Non-Canonical Maternal WNT4 Activates Canonical Zygotic WNT8C for Early Embryonic Development in Chicken

TL;DR

This study shows how maternal WNT4 activates embryonic WNT8C in chickens, helping to establish body patterns during early development.

Contribution

The study reveals a novel maternal-to-zygotic WNT signaling relay in chickens involving WNT4 and WNT8C.

Findings

Maternal WNT4 activates zygotic WNT8C via the JNK pathway in chicken embryos.

WNT8C then activates the canonical WNT/β-catenin pathway necessary for normal development.

Blocking maternal WNT signaling disrupts proper WNT8C activation and embryonic polarity.

Abstract

Early embryonic development requires precise coordination between maternal factors stored in the egg and genes that activate in the developing embryo. WNT signaling proteins play a particularly important role during the maternal-to-zygotic transition; however, the process in birds has remained unclear. This study examined WNT signaling in chicken embryos, revealing that two distinct maternal WNT proteins, WNT4 and WNT6, are predominantly expressed during the early stages of chicken development. By blocking maternal WNT signaling in eggs before laying, the researchers found that a key embryonic WNT protein, WNT8C, failed to activate properly. Further experiments revealed that maternal WNT4 triggers WNT8C expression via the JNK signaling pathway. WNT8C then activates the WNT/β-catenin pathway, which is necessary for normal development. These findings reveal how maternal signals are converted into embryonic gene activity in birds and suggest that this maternal WNT4-to-zygotic WNT8C relay helps establish the embryonic body pattern in chicken.

The proper regulation of signaling pathways, including WNT signaling, during early embryonic development is critical for whole-organism development. In particular, maternally enriched WNTs play critical roles in cell cleavage and axis formation through non-canonical and canonical pathways during early embryogenesis. However, early developmental processes related to maternal WNTs and their underlying mechanisms have remained unstudied in avian species. In this study, we investigated WNT signaling-mediated early development in the chicken embryo. We found that WNT4 and WNT6, different ligands from other species, exhibited expression patterns consistent with maternal enrichment in chicken. The chemical inhibition of maternal WNT signaling in intrauterine embryos led to aberrant zygotic expression of WNT8C, which is important for primitive streak formation. Combined with in vitro functional studies, we demonstrated that WNT4 increased WNT8C expression through the non-canonical JNK pathway and that WNT8C subsequently promoted the canonical β-catenin pathway. Our results indicate that maternal WNT4 activates zygotic WNT8C and potentially regulates embryonic polarity in chicken.

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1. Introduction

Coordinated cell–cell communication is essential for embryonic development in multicellular organisms. Among the major signaling pathways governing developmental processes, WNT signaling represents one of the most fundamental and highly conserved regulatory mechanisms controlling embryogenesis across metazoan species. It orchestrates critical processes, including axis formation, cell fate specification, tissue patterning, and morphogenetic movements. [1,2]. Highly conserved WNT signaling plays central roles in the embryonic development of both vertebrate and invertebrate species, including Lytechinus variegatus, Danio rerio, Mus musculus, Xenopus laevis, and Homo sapiens [3,4,5,6]. WNT signaling operates through two major pathways: canonical and non-canonical. The canonical pathway involves β-catenin stabilization and nuclear translocation, where β-catenin interacts with TCF/LEF transcription factors to activate target gene expression. The non-canonical pathway acts independently of β-catenin and is further divided into the WNT/planar cell polarity (WNT/PCP) and WNT/calcium pathways [7,8,9]. In the WNT/PCP pathway, small GTPase molecules such as Rho, Rac, Cdc42, and Rap1, along with the Jun N-terminal kinase (JNK) pathway, act downstream of non-canonical WNT signaling, leading to cytoskeleton remodeling [10,11,12,13,14,15,16].

The maternal-to-zygotic transition (MZT) represents a complete reprogramming of the newly formed embryo. This process includes the coordinated clearance of maternal transcripts and proteins coupled with chromatin remodeling to enable zygotic genome activation (ZGA). ZGA represents the handover of developmental control from maternally inherited gene products to the embryo’s own genome. This transition occurs through precisely regulated and highly interconnected mechanisms [17,18]. During this critical transition, the WNT signaling pathway participates in diverse developmental events during embryogenesis. Maternally contributed Wnt5 and Wnt11 initiate axis formation in the early Xenopus embryo, and maternally provided β-catenin plays an important role in establishing Spemann’s organizer in frogs and fish [17,18,19,20,21]. The WNT-dependent stabilization of proteins (WNT/STOP) pathway, which is generally considered a β-catenin-independent WNT branch, plays a role in cell cycle progression via maternal WNT during early Xenopus embryogenesis [21,22]. Small GTPases control spindle formation, cell division, and cytokinesis during early embryonic development in Caenorhabditis elegans, Danio rerio, and Sus scrofa [23,24,25]. Moreover, JNK functions in non-canonical WNT signaling to regulate convergent extension movements and gastrulation in Xenopus [14,15] and activates the transcription factors cJun and ATF2 [26,27]. Together, these findings indicate that WNT pathway is involved in various early embryogenesis processes across diverse species.

Avian species have served as valuable research models for embryonic development because it is easy to observe the developmental process within their eggs and they provide practical advantages for experimental manipulation [28,29]. Although advances have been made in studies of post-ovipositional development, poor accessibility has historically limited opportunities for research on intrauterine-stage development [30,31,32,33]. Early embryonic development is critical for lineage segregation and pre-gastrulation development [34]. Therefore, elucidating the molecular mechanisms of intrauterine embryonic development is necessary to provide fundamental information about early avian embryogenesis.

Embryonic development in chicken, a widely used avian model, is classified into the Hamburger and Hamilton (HH) stages in the post-oviposition period and the Eyal-Giladi and Kochav (EGK) stages in the pre-oviposition period. The morphological criteria of the EGK stages cover the intrauterine stages, of which there are 10: from EGK.I to EGK.X [35,36]. In chicken, a wide range of events during embryonic development are associated with multiple signaling pathways, including WNT signaling. For example, WNT8C, Crescent, and Dkk1 are involved in primitive streak formation with cVg1 [37,38]. β-catenin, the main determinant of the canonical WNT pathway, exhibits nuclear localization at the periphery of the chicken embryo from the EGK.VIII to EGK.XI stage [38,39]. Recent RNA sequencing-based studies have revealed several WNT signal pathway genes in chicken intrauterine embryos; in particular, WNT4 and WNT6 with maternal expression patterns show downregulated expression and are replaced by zygotic WNT ligands after EGK.VIII [31,32]. In mammals, combinatorial BMP/WNT signaling controls primitive streak versus extraembryonic fate decisions through the temporal integration of signal duration and intensity, while spatial WNT waves balanced by secreted inhibitors regulate primitive streak formation and patterning [40,41]. However, detailed reports on the expression profiles and exact roles of WNT pathway genes during intrauterine development in avian species have remained limited.

In this report, we examined the detailed function of maternal WNTs during early chicken embryogenesis. The exact downstream pathway of maternal WNTs in chicken was identified by in vivo and in vitro examinations. Our results suggest that WNT8C is a downstream target activated through the JNK pathway by maternal WNT4 and that it is potentially associated with primitive streak formation in chicken. This study provides a basis for understanding WNT-related molecular activity in early avian embryogenesis.

2. Methods

2.1. Experimental Animals and Animal Care

The care and experimental use of White Leghorn (WL) chickens was approved (SNU-150827-1 and SNU-250226-4) by the Institutional Animal Care and Use Committee of Seoul National University, Republic of Korea. All procedures, including chicken maintenance, feeding, reproduction, treatment, and sample collection, adhered to the standard operating protocols of the Animal Genetic Engineering Laboratory at Seoul National University.

2.2. Collection of Intrauterine Eggs and Oocytes from Hens

About 42–59-week-old WL hens were used for the collection of intrauterine eggs and oocytes. Intrauterine eggs were retrieved from WL hens by an abdominal massage technique slightly modified from Eyal-Giladi and Kochav [35]. Briefly, the abdomen of hens was pushed gently until exposure of the shell gland, and the surface of the shell gland expanded when an egg was located there for eggshell formation. After expansion of the surface of the shell gland, massage was used to move the egg gently toward the cloaca until the intrauterine egg was released. All the collected intrauterine embryos were classified according to the morphological criteria of EGK staging. To collect oocytes, WL hens were sacrificed and follicles were collected. The harvested embryos and oocytes were used for RT-PCR, RT-qPCR, and in situ hybridization.

2.3. In Situ Hybridization and Paraffin Section

To make hybridization probes, the total RNA from blastodermal stage embryos was reverse-transcribed, and the cDNA was amplified using the WNT4, WNT6, WNT8C, and EOMES primers (Table S1). The PCR products of the correct size were cloned into the pGEM-T vector (Promega, Madison, WI, USA). After sequence verification, the recombinant plasmids containing the gene were amplified using T7- and SP6- specific primers (T7: 5′-TAA TAC GAC TCA CTA TAG GG-3′ and SP6: 5′-ATT TAG GTG ACA CTA TAG-3′) to prepare the template for labelling the hybridization probes. The digoxigenin (DIG)-labelled sense and antisense transcript hybridization probes were transcribed in vitro using a DIG RNA labelling kit (Roche Diagnostics, Indianapolis, IN, USA). The preparation of hybridization probes and whole mount in situ hybridization were performed following the standard protocol for chickens [42,43]. For longitudinal sections, the intrauterine embryos were embedded in paraffin and sectioned at 5 µm using a HM 355S automatic microtome (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The results of in situ hybridization were visualized and photographed under a Ti-U fluorescence microscope (Nikon, Tokyo, Japan).

2.4. Niclosamide Treatment in Hens

Hens were treated with 50 mg/kg of niclosamide (Sigma, St. Louis, MO, USA) dissolved in Kolliphor EL (Sigma) and diluted in ethanol. Control birds received equal volumes of the vehicle. The niclosamide or the vehicle was injected once every two days into the abdominal cavity of the chickens. The expression of WNT8C in every EGK.X embryo of control and treatment hens was examined by in situ hybridization until 15 days of niclosamide administration. The EGK.X embryos of hens at subsequent days of niclosamide administration were also examined for the expression of WNT8C by in situ hybridization. The oocytes and intrauterine embryos of control and niclosamide-treated chickens were subjected for the expression of WNT signaling-pathway genes by RT-PCR, RT-qPCR, in situ hybridization, and Western blotting until 33 days of niclosamide administration. Throughout the treatment period, hens were monitored for general health, reproductive indices (laying rates, egg quality), and behavior. No significant differences in these parameters were observed between niclosamide-treated and control groups through day 33 of administration, though morphological defects in intrauterine embryos became apparent from day 15 onward.

2.5. RT-PCR and RT-qPCR Analyses

Total RNA was extracted from each sample with Trizol reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s protocol. The RNA quantity was determined by spectrophotometry at 260 nm, and 1 µg of each RNA sample was reverse-transcribed with the Superscript III First-Strand Synthesis System (Invitrogen). The cDNA was diluted fivefold and quantitatively equalized for PCR amplification. RT-PCR and RT-qPCR were conducted to examine the expression of candidate WNT signaling-pathway genes in intrauterine embryos using specific primers along with the chicken beta actin gene (ACTB) for normalization (Table S2). RT-PCR was performed in a Bio-Rad thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), and then the PCR product was loaded onto 1.0% agarose gel for electrophoresis. RT-qPCR was performed using a CFX96 real-time PCR detection system with a C1000 thermal cycler (Bio-Rad Laboratories), and the relative quantification of gene expression was analyzed by the 2^−ΔΔCt^ method [44].

2.6. Western Blot Analysis

Crude protein was isolated from early embryos or DF-1 cells by dissociation in RIPA lysis buffer (Thermo Fisher Scientific) with a protease inhibitor (Sigma) and phosphatase inhibitor (Sigma). Approximately 2 μg of protein was used in each lane for separation in a 15% SDS-PAGE gel. The protein was transferred onto a Hybond 0.45 PVDF membrane (GE Healthcare Bio-sciences, Little Chalfont, UK) and blocked with 5% skim milk (Sigma) for one hour at room temperature. Subsequently, the blocked membrane was incubated overnight at 4 °C for primary antibody attachment with 1:1000 dilution in 3% BSA and 0.05% sodium azide in PBS. Horseradish peroxidase conjugated secondary antibody (Thermo Fisher Scientific) was attached with 1:4000 dilutions in 3% BSA in PBS at room temperature for one hour. The following primary antibodies were used: anti-MYC (MBL life science, Woburn, USA), anti-P-JNK, anti-JNK (Cell Signaling Technology, Danvers, MA, USA), β-catenin (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-α-tubulin (Abcam, Cambridge, UK). Immunoreactive proteins were visualized using an ECL western blotting detection system (GE Healthcare Bio-sciences). All blots were performed in duplicate, and protein levels were quantitated with ImageJ v1.54 (National Institutes of Health, Bethesda, MD, USA) [45].

2.7. Transfection of WNT Overexpressing Vectors and Drugs Treatment in DF-1

DF-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 1× antibiotic/antimycotic at 37 °C with 5% CO_2_. Approximately, 6 × 10^5^ DF-1 cells/well were seeded in a six-well plate containing 2 mL of culture medium overnight. The cells were transfected with WNT4 and WNT6 overexpression vectors under a CMV promoter using the Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) transfection system according to the manufacturer’s protocol. Simultaneously, DF-1 cells were transfected with empty vectors as the control. In addition, the transfection medium containing WNT4 overexpression vectors was supplemented with the following drugs at the indicated concentration: SP600125 (10 µM) (Sigma). 18 h after drug treatment, control and experimental cells were harvested and subjected to RT-PCR, RT-qPCR, and Western blotting analyses.

2.8. Knockdown Analysis Through siRNA Transfection

After WNT4 overexpression in DF-1 cells, the cells (6 × 10^5^) were transfected with two siRNAs (100 pmol/L) designed for the knockdown of WNT8C. The sense and antisense oligos used for the knockdown of WNT8C are shown in Table S3. The siRNAs were transfected using the RNAiMAX (Invitrogen) transfection system according to the manufacturer’s protocol. Simultaneously, parental DF-1 cells were also transfected with the two siRNAs targeting WNT8C. Negative control siRNA with no complementary sequence in the chicken genome was used as a control. The siRNA-treated cells were analyzed after 24 h by RT-PCR, RT-qPCR, and immunocytochemistry.

2.9. Immunocytochemistry

Control and treated DF-1 cells fixed in 4% paraformaldehyde were washed with 1× PBS two times for 5 min each. The cells were blocked with 5% (v/v) goat serum in PBS for 1 h at room temperature. The samples were then incubated overnight at 4 °C with β-catenin primary antibody diluted 1:200 in blocking buffer. The samples were then incubated for 1 h at room temperature with secondary antibodies labelled with fluorescein isothiocyanate (FITC). Washed samples were mounted with ProLong Gold antifade reagent and 4′, 6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) and visualized under a Ti-U fluorescence microscope (Nikon, Tokyo, Japan).

2.10. Statistical Analyses

Significant differences between the two groups were examined statistically using Student’s t-test (two-tailed). One-way analysis of variance (ANOVA) was followed by Tukey’s post hoc test for multiple comparisons. A p value < 0.05 was considered to indicate statistical significance (*** p < 0.001, ** p < 0.01, and * p < 0.05).

3. Results

3.1. Expression Profiling of WNT Signaling Pathway-Related Genes in Chicken Intrauterine Embryos

We collected chicken oocytes and intrauterine embryos (EGK.I, EGK.III, EGK.VI, EGK.VIII, and EGK.X) (Figure 1a) based on the criteria of Eyal-Giladi and Kochav [35,36]. All samples were subjected to reverse-transcription polymerase chain reaction (RT-PCR), quantitative reverse-transcription PCR (RT-qPCR), and in situ hybridization to determine the expression profiles of 11 candidate WNT signaling pathway genes identified by RNA sequencing of early chicken embryos in our previous study [31]. As shown in Figure 1b, WNT ligands WNT4 and WNT6 and the WNT ligand receptor FZD1 were expressed from the oocyte to EGK.VI stages and then downregulated after EGK.VIII; this expression pattern is consistent with a maternal origin. The expression levels of other WNT ligand genes, such as WNT8C, WNT3A and WNT5B, were not detected in oocytes but were observed after EGK.VIII and increased consistently until EGK.X. Moreover, CTNNB1 (β-catenin), LEF1, and FZD7 showed an expression pattern similar to that of the previous genes. The RT-PCR and RT-qPCR expression results were correlated for the WNT genes in the samples examined (Figure 1b–d). These results indicate that WNT pathway components can be categorized as maternal or zygotic WNTs. WNT4, WNT6, and FZD1 were categorized as maternal WNTs (Figure 1b,c). WNT8C, WNT3A, WNT5B, CTNNB1, LEF1, FZD7, WNT5A, and WNT11 were categorized as zygotic WNTs (Figure 1b,d).

3.2. Localization of WNT4 and WNT6 in Chicken Intrauterine Embryos

To observe the expression patterns of oocytes and early embryos, in situ hybridization of whole-mount embryos and longitudinal sections was conducted with WNT4 and WNT6 mRNA transcripts (Figure 2). Consistent with previous RT-PCR and RT-qPCR results, WNT4 mRNA was detected in high amounts in oocytes (Figure 2a,a’). Thereafter, ooplasmic determinants of WNT4 formed along the lines in the EGK.I embryo (Figure 2b,b’) and were found in cellularization from EGK.III (Figure 2c,c’). Before oviposition, WNT4 exhibited a heterogeneous expression pattern in all cell layers at EGK.VI (Figure 2d,d’). WNT4-positive (black arrows) and -negative cells (red arrows) were well-distributed throughout all layers. These transcripts were continuously detected during the cleavage period and early area pellucida formation (EGK.I–EGK.VI). As development progressed, WNT4 was predominantly expressed on the outer side of the embryo, the future area opaca, and the marginal zone at EGK.VIII (Figure 2e,e’). At EGK.X, WNT4 mRNA expression was rarely detected (Figure 2f,f’). WNT6 mRNA expression was detected near the oocyte germinal vesicle (Figure 2g,g’). Before EGK.VI, maternal ooplasmic determinants of WNT6 were found during cellularization (Figure 2h,h’,i,i’). However, they became localized in a heterogeneous manner in the EGK.VI embryo (Figure 2j,j’) and then gradually disappeared until the EGK.X stage (Figure 2k,k’,l,l’) in a similar manner to WNT4. Collectively, these results imply that WNT4 and WNT6 transcripts exhibited maternal expression patterns and were expressed heterogeneously during early embryonic development in chicken.

3.3. Maternal WNTs Control Transcriptional Regulators, JNK and β-Catenin, in Chicken Intrauterine Embryos

To observe WNT functions during intrauterine development in chicken, hens were treated with intraperitoneal injections of niclosamide, which globally blocks the upstream WNT signaling by inhibiting interaction between WNT ligands and FZD receptors [46]. Niclosamide was administered once every 2 days, and oocytes and intrauterine embryos were collected from vehicle- and niclosamide-treated hens. Niclosamide treatments were well tolerated by the chickens, with no significant differences between the groups in dropout due to side effects. Previous studies have shown that maternally inherited WNTs activate axis formation in other species [19,20] and that WNT8C is strongly expressed in the marginal zone of EGK.X embryos and controls post-ovipositional development [37]. Accordingly, we used WNT8C as a marker to examine the effects of niclosamide on the normal development of EGK.X embryos. WNT8C mRNA expression and localization began to alter in the EGK.X embryos of niclosamide-treated hens by day 5 and completely diminished from day 15 compared to the control (Figure 3a). This abnormal expression of WNT8C and morphological damage were observed in EGK.X embryos of hens treated with niclosamide at 17 days (Figure 3b). The reduced expression level of EOMES in the EGK.X stage and later inferred aberrant development at the HH7 stage were observed in the embryos of niclosamide-treated hens (Figure 3c–e). We examined WNT gene expression in EGK.X embryos of niclosamide-treated hens using RT-PCR and RT-qPCR (Figure 3f,g). The results showed that the zygotic WNT ligand (WNT8C), non-canonical WNT pathway genes (cJUN and ATF2), and canonical WNT pathway gene (LEF1) were all downregulated in EGK.X embryos after niclosamide treatment compared to the control group, whereas the expression level of genes related to mTOR signaling, which is another target of niclosamide [47], was not significantly different in EGK.X stage embryos after niclosamide treatment (Figure S1). These results suggest that niclosamide treatment in hens blocked maternal WNT, as well as further development.

Abnormal intrauterine embryos were observed in chickens that received niclosamide from day 15 (Figure 4a). Western blot analysis was conducted to investigate the downstream molecular interaction of maternal WNTs and zygotic WNT8C in the intrauterine embryos of chickens that received niclosamide. As non-canonical JNK signaling and canonical β-catenin can activate transcriptional responses by WNTs in Drosophila and mammalian cells [7,48,49], we examined the expression profiles of JNK and β-catenin in oocytes and EGK.VI, EGK.VIII, and EGK.X embryos. JNK and β-catenin were detected from the oocyte to EGK.X stages; however, JNK phosphorylation was first detectable after EGK.VI (Figure 4b). Next, to confirm the effects of blocking maternal WNTs by niclosamide, we examined the expression difference between control- and niclosamide-treated embryos. In the oocyte, JNK and β-catenin expression did not differ between the control and niclosamide treatment groups (Figure 4c). After fertilization, JNK was consistently expressed in control and treated samples during the intrauterine stage, but levels of JNK phosphorylation decreased in niclosamide-treated embryos at all stages (Figure 4d–f). β-catenin expression did not differ between control and niclosamide-treated embryos until EGK.VI but was downregulated from the EGK.VIII stage (Figure 4d–f). Collectively, these results indicate that the expression levels of JNK phosphorylation and β-catenin were controlled by the inhibition of maternal WNTs during early chicken embryogenesis, and these downstream pathways could increase zygotic WNT8C gene activation by maternal WNTs.

3.4. Chicken WNT4 Regulates Both Non-Canonical and Canonical WNT Pathways, as Well as WNT8C Expression In Vitro

Next, we investigated the genetic regulation of zygotic WNT ligand expression by maternal WNTs. Owing to the practical difficulties in functional studies of pre-oviposited embryos, we manipulated the WNT pathways following the overexpression of WNT ligands to identify the vertical relationship among WNT ligands in chicken DF-1 cells as an alternative. First, to determine which maternal WNT ligands could activate the zygotic expression of WNT8C, we treated DF-1 cells with WNT4 or WNT6 overexpression vectors (Figure 5a and Figure S2a). RT-PCR and RT-qPCR detected the overexpression of WNT4 and WNT6. We also found that WNT4 overexpression slightly increased WNT6 mRNA expression, but the reverse did not occur (Figure S2b,c). WNT8C mRNA expression was increased significantly only in WNT4-overexpressed DF-1 cells (Figure S2b,c). These results demonstrate that only maternal WNT4 acts as a major activator of zygotic WNT8C in chicken.

We then investigated downstream non-canonical and canonical WNT pathway genes in WNT4-overexpressed DF-1 cells. Western blotting analysis detected cMyc, confirming WNT4 protein expression (Figure 5b). The phosphorylated JNK pathway and β-catenin were significantly upregulated as WNT4 was overexpressed (Figure 5b,c), which is consistent with the upregulation of WNT8C expression (Figure 5d). The expression levels of cJUN and ATF2, downstream transcription factors of the non-canonical JNK pathway, were upregulated 9.76- and 2.16-fold, respectively, following WNT4 overexpression (Figure 5e). The expression of LEF1, a downstream transcription factor of the canonical pathway, increased 4.85-fold following WNT4 overexpression (Figure 5e). Collectively, these results indicate that WNT4 can induce WNT8C expression in DF-1 cells via non-canonical and canonical WNT pathways.

3.5. WNT4-Induced WNT8C Promotes Canonical β-Catenin Signaling

Our overexpression results implied that WNT4 can induce both non-canonical and canonical WNT signaling. However, previous studies have indicated that canonical β-catenin signaling can be activated by WNT8C in the area opaca [37,39,50]. To further confirm the role of WNT4 in the activation of the canonical WNT pathway, we conducted a small interfering RNA (siRNA)-mediated knockdown assay. We used two target siRNAs to knock down WNT8C in WNT4-overexpressed DF-1 cells. The RT-PCR results showed that only WNT4-overexpressed DF-1 cells, with or without control siRNA, expressed WNT8C (Figure 6a). RT-qPCR results showed that two target-siRNAs significantly decreased WNT8C expression, i.e., a 72% decrease by siRNA #1 and a 70% decrease by siRNA #2, compared to that of WNT4-overexpressed control DF-1 cells (Figure 6b). After the maximum knockdown of WNT8C was achieved using siRNA #1 in WNT4-overexpressed DF-1 cells, we examined canonical pathway activity via LEF1 gene expression by RT-qPCR and β-catenin localization by immunocytochemistry to determine whether the canonical WNT pathway was affected by WNT8C knockdown. WNT8C knockdown in WNT4-overexpressed cells significantly reduced the expression level of LEF1 (62%) compared to that of control cells (Figure 6c). Compared to DF-1 cells, β-catenin was localized to the nucleus in WNT4-overexpressed cells; however, after WNT8C knockdown, β-catenin moved to the cytoplasm (Figure 6d). Taken together, these results indicate that WNT8C is a molecular component downstream of WNT4 and that the canonical WNT pathway is mediated by WNT8C in chicken.

3.6. WNT4 Activates WNT8C Expression Through the JNK Pathway in Chicken

According to our in vivo and in vitro results, we posit a new mechanism by which maternal WNT4 may activate the JNK pathway and induce the expression of zygotic WNT8C. To validate whether the JNK pathway induces zygotic WNT8C in WNT4-overexpressed DF-1 cells, we employed a chemical JNK pathway inhibitor, SP600125 [51]. We first performed Western blot analysis to confirm WNT4 overexpression and SP600125 activity. SP600125 effectively downregulated JNK phosphorylation without altering JNK expression compared to WNT4-overexpressed DF-1 cells (Figure 7a,b). The mRNA expression level of WNT8C was also significantly decreased in WNT4-overexpressed DF-1 cells treated with SP600125, as determined by RT-PCR and RT-qPCR (Figure 7c). Together, these data suggest that the WNT/JNK pathway is the downstream activator, via WNT4, of zygotic WNT8C expression in chicken.

4. Discussion

In this study, we examined the role of maternal WNTs in the early embryogenesis of chicken. According to the expression patterns in oocyte- and intrauterine-stage embryos, expressed WNT ligands can be divided into two groups: maternal and zygotic WNTs. In chicken, WNT4 and WNT6 exhibit maternal expression patterns; these differ from the Xenopus embryo, for example, in which Wnt5 and Wnt11 are maternally contributed to directly specify the dorsal axis and early embryonic development [19,20]. In zebrafish, maternal wnt8a plays a critical role in establishing dorsoventral polarity through β-catenin stabilization [52]. Zygotic WNTs in chicken, such as WNT5A, WNT5B, WNT11, and WNT8C, were upregulated during area pellucida formation. β-catenin, an important mediator of WNT signaling, was also upregulated after EGK.VIII. This upregulation is crucial for modulating future axis formation, potentially through interactions with zygotic WNTs that have orthologs in other species, playing similar roles. In mice, WNT4 and WNT6 are known to be involved in Mullerian duct formation and extraembryonic endoderm formation, respectively [53,54,55]. The chicken’s utilization of WNT4 and WNT6 as the predominant maternal ligand may be associated with its intermediate position among those species in terms of evolution. This species-specific variation in maternal WNT identity reflects evolutionary adaptations to distinct developmental strategies. In avian species, the rapid clearance of maternal transcripts during MZT may result in the transition to alternative zygotic WNT ligands for resistance to zygotic microRNAs [32]. Despite utilizing different WNT ligands, these vertebrate lineages converge on the common developmental logic of maternal-to-zygotic WNT transitions governing early axis formation.

Maternal WNT ligands are expressed in the α ooplasm from the oocyte stage [56] and participate in cellularization until EGK.III. Maternal WNT transcript expression occurred at peak levels at EGK.VI in a “salt-and-pepper” manner, which is typical of lineage segregation [57], as development progressed. These maternal WNT ligands were only expressed on the outer side of the embryo, at reduced levels, and replaced with zygotic WNT ligands, especially WNT8C, after EGK.VIII. This temporal transition reflects the broader MZT program that orchestrates the handover from maternal to embryonic genome control across vertebrates through coordinated mechanisms of maternal transcript clearance and zygotic genome activation [17]. This process was probably mediated by the miR-302 family during maternal-to-zygotic transition [32]. According to the expression pattern, which is similar to that of maternally-inherited genes in Danio rerio [58] and Xenopus laevis [59], WNT4 and WNT6 exhibit characteristics consistent with a predominantly maternal origin. However, definitive classification requires the transcriptional inhibition of zygotic expression using agents such as α-amanitin [60,61] or allele-specific expression analysis [62,63], as employed in previous studies. These approaches remain technically challenging for chicken intrauterine embryos due to their inaccessibility and fragility. A previous allele-specific expression study in chicken successfully identified other maternal genes but did not detect WNT4 or WNT6 [63], due to the absence of SNPs in these regions. Our maternal classification therefore relies on converging circumstantial evidence: high expression in oocytes, progressive decline coinciding with MZT timing (EGK.VI-VIII), presence before the major wave of zygotic genome activation, and expression dynamics matching those of confirmed maternal transcripts in other vertebrates [56,57]. These results imply that the transition of WNT ligands occurs during intrauterine development in chicken.

The expression of WNT8C was completely decreased in the EGK.X of hens at the 15th day of niclosamide treatment. The time point at which the effects of niclosamide fully appeared was similar to the total 17 days required for the development from the primordial follicle to the ovulatory follicle in chickens [64]. This may imply that the accumulated administration of niclosamide from the small follicle is needed to regulate maternal factors. Niclosamide may have pleiotropic effects beyond WNT inhibition. These effects include actions on mitochondrial oxidative phosphorylation, STAT3 signaling, and mTOR-independent pathways in hens [47], which complicates definitive mechanistic attribution. However, our in vivo inhibition study results indicate that maternal WNT ligands could regulate the expression of zygotic WNT8C, as well as canonical and non-canonical WNT pathways, whereas the expression levels of genes related to mTOR signaling, which is another target of niclosamide, were unchanged, probably because this pathway was not involved in early chicken embryogenesis [31]. Moreover, the convergence of our in vivo niclosamide results with independent in vitro findings, including WNT4 overexpression-induced WNT8C upregulation, WNT8C knockdown-mediated β-catenin relocalization, and JNK inhibitor effects, strengthens the interpretation that observed developmental defects probably result from WNT pathway disruption.

Maternal WNTs-blocked embryos by niclosamide failed to show orderly cellularization and primitive streak formation. The WNT8C-mediated canonical pathway is a potential regulator of primitive streak formation in chicken [37,65]. During avian gastrulation, primitive streak formation involves coordinated tissue flows creating distinct cellular compartments that direct mesendoderm precursors to the midline [66]. These modular morphogenetic processes demonstrate remarkable plasticity and can be reconfigured by manipulating pathways [67]. Nuclear β-catenin was first localized to the periphery of the embryo and to the future area opaca after EGK.VIII [39]; small GTPase pathway-related genes were expressed during the chick cleavage stage [31]. Previous reports have clarified that a non-canonical WNT pathway, such as maternal WNT/STOP signaling and small GTPase signaling, can regulate cell division and cell cycle progression in various species [8,21,23,24,25]. These findings suggest that maternal WNT ligands in the chicken embryo may control both the non-canonical pathway during cell division and the canonical pathway that prepares the embryonic context for polarity establishment. WNT8C serves as a critical molecular marker at the late intrauterine and pre-primitive-streak stage [38], but primitive streak formation also requires additional factors, including Brachyury, Chordin, and other canonical signaling components [37]. Our observation of reduced EOMES expression and aberrant development at HH7 following niclosamide treatment supports the interpretation that disruption of the maternal WNT pathway cascade impairs the preparatory events necessary for normal post-oviposition development. However, the comprehensive mechanistic links between intrauterine WNT signaling and primitive streak morphogenesis remain to be fully elucidated. Moreover, maternal WNTs have been shown to directly regulate WNT8C expression for future embryonic development in a different manner in Xenopus, i.e., to regulate embryonic axis patterning by the direct effect of maternal WNTs [19,20].

In addition, our data suggest that the maternal WNTs-JNK-WNT8C cascade operates during the pre-primitive streak stage to establish developmental competence for subsequent axis formation, rather than directly specifying primitive streak identity. The JNK signaling pathway and β-catenin are transcriptional regulators that may be involved in the regulation of WNT8C expression [7,48]. In chicken, β-catenin translocates to the nucleus after EGK.VIII [39], and JNK signaling is activated after EGK.VI. We confirmed that both JNK phosphorylation and β-catenin can be downregulated in early embryos by niclosamide treatment. Furthermore, our in vitro analysis revealed that only WNT4 drives the upregulation of JNK phosphorylation and WNT8C, as well as the nuclear localization of β-catenin. According to the siRNA-mediated knockdown results, WNT8C switched to the β-catenin canonical pathway following stimulation by maternal WNT4 in chicken. In findings consistent with those of previous studies of WNT8C and β-catenin in chicken [37,39], we demonstrate that chicken maternal WNT4 could stimulate the canonical pathway through zygotic WNT8C expression to further regulate embryonic development at EGK.VIII. β-catenin subcellular localization following JNK inhibition was not examined. This represents a partial characterization of the WNT4-JNK-WNT8C-β-catenin cascade, and the specific effect of the JNK pathway blockade on β-catenin nuclear translocation remains to be directly demonstrated in the future. Furthermore, elucidating the direct role of c-JUN/ATF2 in regulating WNT8C will complete this cascade. On the other hand, it does not appear that WNT6 is involved in the regulation of WNT8C. WNT6 may play redundant roles with WNT4, contribute to distinct spatial or temporal functions not captured in our assays, or serve auxiliary roles during specific developmental windows. Future loss-of-function approaches targeting WNT6 specifically will be necessary to resolve its individual contribution to early chicken embryogenesis. While the in vitro DF-1 cell system does not fully recapitulate the complex signaling dynamics of intrauterine embryonic tissues, it has been demonstrated to be useful for demonstrating WNT4’s capacity to activate JNK and β-catenin pathways cell-autonomously.

Collectively, the activation of the JNK pathway by WNT4 induces zygotic WNT8C expression. Although previous studies have demonstrated the influence of crosstalk between non-canonical and canonical pathways on β-catenin stability [68,69,70], our results offer a new perspective on the direct transcriptional relationship between WNT ligands in early embryogenesis in avian species. This mechanism resonates with findings that combinatorial BMP/WNT signaling controls primitive streak fate decisions through the temporal integration of signal duration and intensity, while spatial WNT waves balanced by secreted inhibitors regulate primitive streak patterning extent and duration [40,41]. These studies collectively support the concept that morphogen signaling operates through complex spatiotemporal integration mechanisms during early vertebrate development. We therefore conclude that WNT4 triggers the expression of WNT8C, which can be mediated by WNT/JNK signaling during intrauterine development, to prepare for the primitive streak formation after oviposition. However, the direct evidence of maternal WNT functions remains to be revealed through the specific inhibition of maternal WNT ligands or underlying pathways, since niclosamide affects cellular metabolism globally. Unlike other model animals, the manipulation system for early embryogenesis in avian species has not been established until now. Future studies should overcome these technical limitations of early embryo manipulation in chickens by conducting gene-specific functional studies in vitro using avian pluripotent stem cells [71] rather than the DF-1 cell line and by applying recent embryo modeling technologies [72,73,74].

5. Conclusions

Our findings demonstrate that WNT4 and WNT6, different ligands from other species, exhibit maternal expression patterns and are expressed during the intrauterine stage in chicken. The WNT signaling pathway regulates post-ovipositional development, resulting in a transition from the non-canonical to the canonical pathway in the EGK.VIII embryo in chicken. Furthermore, we propose that maternal WNT4 potentially regulates future embryonic polarity in an avian-specific manner through the induction of zygotic WNT8C ligands via the WNT/JNK pathway (Figure 8). Our results provide a new perspective on the role of maternal WNTs in activating zygotic WNT expression during embryonic development, contributing to the growing understanding of how maternal factors orchestrate the maternal-to-zygotic transition and how evolutionarily conserved signaling pathways are deployed in species-specific developmental contexts.