Rad), and the relative protein levels were normalized to those of intracellular β-actin were expressed as the percentage compared with the respective controls (without treatment). The relative mRNA level of the target gene was first calculated as the ratio to that of β-actin and then expressed as the percentage compared with the respective controls (without treatment). The data were analyzed by Student’s t-test (for 2 groups) or by 1-way analysis of variance followed by Dunnett’s test using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). To validate our results, all experiments were repeated at least 3 times.

Results

Spatial Expression of NPBWR2 in Chicken Pituitaries

The qPCR results indicate that NPBWR2 mRNA predominantly expressed in the cephalic lobe of chicken pituitaries, which comprise corticotrophs (ie, ACTH-producing cells), as reported previously (54); these results were validated by IHC assays (Fig. 1A-1E). In contrast, GH immunoreactivity was only observed in the caudal lobe (data not shown), as reported by Meng et al (39). Since NPW mRNA was abundant in the chicken hypothalamus [Supplemental Figure2 (55)] (37), and the spatial expression characteristics of NPBWR2 indicated that the hypothalamus of chickens had the ability to secrete NPW peptide, which reached the pituitary gland and then activated its receptor NPBWR2 to exert its functions, such as regulating ACTH synthesis/secretion.

Figure 1. — The location of *POMC* and *NPBWR2* in the chicken pituitary. (A and B) Quantitative polymerase chain reaction was used to examine the messenger RNA (mRNA) expression levels of *POMC* and *NPBWR2* in the caudal (Ca) and cephalic (Ce) lobes of pituitaries from 3-week-old chicks. The relative mRNA level of each gene was first calculated as the ratio to that of *β-actin* and then expressed as the percentage compared with the expression in the Ce lobe. Each data point represents the mean ± SE of the mean of 10 individuals. *******p < 0.001 vs. Ca lobe. (C-E) adrenocorticotropin-immunoreactive cells (dark color) are localized in the Ce lobe of 3-week-old chick pituitaries. Scale bar = 500 μm (C) and 50 μm (D and E).

cNPW23 Inhibits ACTH Synthesis and Secretion in the Chick Pituitary

To further verify the previously discussed hypothesis, in the presence of cCRH or cAVT and both of them together, the qPCR results showed that both 5 nM cCRH and 10 nM cAVT, separately or together, could significantly increase the expression of POMC mRNA in cultured chicken pituitary cells, which was significantly inhibited by cNPW23 (10 nM). We found that the effect of cNPW23 on POMC expression in the absence of CRH and cAVT was not statistically significant (Fig. 2).

Figure 2. — Effects of cNPW23 on *POMC* synthesis in cultured chicken pituitary cells. Quantitative polymerase chain reaction analyses showed that *POMC* synthesis was stimulated by cCRH (5 nM, 24 hours) (A), cAVT (10 nM, 24 hours) (B), and cCRH (5 nM, 24 hours) + cAVT (10 nM, 24 hours) (C) in cultured chicken pituitary cells and could be inhibited by cNPW (10 nM). (D) The effect of NPW (1-10 nM, 24 hours) on *POMC* expression in cultured chicken pituitary cells. The relative *POMC* levels were normalized to those of *β-actin* in pituitary cell lysate and then expressed as a percentage of the levels in the cCRH (A), cAVT (B), cAVT + cCRH (C), or control (D) groups. Each data point represents the mean ± SE of the mean of 4 replicates. *p < 0.05 and **p < 0.01 vs. cCRH (A), cAVT (B), or cAVT + cCRH (C) treatment group.

Then, we detected whether NPW affects ACTH secretion, as shown in Figure 3A and 3B, in cultured chicken pituitary cells, 5 nM cCRH significantly stimulated ACTH secretion, and this stimulatory effect was attenuated by cNPW23 (1-100 nM, 4 hours) in a dose-dependent manner. Meanwhile, we found that even 1 nM cNPW23 could remarkably depress cCRH-stimulated ACTH release in cultured chick pituitary cells. Furthermore, 10 nM cAVT notably stimulated ACTH secretion, and this effect could be inhibited by cNPW23 (1-100 nM, 4 hours); however, this inhibitory effect was not as obvious as that on cCRH-stimulated ACTH release (Fig. 3C and 3D). We also found that cNPW23 inhibited the secretion of ACTH stimulated by the presence of both cCRH and cAVT (Fig. 3E). These results indicated that cNPW23 has a significant inhibitory effect on cCRH- and cAVT-stimulated ACTH secretion.

To clarify whether the cAMP signaling pathway participates in NPW-induced inhibition of pituitary ACTH secretion, we used Western blotting to examine the effect of cNPW23 (10 nM, 4 hours) on pituitary ACTH release in the presence of forskolin (2 μM). Consistent with expectations, forskolin-stimulated ACTH secretion in cultured chick pituitary cells, and this activity was significantly blocked by 10 nM cNPW23 (Fig. 3F).

To verify our hypothesis, the expression of POMC and the ACTH concentration in plasma were detected after subcutaneous injection of different polypeptides into chickens. The results showed that (1) POMC mRNA in the chicken pituitaries was significantly increased after subcutaneous injection of cCRH and cAVT (separately or together) after 1 hour compared with POMC mRNA levels in the control group, while it was slightly decreased after injection of cNPW23, albeit with no significant difference; (2) there was a significant decrease in pituitary POMC expression after simultaneous coinjection of cNPW23 with cCRH and cAVT (separately or together), compared with injection cCRH or cAVT (separately or together) alone (Fig. 4A); (3) after subcutaneous injection, the concentration of ACTH in the plasma was increased by cAVT and cCRH (respectively or together) and could be inhibited by cNPW23 (Fig. 4B) [for the effect on corticosterone, see Supplementary Figure 3 (56)]. These results indicated that NPW could inhibit ACTH in vivo.

Figure 4. — Effects of subcutaneous injection of polypeptides on the expression of *POMC* in the pituitary and adrenocorticotropin (ACTH) in plasma in 4-week-old chickens. Subcutaneous injection with different polypeptides: cNPW23 [10 nM/100 g bodyweight (BW)/day, 0.5 mL], cCRH (5 nM/100 g BW/day, 0.5 mL), and cAVT (10 nM/100 g BW/day, 0.5 mL) 1 hour later, the results of the expression of *POMC* in the pituitary gland (A) and the concentration of ACTH in plasma (B). Each data point represents the mean ± SE of the mean of 6 replicates. **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01, and ***###***p < 0.001 vs. the cCRH, cAVT, or cAVT + cCRH treatment group.

Coexpression of NPBWR2 and ACTH in Chicken Pituitary

In our previous studies, we found that NPW could regulate pituitary ACTH secretion through its specific receptor, NPBWR2. To obtain more direct evidence, single-cell sequencing was used to identify whether NPBWR2 and POMC colocalized in chicken pituitary glands (n = 4) (Fig. 5). By analyzing the expression of POMC and NPBWR2 in chicken pituitary cells, it was found that POMC was abundantly expressed in the chicken pituitary gland (Fig. 5A and 5D, red), whereas many cells expressed NPBWR2 (Fig. 5B and 5E, green); astonishingly, many cells showed coexpression (Fig. 5C and 5F, yellow).

Figure 5. — UMAP maps showing POMC and NPBWR2 coexpression in chicken pituitary cells. Dark red and green indicate high expression levels. *POMC* (A and D, red) and *NPBWR2* (B and E, green) expression in chicken pituitary cells was examined by single-cell sequencing, and the coexpression of *POMC* and *NPBWR2* is indicated by yellow fluorescence (C and F).

To more intuitively explain the colocalization of NPBWR2 and ACTH in chicken pituitary cells, we carried out morphological experiments (Fig. 6). First, the the pituitary pituitary tissue was embedded in paraffin and sliced into 5-μm thick sections, and synthetic NPBWR2 mRNA probe successfully bound to pituitary gland sections from 3-week-old chickens (the red area indicates the localization of the NPBWR2 mRNA probe, and white arrows indicate the positive cells) (Fig. 6B, 6F, 6J, and 6N). Subsequently, the IF experiment was continued on the same section. The location of ACTH-immunoreactive cells was marked by ACTH antibody (the green area indicates ACTH-immunoreactive cells, and white arrows show the positive cells) (Fig. 6C, 6G, 6K, and 6O). All sections were later counterstained with DAPI (the blue area is the cell nucleus stained with DAPI) (Fig. 6A, 6E, 6I, and 6M). The merged image shows that NPBWR2 and ACTH expression are colocalized in cells (the yellow area shows coexpressed cell localization, and white arrows show coexpressed cells) (Fig. 6D, 6H, 6L, and 6P). Other cells expressing ACTH or NPBWR2 were also found. The expression of NPBWR2 or ACTH in the caudal lobe of chicken pituitary gland cells was minimal. These results indicated that NPBWR2 was expressed in the cephalic lobe of chicken pituitary glands and that ACTH was also expressed in the same region; that is, NPBWR2 and ACTH are coexpressed in cells, which means that NPW is potentially a CRIF.

Figure 6. — cNPBWR2 and adrenocorticotropin (ACTH) colocalize in chicken pituitary glands. The fluorescence in situ hybridization results showed *cNPBWR2* expression in the cephalic lobe (Ce) of chicken pituitary cells (B, red). Immunofluorescence showed ACTH cells located in the cephalic lobe (Ce) of chicken pituitary cells (C, green). The merged figures showed that *cNPBWR2* and ACTH were coexpressed in cells (D, yellow). The nuclei were stained with diaminopyrolylindole 4,6-diamino, 2-pyrolylindole (A, blue). (E-H) was enlarged images of (A-D). (IH and L) *cNPBWR2* and ACTH expression in the caudal lobe (Ca) was low to nonexistent. (M-P) was enlarged images of (I-L). Scale bar = 100 μm (A-D, I-L) or 50 μm (E-H, M-P).

DEX Stimulates NPW-NPBWR2 Expression in the Chicken Hypothalamus-Pituitary Axis

These results suggest that NPW is an important factor inhibiting ACTH secretion/synthesis in chickens and that its function is mainly mediated by NPBWR2. Then, we treated cultured pituitary cells with DEX for 6, 12, 24, and 48 hours and detected the relative expression of genes by qPCR (Fig. 7). It was found that 100 nM DEX significantly increased the expression of NPBWR2, which was significantly higher at 6 hours and gradually diminished at 12 to 48 hours (but was still significant) compared with the expression in the control (Fig. 7A). Meanwhile, it was also found that 100 nM DEX had an obvious inhibitory effect on POMC expression after treatment for 6 hours (POMC mRNA expression was approximately 75% that of the control), which was more pronounced after 12 hours and maintained this increase at 24 and 48 hours (Fig. 7B). Afterward, we selected 6 hours as the treatment time and detected the expression of NPBWR2 and POMC after treatment with different concentrations of DEX (0.1-100 nM). We found that 0.1 nM DEX significantly increased the expression of NPBWR2 and showed an intensive dose-dependent effect at increasing concentrations (Fig. 7C). Moreover, we found that the inhibitory effect of DEX on POMC expression was significant at 1.0 nM DEX but did not increase at higher concentrations of DEX (10 nM and 100 nM) (Fig. 7D). To further verify the aforementioned conclusion, cultured chicken pituitary cells were treated with 10 nM DEX for 4 and 24 hours, and then samples were collected for transcriptome sequencing, the results of which showed that 10 nM DEX could significantly stimulate the expression of NPBWR2 and inhibit the expression of POMC in cultured chicken pituitary cells at both time points (Fig. 7E and 7F). In addition, 10 nM DEX had no significant effect on NPW and NPB expression (Fig. 7G and 7H).

Figure 7. — Expression of the *NPW-NPBWR2* system after glucocorticoid treatment in cultured chicken pituitary cells. Dexamethasone (DEX; 100 nM) had an obvious stimulatory effect on *NPBWR2* expression (A) and an inhibitory effect on *POMC* expression (B) after treatment for 6 to 48 hours. DEX (0.1 nM-100 nM, 6 hours) stimulated *NPBWR2* expression (C) and inhibited *POMC* expression (D). Each data point represents the mean ± SE of the mean of 4 replicates. **p < 0.01 vs. control group. Transcriptome sequencing results showed that 10 nM DEX stimulated *NPBWR2* gene expression (E) but inhibited *POMC* (F), *NPW* (G), and *NPB* (H) gene expression after treatment for 4 or 24 hours. Each data point represents the mean ± SE of the mean of 4 replicates. **p < 0.01 vs. control group. The RNA-sequencing results of *NPBWR2* (E) and *POMC* (F) in the pituitary gland and *NPW* (G) and *NPB* (H) in the hypothalamus were shown, and the gene expression patterns were presented as transcripts per kilobase per million mapped reads values. The P-value was based on the adjusted P-value. Genes that were not differentially expressed were defined as not significant.

Additionally, subcutaneous injection of DEX was carried out to verify whether GCs had a negative feedback regulating effect on the NPW-NPBWR2 system in vivo. The results showed that DEX [4 mg/kg body weight (BW)] had no significant effect on the expression of POMC in the chicken pituitary in the short-term group (3 hours after injection), but the expression of NPBWR2 was significantly increased compared with that in the control group (8.6-fold). Meanwhile, after treatment with DEX (2 mg/kg BW) for 7 days in the long-term group, the expression of POMC in the chicken pituitary decreased to approximately 60.1%, and the expression of NPBWR2 was increased by approximately 18.4-fold (Fig. 8A and 8B). In addition, we found that the expression of NPW in the hypothalamus was increased approximately 2.3 times to that in the control group in the long-term group (Fig. 8C). Meanwhile, we also detected the concentrations of ACTH and corticosterone in the plasma and showed that ACTH and corticosterone were inhibited by injection with DEX both in the short- and long-term groups (Fig. 8D and 8E), suggesting that ACTH and corticosterone could be inhibited by GCs. These results indicated that GCs could regulate the expression of POMC and NPBWR2 in the chicken pituitary gland, suggesting that GCs could control the expression of the NPW-NPBWR2 system in the hypothalamus and pituitary glands.

Figure 8. — Effect of subcutaneous injection of glucocorticoid on the hypothalamus-pituitary-adrenal gland axis in 4-week-old chickens. The expression of *NPBWR2* (A) and *POMC* (B) in the pituitary gland and *NPW* (C) in the hypothalamus after subcutaneous injection of dexamethasone [0.4 mg/100 g body weight (BW)/day, 0.5 mL for short-term evaluation (3 hours); 0.2 mg/100 g BW/day, 0.5 mL for long-term evaluation (7 days)]. The concentrations of adrenocorticotropin (D) and corticosterone (E) in plasma after subcutaneous injection of dexamethasone. Each data point represents the mean ± SE of the mean of 6 replicates. *p < 0.05, **p < 0.01, and *******p < 0.001 vs. control group.

Temporal Expression Profiles of NPW-NPBWR2 in the Chicken Hypothalamus-Pituitary Axis

Using transcriptome sequencing, we detected the expression of NPW, NPBWR2, and POMC in multiple tissues from chickens. The results showed that NPW was widely distributed in chickens but mainly highly expressed in the hypothalamus (Fig. 9A), similar to previous work, whereas POMC was highly expressed in the pituitary gland (Fig. 9E). Meanwhile, we found that the NPW-specific receptor NPBWR2 was also mainly localized to and highly expressed in the pituitary glands of chickens (Fig. 9C).

Figure 9. — Temporal expression atlas of *NPW-NPBWR2* on the hypothalamus-pituitary-adrenal gland axis in chickens. The expression of *NPW* (A), *NPBWR2* (C), and *POMC* (E) in multiple chicken tissues as detected by transcriptome sequencing (n = 8). The expression of *NPW* in chicken hypothalamus (B) and of *NPBWR2* (D) and *POMC* (F) in chicken pituitaries at different developmental stages, including embryonic (E) day 12, 16, and 19; 3-, 6-, 10-, 15-week-old; and adult (Ad) chickens. (B, D, F) The messenger RNA (mRNA) levels were examined by quantitative polymerase chain reaction, and the relative mRNA level of each gene was first calculated as the ratio to that of *β-actin* (as an internal control) and then expressed as the percentage compared with that of adult chickens. Each data point represents the mean ± SE of the mean of 8 individuals.

The mRNA expression levels of NPW in the hypothalamus and NPBWR2 and POMC in the pituitary gland from embryos to adults were examined using qPCR. As shown in Figure 9B, in the chicken hypothalamus, NPW mRNA expression was minimal at embryonic day 12 (E12), increased from E16 to E6 weeks of age after hatching (6 weeks), and was maintained at a high level through adulthood. Meanwhile, we noticed that the mRNA level of NPW slightly decreased at 10 weeks, peaked at 15 weeks, and then decreased in adults. In the developing chicken pituitary glands, the expression level of NPBWR2 remained minimal at E12 but increased significantly from E16 to E19 and was maintained at a high level from E16 to adulthood. Although the NPBWR2 mRNA level in the pituitary gland decreased temporarily at 3 weeks, similar to hypothalamic NPW mRNA, a slight decrease was noted at 10 weeks, and its strongest expression was observed at 15 weeks (Fig. 9D). Interestingly, as shown in Figure 9F, in the chicken pituitary gland during embryonic development, the temporal expression atlas of POMC was found to be almost completely in accordance with the expression of NPW in the hypothalamus and NPBWR2 in the pituitary gland. POMC was expressed as early as E12 but was extremely low, but its expression level surged from E16 to E19, slightly fluctuated between 3 and 10 weeks, peaked at 15 weeks, and was maintained at a high level in adults (Fig. 9F). Collectively, this evidence suggests that the temporal profiles of NPW in the hypothalamus are similar to those of NPBWR2 and POMC in the pituitary gland in chickens.

Discussion

As a negative signal from the hypothalamus, CRIFs are important for inhibiting the activity of the HPA axis. However, years of research on identifying CRIFs have still not made substantial progress. In vitro studies of CRIFs found that rat prepro-thyrotropin-releasing hormone (TRH)-(178-199), released from TRH prohormone, was capable of inhibiting ACTH secretion in the mouse AtT-20 cell line (57, 58), rat anterior pituitary cell (57, 59), and human tumor corticotrophs (59), thus raising the possibility that this peptide might function as a physiological CRIF. However, these findings were refuted by Nicholson et al, because no significant inhibitory effect of prepro-TRH-(178-199) on basal or stimulated ACTH secretion was noted in normal pituitary corticotrophs (60). Moreover, the amino acid sequence alignment of TRH precursors showed that the peptide fragments corresponding to rat prepro-TRH-(178-199) were not conserved among species [Supplementary Figure 1 (61)], clearly demonstrating that this non-TRH peptide was unlikely to be the physiological CRIF in numerous species. Additionally, dopamine and some polypeptides, such as SST and atrial natriuretic peptide, can reduce ACTH activity in tumor cell lines or in vivo, but no similar effect has been found in normal anterior pituitary cells, so they were not considered a physiological CRIF (8).

It was reported that NPW could modulate the neuronal activities of the paraventricular nucleus of the hypothalamus through the autonomic nervous system, particularly under stress-related conditions (62). In contrast to our findings in chickens, although NPW was reported to modulate the HPA axis in mammals, it was found that ICV (25) and intraperitoneal injection (63) of NPW or NPB could increase plasma ACTH and corticosterone concentrations in rats, suggesting a stimulatory role of NPB/NPW in regulating the HPA axis. However, in rats, these NPB/NPW-related effects were blocked by a CRH antagonist, indicating that the action of NPW on the HPA axis may be related to the function of the CRH receptor. Since NPBWR2 was lost in rats, the functions of NPB/NPW were mediated only by NPBWR1, suggesting that the differentiated effects in modulating the HPA axis between chickens and rats might be relevant to the distinct functional receptor subtype. ICV injection of NPB/NPW stimulated plasma corticosterone levels, which could be partially eliminated by CRH antiserum, suggesting that NPB/NPW-mediated regulation of HPA axis activity is the result of neural signals in the hypothalamus but does not directly target the pituitary gland in rats. This notion has also been verified in cultured rat pituitary cells, since NPB/NPW did not affect ACTH secretion in vitro. In this study, we found that NPW is potent in impeding the synthesis and secretion of ACTH by directly acting on pituitary NPBWR2 in chickens, indicating that NPW might be a potential CRIF in chickens. Soon afterward, this speculation was partially confirmed by in vivo injection assays, since it was proven that NPW could effectively mediate the inhibition of ACTH and corticosterone levels in chicken plasma. Taken together, these results clearly indicate that the actions and functional routes of the NPB/NPW system are distinct between chickens and mammals.

Both CRH and AVT could induce pituitary hormone ACTH release in chickens (49), and we found that NPW can inhibit the activity of the HPA axis by directly suppressing pituitary ACTH secretion and synthesis in chickens. In a previous study, it was reported that the action of CRH on ACTH release is mediated by the cAMP/protein kinase A (PKA) signaling pathway in rat pituitary cells (64). In this study, we demonstrated that NPBWR2, activated by binding NPW, reduces the cAMP levels stimulated by cCRH/cAVT in the chicken pituitary gland and subsequently decreases stimulating factor-induced ACTH secretion. These data indicated that the roles of NPW in inhibiting ACTH synthesis/secretion were mediated by the cAMP/PKA signaling pathway in chicken pituitary cells. Considering the widespread expression of NPW in chicken tissues, it was speculated that the suppressive effect of NPW on ACTH secretion/synthesis might be triggered by endocrine and autocrine/paracrine routes, similar to the inhibitory mode of GH/PRL release.

Transcriptome analysis and single-cell sequencing showed that POMC and NPBWR2 were abundantly expressed and exhibited a high level of coexpression in adult chicken pituitary cells. Furthermore, we observed that the NPBWR2 mRNA signal was mainly and profoundly coexpressed with the ACTH-immunopositive signal in chicken pituitary cells. In other words, NPBWR2 was predominantly and abundantly expressed in chicken pituitary corticotrophs. Using transcriptome sequencing, we noted that NPW was widely expressed in multiple tissues in chickens, including the hypothalamus, whereas NPBWR2 and POMC were highly expressed in the pituitary; this is different from the expression pattern observed in mammals but similar to our previous qPCR results in chickens (37, 65). Afterward, temporal expression profiles of hypothalamic NPW, pituitary NPBWR2, and POMC in chickens were also detected in our study. As mentioned previously, there was a striking increase in the number of corticotrophs on E14 (66). Accordingly, we found that POMC mRNA levels were extremely low at E12, increased abruptly at E16, and maintained a high expression level from E16 to adulthood (Fig. 9). Interestingly, the temporal expression profiles of the NPW-NPBWR2 pair in the hypothalamus-pituitary axis were found to be approximately synchronous with those of POMC in the pituitary; collectively, this evidence further demonstrated that NPW was the likely physiological CRIF in chickens.

In the HPA axis, adrenal gland cortex cells secrete GCs that have feedback regulation on the hypothalamus and pituitary glands (59), such as suppressing the secretion/synthesis of CRH in the hypothalamus and ACTH in the pituitary; these feedback loops are the key to maintaining the stability of HPA axis activity (67, 68). Combined with the previously discussed results, the in vitro and in vivo experimental results all confirmed that GCs could feedback to increase the inhibitory signal mediated by NPW-NPBWR2, thereby attenuating the activity of the chicken HPA axis, further confirming that NPW is the physiological CRIF in chicks. Interestingly, these results were also consistent with a previous finding in rat stomach mucosa (13), clearly demonstrating that GCs might enhance the inhibitory signal not only in an endocrine manner but also in an autocrine/paracrine manner to regulate the chicken HPA axis. Nonetheless, the drawback was that these experiments were only the results of acute reactions, and further research is needed to further explore the regulation of NPW on ACTH and adrenal gland function in vivo.

All of the previously discussed experimental results from our present studies clearly indicated that NPW has the potential to act as a physiological CRIF in chickens (Fig. 10). In chicken corticotrophs, hypothalamic NPW could directly target the pituitary to inhibit CRH-/AVT-induced ACTH synthesis/secretion via an NPBWR2-triggered decrease in intracellular cAMP levels. As a negative feedback signal, GCs produced from the adrenal gland could increase NPW-NPBWR2 expression in the hypothalamus-pituitary axis, thus depressing chicken HPA activity. In addition, similar to its role in suppressing GH/PRL release, NPW signaling from other peripheral tissues may inhibit ACTH synthesis/secretion in chickens in an endocrine and autocrine/paracrine manner. Nevertheless, in rats, it has been reported that NPB/NPW acts on the hypothalamus and modulates the release of neuroendocrine signals to the anterior pituitary rather than directly affecting hormone secretion in the pituitary (24, 25, 63, 69-72); this varies from NPB/NPW activity in chickens, suggesting the possibility that the NPB/NPW system might exert different roles via distinct routes in different species. However, our recent study revealed that the roles of the NPB/NPW system were mediated by NPW through its receptor NPBWR2 in chickens. Given the absence of an NPBWR2 analog in some rodents (eg, rats), the results obtained in these rodent studies on NPB/NPW system-related roles may not be sufficient as a reference for all species, especially in animals that express an NPBWR2 analog (9). Interestingly, NPBWR2 is present in a large number of species, raising the possibility that NPW might be the physiological CRIF in these species, including humans, pigs, or fish (73, 74), and even in some NPBWR2-containing rodents, such as guinea pigs (75, 76).

Figure 10. — The proposed model for NPW activity on the chicken pituitary in regulating adrenocorticotropin (ACTH) secretion. In chicken corticotrophs, hypothalamic corticotropin-releasing hormone (CRH) and arginine vasotocin (AVT) strongly stimulated ACTH secretion via activation of CRHR1 and AVPR coupled to AVPR1A/1B, respectively, which activate the 3′,5′-cyclic adenosine 5′-monophosphate (cAMP)/protein kinase A signaling pathway. As a potential chicken corticotropin-releasing inhibitory factor, hypothalamic NPW was able to inhibit CRH- or AVT-induced ACTH secretion, and this inhibitory action was likely mediated by NPBWR2, whose activation could decrease intracellular cAMP levels induced by CRH and subsequently depress ACTH secretion. The release of ACTH to the adrenal gland cortex stimulated the release of corticosteroid into the blood, which in turn played a negative feedback regulatory role in the release of hormones for the pituitary gland and hypothalamus.

In summary, we provide the first in vivo and in vitro evidence that NPW can inhibit pituitary ACTH synthesis/secretion via NPBWR2-mediated inhibition of the cAMP/PKA signaling cascade. Single-cell RNA-sequencing, IF staining, and FISH found that NPBWR2 was expressed abundantly in corticotrophs (ACTH-producing cells) and located mainly in the cephalic lobe of the chicken pituitary. DEX could stimulate NPW-NPBWR2 in the hypothalamus-pituitary of chickens, which was accompanied by a decrease in pituitary POMC mRNA levels. Moreover, we also found that the temporal expression profiles of the NPW-NPBWR2 pair in the hypothalamus-pituitary axis and POMC in the pituitary were almost identical in chickens. Taken together, this evidence clearly shows that NPW can act as a physiological CRIF in chickens and could directly act on the chicken pituitary to suppress ACTH synthesis/secretion via NPBWR2-mediated cAMP/PKA signal inhibition. Undoubtedly, this discovery not only allows us to reconstruct the regulatory mechanism of the HPA axis in chickens, but also provides a clue to the search for physiological CRIF in other species, such as humans.

Acknowledgments

This work was supported by National Natural Science Foundation of China (U1901206, 32072706, 32072705, 31702111) and Applied Basic Research Project from Science & Technology Department of Sichuan Province (2019YJ0146, 2020YJ0127, 2019YJ0021).

Contributor Information

Meng Liu, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.

Guixian Bu, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China; College of Life Science, Sichuan Agricultural University, Ya’an, China.

Yiping Wan, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.

Jiannan Zhang, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.

Chunheng Mo, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China; Key Laboratory of Birth Defects and Related Diseases of Women and Children of Ministry of Education, State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University, Chengdu, China.

Juan Li, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.

Yajun Wang, Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China.

Author Contributions

M.L., G.B., Y.W., J.Z., C.M., and J.L. conducted the experiments. M.L. and G.B. joined the analysis and interpretation of data. M.L. and G.B. designed and drafted the manuscript. Y.W. revised the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare that there are no conflicts of interest that could have appeared to influence the work reported in this paper.

Data Availability Statement

Some or all data sets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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63. Hochol A, Belloni AS, Rucinski M, et al. Expression of neuropeptides B and W and their receptors in endocrine glands of the rat. Int J Mol Med. 2006;18(6):1101-1106. [PubMed] [Google Scholar]
64. Aguilera G, Harwood JP, Wilson JX, Morell J, Brown JH, Catt KJ. Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells. J Biol Chem. 1983;258(13):8039-8045. [PubMed] [Google Scholar]
65. Bu G, Cui L, Lv C, et al. Opioid peptides and their receptors in chickens: structure, functionality, and tissue distribution. Peptides. 2020;128:170307. [DOI] [PubMed] [Google Scholar]
66. Jozsa R, Scanes CG, Vigh S, Mess B. Functional differentiation of the embryonic chicken pituitary gland studied by immunohistological approach. Gen Comp Endocrinol. 1979;39(2):158-163. [DOI] [PubMed] [Google Scholar]
67. Allen DB. Growth suppression by glucocorticoid therapy. Endocrinol Metab Clin North Am. 1996;25(3):699-717. [DOI] [PubMed] [Google Scholar]
68. Baxter JD. Mechanisms of glucocorticoid inhibition of growth. Kidney Int. 1978;14(4):330-333. [DOI] [PubMed] [Google Scholar]
69. Shimomura Y, Harada M, Goto M, et al. Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8. J Biol Chem. 2002;277(39):35862-35869. [DOI] [PubMed] [Google Scholar]
70. Hochol A, Tortorella C, Ricinski M, Ziolkowska A, Nussdorfer GG, Malendowicz LK. Effects of neuropeptides B and W on the rat pituitary-adrenocortical axis: in vivo and in vitro studies. Int J Mol Med. 2007;19(2):207-211. [PubMed] [Google Scholar]
71. Ziolkowska A, Rucinski M, Tyczewska M, Malendowicz LK. Neuropeptide B (NPB) and neuropeptide W (NPW) system in cultured rat calvarial osteoblast-like (ROB) cells: NPW and NPB inhibit proliferative activity of ROB cells. Int J Mol Med. 2009;24(6):781-787. [DOI] [PubMed] [Google Scholar]
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73. Ye C, Xu S, Hu Q, et al. Global view of neuropeptides and their receptors in the brain and pituitary of grass carp (Ctenopharyngodon idellus). Aquaculture. 2019;512:7343601-7343622. [Google Scholar]
74. Yang L, Sun C, Li W. Neuropeptide B in Nile tilapia Oreochromis niloticus: molecular cloning and its effects on the regulation of food intake and mRNA expression of growth hormone and prolactin. Gen Comp Endocrinol. 2014;200:27-34. [DOI] [PubMed] [Google Scholar]
75. Scanes CG. Opening a new door: neuropeptide W (NPW) is a novel inhibitory secretagogue for GH and prolactin acting via the Gi protein-coupled NPBWR2. Endocrinology. 2016;157(9):3394-3397. [DOI] [PubMed] [Google Scholar]
76. Yang S, Ma Z, Suo C, Cheng L, Su J, Lei Z. Cloning and mRNA expression of NPB and its effect on hormone secretion of the reproductive cells in the pig. Gen Comp Endocrinol. 2018;261:97-103. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Some or all data sets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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[CIT0076] 76. Yang S, Ma Z, Suo C, Cheng L, Su J, Lei Z. Cloning and mRNA expression of NPB and its effect on hormone secretion of the reproductive cells in the pig. Gen Comp Endocrinol. 2018;261:97-103. [DOI] [PubMed] [Google Scholar]

PERMALINK

Evidence for Neuropeptide W Acting as a Physiological Corticotropin-releasing Inhibitory Factor in Male Chickens

Meng Liu

Guixian Bu

Yiping Wan

Jiannan Zhang

Chunheng Mo

Juan Li

Yajun Wang

Abstract

Materials and Methods

Animals and Tissues

Chemicals, Peptides, Antibodies, and Primers

Table 1.

Total RNA Extraction for RNA-sequencing and Assays on Gene Expression

Effect of cNPW23 on ACTH Secretion and of Dexamethasone on NPBWR2 Expression in Cultured Chick Pituitary Cells

Subcutaneous Injection Experiments

Single-cell Sequencing and RNA Sequencing

Immunohistochemical Staining, Immunofluorescent Staining, and Fluorescence In Situ Hybridization

Data Analysis

Results

Spatial Expression of NPBWR2 in Chicken Pituitaries

Figure 1.

cNPW23 Inhibits ACTH Synthesis and Secretion in the Chick Pituitary

Figure 2.

Figure 3.

Figure 4.

Coexpression of NPBWR2 and ACTH in Chicken Pituitary

Figure 5.

Figure 6.

DEX Stimulates NPW-NPBWR2 Expression in the Chicken Hypothalamus-Pituitary Axis

Figure 7.

Figure 8.

Temporal Expression Profiles of NPW-NPBWR2 in the Chicken Hypothalamus-Pituitary Axis

Figure 9.

Discussion

Figure 10.

Acknowledgments

Contributor Information

Author Contributions

Conflict of Interest

Data Availability Statement

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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