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. 2026 Jul 19;122(3):e70194. doi: 10.1002/arch.70194

Temporal Dynamics of Extracellular Signal‐Regulated Kinase Phosphorylation and Associated Signaling Genes During Embryonic Development in Bombyx mori

Shi‐Hong Gu 1,✉
PMCID: PMC13380809  PMID: 42471771

ABSTRACT

This study investigated the temporal dynamics of extracellular signal‐regulated kinase (ERK) phosphorylation, together with the expression profiles of its putative upstream ligands and downstream target genes, during embryonic development in Bombyx mori. The results revealed distinct ERK phosphorylation dynamics: significantly higher levels of phosphorylated ERK were observed between Days 1 and 6 after oviposition in developing eggs (non‐diapause and HCl‐treated eggs), whereas diapause eggs exhibited low levels. The temporal expression patterns of genes in the prothoracicotropic hormone (PTTH)/torso and epidermal growth factor (EGF) signaling pathways—spitz (spi), rhomboid (rho), and Egf receptor (Egfr)—did not differ between diapause and HCl‐treated eggs. In contrast, the EGF pathway gene vein (vn) and genes in the transforming growth factor (TGF)‐β signaling pathway—including the ligand myoglianin (myo) and its receptor babo—displayed distinct temporal expression patterns. Similarly, Jelly belly (Jeb)/anaplastic lymphoma kinase (Alk), fibroblast growth factor (Fgf)/Fgf receptor (Fgfr), neurotrophin (NT), and Toll‐receptor ligands (spätzle family members (spz2, spz4, and spz5)) showed differential expression, with levels significantly elevated during the middle and late stages of embryonic development in HCl‐treated eggs. Non‐diapause and chilled eggs also exhibited increasing expression patterns of these genes, similar to those observed in HCl‐treated eggs. Additionally, ERK target genes pointed and huckebein (hkb) showed higher and broader expression peaks in developing eggs compared to diapause eggs. This study provides a comprehensive analysis of the transcriptional regulation of upstream and downstream ERK signaling components during embryonic development and reveals new insights into the mechanisms underlying embryonic diapause.

Keywords: embryonic diapause, FGF, huckebein, Jeb/Alk, MAPK, pointed, RTK, TGF‐β

Upstream signals regulate silkworm embryonic development through phosphorylation‐dependent signaling networks. Dashed lines indicate pathways investigated in the present study, including transcriptional upregulation of the TGF‐β, Fgf, Jeb, NT, Egf, and Toll signaling pathways, as well as the ERK target genes pointed and hkb, in developing eggs.

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Summary

  • Expression of vn, myo, Jeb/Alk, Fgf/Fgfr, NT, and spzs is elevated in developing eggs.

  • ERK target genes (pointed and hkb) have higher and broader peaks in developing eggs.

  • This study provides the first comprehensive analysis of ERK‐related genes during B. mori embryogenesis.

1. Introduction

To cope with harsh and unpredictable environmental conditions, animals have evolved a range of physiological and developmental strategies that enhance survival during periods of stress, such as extreme temperatures, desiccation, or prolonged shortages of food and water (Hahn and Denlinger 2007, 2011; Schebeck et al. 2024; Izadi 2025). In insects, one of the most important adaptive responses to such environmental challenges is diapause. Diapause is a hormonally regulated state of developmental arrest accompanied by profound physiological adjustments, including metabolic suppression and increased tolerance to environmental stress (Hand et al. 2016; Denlinger 2023). This strategy is widespread across insect taxa and can occur at different stages of the life cycle, including the egg, larval, pupal, and adult stages, depending on the species (Koštál 2006; Denlinger 2022).

In the silkworm Bombyx mori, embryonic diapause occurs during the early stages of development and is one of the best‐characterized diapause systems in insects (Yamashita and Yaginuma 1991; Xu et al. 1995; Shiomi et al. 2015). The induction of this developmental arrest is maternally controlled by diapause hormone (DH), a neuropeptide produced in the subesophageal ganglion during the pupal stage of the female moth. After secretion into the hemolymph, DH acts on the ovaries and induces physiological changes that prepare the developing oocytes for diapause (Yamashita 1996).

In a recent study, we examined transcriptional changes associated with the initiation of diapause (Gu and Lin 2026). Our results showed that the glycerol‐3‐phosphate dehydrogenase 1 (Gpdh1) gene—encoding an NAD+‐dependent enzyme involved in glycerol synthesis (Mráček et al. 2013)—is strongly upregulated in diapause‐destined eggs and likely contributes to the initiation of embryonic diapause. Our results further suggest that diapause‐destined females undergo pronounced metabolic reprogramming during the pupal stage. In particular, reduced insulin signaling, together with increased expression of Gpdh1, bombyxin‐Z1, trehalose transporter 1 (Tret1), and trehalase 2 (Treh2), is associated with enhanced glycogen accumulation in the ovaries. This metabolic shift may prime developing oocytes for diapause by promoting elevated Gpdh1 expression in the eggs produced by these mothers, thereby facilitating the initiation of embryonic diapause (Gu and Lin 2026).

Upon diapause initiation, embryonic development is arrested at the G2 phase of the cell cycle, after the formation of the cephalic lobe and telson and the segmentation of mesodermal tissues (Nakagaki et al. 1991). Under constant conditions of 25°C, this dormant state can be maintained for up to 1 year. Diapause termination can be induced by exposing the eggs to low temperatures (approximately 5°C) for 2–3 months; embryonic development then resumes when the eggs are returned to 25°C (Yamashita and Yaginuma 1991; Moribe et al. 2001).

Alternatively, diapause can be prevented experimentally by treating diapause‐destined eggs approximately 20 h after oviposition with hydrochloric acid (HCl; specific gravity 1.075 at 15°C) for 5 min at 46°C. This treatment disrupts diapause‐inducing signaling and allows embryogenesis to proceed normally until larval hatching (Yamashita and Yaginuma 1991; Lin et al. 2009; Gu et al. 2019). Because of its effectiveness, HCl treatment has been widely used in sericulture for decades to ensure a continuous supply of silkworm larvae.

Multiple protein kinase–mediated signaling pathways have been implicated in the regulation of embryonic development in B. mori. Among these, extracellular signal‐regulated kinase (ERK), a member of the mitogen‐activated protein kinase (MAPK) family, and glycogen synthase kinase (GSK)‐3β, a multifunctional kinase involved in glycogen metabolism and protein synthesis, have been identified as key regulators of early embryogenesis (Iwata et al. 2005; Fujiwara and Shiomi 2006; Fujiwara, Tanaka, et al. 2006; Lin et al. 2009; Gu and Chen 2017). In our previous studies, we further demonstrated that phosphorylation of the translational repressor 4E‐binding protein (4E‐BP)—a well‐established indicator of target of rapamycin (TOR) signaling activity (Hay and Sonenberg 2004; Bhaskar and Hay 2007)—occurs at an early stage of development in eggs in which diapause was prevented by HCl treatment (Gu et al. 2011). Subsequent analyses revealed differential activation of insulin/Akt and protein kinase C (PKC) signaling pathways when diapause eggs were compared with developing eggs, suggesting that multiple signaling networks participate in the regulation of the developmental transition associated with diapause (Gu et al. 2019, 2020).

Among the various protein kinases implicated in silkworm embryonic development, ERK has attracted particular attention due to its central role in embryogenesis. Previous studies have shown that elevated ERK phosphorylation correlates with the progression of embryonic development (Fujiwara, Shindome, et al. 2006). In contrast, ERK activity is markedly reduced during diapause, suggesting that suppression of the MAPK/ERK signaling pathway may contribute to the arrest of embryonic development (Fujiwara, Shindome, et al. 2006). ERK signaling functions downstream of receptor tyrosine kinases (RTKs) and integrates signals from multiple extracellular ligands to regulate diverse cellular processes, including cell proliferation, differentiation, pattern formation, and survival (Lemmon and Schlessinger 2010; Sopko and Perrimon 2013). Although ERK signaling has been implicated in embryonic development, the upstream ligands that regulate ERK activation and the downstream target genes involved in this process in B. mori remain largely unknown.

A variety of conserved signaling pathways regulate developmental processes in insects, including epidermal growth factor (EGF), fibroblast growth factor (FGF), Jelly belly (Jeb), transforming growth factor (TGF)‐β, and the Toll/spätzle (spz) pathway (Wu and Hill 2009; Muha and Müller 2013; Sopko and Perrimon 2013). Ligands such as spitz (spi) and vein (vn), along with regulators such as rhomboid (rho), activate EGF signaling, which is essential for numerous developmental processes in insects (Simcox et al. 1996; Yarnitzky et al. 1997; Wang et al. 2000; De Celis 2003). Similarly, Jeb activates anaplastic lymphoma kinase (Alk), which regulates multiple developmental and physiological processes (Englund et al. 2003; Bazigou et al. 2007). FGF signaling plays crucial roles in embryonic development, including mesoderm migration and tracheal morphogenesis (Muha and Müller 2013). Components of the TGF‐β signaling pathway, including the ligand myoglianin (myo) and its receptor babo, regulate cell proliferation, differentiation, and developmental patterning (Wu and Hill 2009). In addition, neurotrophin (NT) and Spz family proteins function as extracellular signaling molecules involved in developmental regulation and immune responses (Lemaitre et al. 1996; Schwer 2001; Lewis et al. 2013). Many of these pathways converge on the MAPK/ERK (Mulder 2000; Zhang 2017), suggesting that they may contribute to the regulation of ERK activity during embryogenesis. However, relatively few studies have investigated the expression of ERK‐related upstream ligand genes during diapause.

In addition to upstream signaling pathways, downstream transcription factors activated by ERK signaling are important mediators of developmental regulation. The ETS transcription factor pointed is a well‐established nuclear target of the MAPK/ERK pathway and plays key roles in regulating transcriptional responses during development (Brunner et al. 1994; O'Neill et al. 1994). Another transcription factor, huckebein (hkb), has been implicated in early embryonic patterning and terminal structure formation in insects (Bronner and Jäckle 1991; Reuter and Leptin 1994). Although these genes act as downstream components of ERK signaling in insects, their potential connection to diapause remains poorly understood.

Recent studies indicated that multiple signaling pathways may contribute to the regulation of chilling‐induced diapause termination in B. mori. For instance, transcriptional changes in the insulin, prothoracicotropic hormone (PTTH), and EGF signaling pathways have been linked to increased ERK phosphorylation during chilling‐induced diapause termination (Gu and Lin 2024; Gu 2025). However, it remains unclear whether these pathways also participate in embryonic development and how their expression patterns differ between diapause and developing eggs. Examining the signaling molecules that regulate ERK activation during embryogenesis is therefore crucial for understanding the molecular mechanisms controlling both developmental progression and diapause in B. mori.

In the present study, ERK phosphorylation dynamics and the expression patterns of ERK pathway–related genes were compared between diapause eggs and HCl‐treated developing eggs. The analyzed genes included potential upstream regulators of ERK signaling, including PTTH/torso, EGF signaling components (vn, spi, rho, and the Egf receptor (Egfr)), Jeb/Alk, Fgf/Fgf receptor (Fgfr), TGF‐β signaling components (myo and babo), neurotrophin (NT), and members of the spz gene family (spz1–5). In addition, downstream ERK target genes (pointed and hkb) were examined to evaluate their association with embryonic development. Non‐diapause and chilled eggs were further utilized to verify that the observed increases in gene expression are associated with embryogenesis. These findings suggest that multiple growth factor signaling pathways are transcriptionally linked to embryonic development in B. mori. However, these associations are based on expression correlations and do not establish direct causal relationships between these signaling molecules and ERK phosphorylation.

2. Materials and Methods

2.1. Experimental Animals

A hybrid univoltine strain (Guofu × Nongfong) of the silkworm, B. mori, was used as a diapause‐egg producer. Larvae were reared on fresh mulberry leaves at 25°C under a 12L:12D photoperiod. Eggs laid during the first 3 h were pooled and then divided into three experimental groups. The first group of eggs was incubated at 25°C under a 12L:12D photoperiod to maintain diapause state. The second group was treated with HCl (with a specific gravity of 1.075 at 15°C) for 5 min at 46°C at 20 h after oviposition to prevent diapause initiation. The third group was exposed to 5°C in the dark 15 days after oviposition to induce diapause termination (Gu 2025). After a chilling period of 90 days, the chilled eggs were transferred to 25°C, and they hatched around Day 9 or 10 post‐transfer. In addition, a polyvoltine strain (P1), which produces non‐diapause eggs, was used as previously reported (Gu et al. 2017, 2020). When non‐diapause eggs from the polyvoltine strain were incubated at 25°C, the eggs hatched on Days 9–10 after oviposition. Successful embryonic development was verified by hatchability rates exceeding 95% in chilled, non‐diapause, and HCl‐treated eggs. Samples were collected by rapid freezing at −80°C, and all samples were stored at −80°C until further use.

2.2. Antibodies

Anti‐phospho‐ERK and anti‐total‐ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)‐linked goat anti‐rabbit antibody was purchased from PerkinElmer Life Sciences (Boston, MA, USA).

2.3. Western Blot Analysis

Eggs from different developmental stages were homogenized in 20 μL of lysis buffer (10 mM Tris and 0.1% Triton X‐100) at 4°C (Lin et al. 2009; Gu et al. 2011). Lysates were boiled in an equal volume of sodium dodecyl sulfate (SDS) sample buffer for 4 min followed by centrifugation at 15,800g for 3 min to remove particulate matter. A 10 μL aliquot of supernatant (approximately 12 μg of total protein) was loaded onto SDS gels. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Mini Trans‐Blot Electrophoretic Transfer Cell System (Bio‐Rad, Hercules, CA, USA), and then washed with Tris‐buffered saline (TBS) for 10 min at room temperature. Blots were blocked at room temperature for 1 h in TBS containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat powdered dry milk, followed by washed three times (5 min each) with TBST. Blots were incubated overnight at 4°C with the primary antibody in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and incubated with the HRP‐linked secondary antibody in TBST with 1% BSA. Following three additional washes, the immunoreactivity was visualized by chemiluminescence using Western Lightning Chemiluminescence Reagent Plus from PerkinElmer Life Sciences. Chemiluminescent signals were scanned and quantified using the AlphaImager Imaging System and AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA).

2.4. RNA Extraction and Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR)

Total RNA was isolated from eggs (29 mg, ~50 eggs per sample, with 4 independent biological replicates) on the specified days using 600 μL of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. RNA concentration was measured with a NanoPhotometer Pearl (Implen GmbH, Munich, Germany). First‐strand cDNA was synthesized using the iScript cDNA synthesis kit (Bio‐Rad, Hercules, CA, USA).

qRT‐PCR was conducted in a 20 μL reaction containing 10 μL of SYBR Green Real‐Time PCR Master Mix (Bio‐Rad), 2 μL of first‐strand cDNA, 2 μL of forward and reverse primers, and 4 μL of nuclease‐free water. Reactions were performed on an iQ5 Real‐Time PCR Detection System (Bio‐Rad) according to the manufacturer's instructions. Primers were designed following the SYBR Green Master Mix guidelines, ensuring no primer dimers and an amplicon length ≤ 200 bp. Melting curve analysis was performed for all reactions to confirm amplification specificity. Thermal cycling conditions included an initial denaturation at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, annealing at 59.5°C for 30 s, and extension at 55°C for 1 min. Across all assays, amplification specificity was verified by observing a single peak in the melting‐curve profiles. Each plate also included no‐template controls, in which cDNA was replaced with nuclease‐free water, and these controls consistently produced no detectable amplification.

Because previous studies showed that B. mori ribosomal protein 49 (rp49) is the most stable gene during development (Kobayashi et al. 2014), it was chosen as a reference gene in the current study. Transcript levels were normalized to rp49, and relative gene expression was calculated using the ΔΔC T method. The qRT‐PCR was performed using primers listed in Table S1.

2.5. Statistical Analysis

Data are presented as the mean ± standard deviation (SD) of four independent biological replicates. Two‐group comparisons were performed using Student's t‐test, and one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied for multiple group comparisons. Statistical significance was defined as p < 0.05.

3. Results

3.1. Comparison of ERK Phosphorylation Between Diapause and Developing Eggs

In the first experiment, temporal changes in ERK phosphorylation were investigated in diapause and HCl‐treated eggs. Figure 1A showed the dynamics of ERK phosphorylation in diapause eggs during the first 9 days after oviposition. On the first day after oviposition, ERK phosphorylation levels were low but increased on the second day. By Day 2, the levels began to decline and remained low during Days 3–5. After Day 5, ERK phosphorylation decreased further and was maintained at very low levels. Figure 1B showed changes in ERK phosphorylation in HCl‐treated eggs during embryonic development. On the first day after oviposition, ERK phosphorylation levels were low, but they increased by the second day. From Days 2 to 5, phosphorylation levels remained high. After Day 6, ERK phosphorylation decreased dramatically. These results indicated distinct temporal patterns of ERK phosphorylation between diapause and HCl‐treated eggs.

Figure 1.

Figure 1

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Changes in ERK phosphorylation in diapause eggs (A), HCl‐treated eggs (B), non‐diapause eggs (C), and eggs in which diapause was terminated by chilling (D). Egg lysates from each developmental stage were analyzed by immunoblotting using anti‐phospho‐ERK (P‐ERK) and anti‐total ERK (T‐ERK) antibodies. The left panels in (A–D) show representative blots from four independent experiments. Quantified ERK phosphorylation levels (P‐ERK/T‐ERK ratios) were normalized to levels at Day 0 after oviposition (A and C), after HCl treatment (B), or after transfer to 25°C (D). Different letters above the bars indicate significant differences (ANOVA followed by Tukey's multiple comparison test, p < 0.05) (right panels in A–D).

To further confirm the association between ERK phosphorylation and embryogenesis, changes in ERK phosphorylation levels were examined in protein samples from non‐diapause eggs. As shown in Figure 1C, ERK phosphorylation increased on Day 1 and continued to rise on Day 2. High levels were maintained from Days 2 to 7, followed by a decline to low levels on Day 8. These results indicated that ERK phosphorylation in non‐diapause eggs showed a later and broader peak than in diapause eggs.

ERK phosphorylation was also examined in eggs in which diapause was terminated by chilling. As shown in Figure 1D, relatively high levels of ERK phosphorylation were detected during the first 7 days after the eggs were transferred to 25°C. On Day 7, phosphorylation levels declined sharply and remained low thereafter. These results suggested that elevated ERK phosphorylation is associated with embryonic development.

3.2. Changes in Gene Expression of PTTH/Torso and EGF Signaling Pathways in Diapause and HCl‐Treated Eggs

Figure 2A showed a comparison of temporal changes in PTTH gene expression between diapause and HCl‐treated eggs, with no significant difference between the two conditions. In addition, as shown in Figure 2B, no significant difference in temporal expression patterns of PTTH receptor gene torso was detected between diapause and HCl‐treated eggs. Similar to PTTH/torso, EGF signaling pathway genes spi and rho also showed comparable temporal expression patterns (Figure 2C,E), indicating that the expression of PTTH/torso, spi, and rho may not be associated with embryonic development. For the Egfr gene, both diapause and HCl‐treated eggs exhibited decreasing expression levels, with higher expression observed in HCl‐treated eggs. However, differential vn gene expression patterns were observed between diapause and HCl‐treated eggs. In HCl‐treated eggs, vn expression remained low during the first 4 days after oviposition but increased during later stages of embryonic development (Figure 2D).

Figure 2.

Figure 2

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Changes in expression levels of PTTH (A), torso (B), spi (C), vn (D), rho (E), and Egfr (F) in diapause and HCl‐treated eggs. Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Gene expression levels relative to rp49 were normalized to the mean of Day 0 diapause eggs. Each data point represents the mean ± SD (n = 4). Squares indicate diapause eggs, and circles indicate HCl‐treated eggs. Asterisks indicate significant differences between diapause and HCl‐treated eggs from the same day (by Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001).

3.3. Changes in the Expression of Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and the spz Family Genes in Diapause and HCl‐Treated Eggs

As shown in Figure 3, temporal expression patterns of Jeb/Alk and Fgf/Fgfr differed significantly between diapause and HCl‐treated eggs, with markedly higher expression levels observed during the middle and late stages of embryonic development in HCl‐treated eggs, in contrast to very low levels seen in diapause eggs. In HCl‐treated eggs, myo expression increased during early and mid‐embryonic development, with some fluctuations, followed by a sharp rise at later stages, whereas in diapause eggs, it remained consistently low, except on Day 1. For babo expression, although both HCl‐treated and diapause eggs exhibited gradually decreasing expression patterns, the levels remained higher in HCl‐treated eggs compared to diapause eggs.

Figure 3.

Figure 3

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Changes in expression levels of Jeb (A), Alk (B), Fgf (C), Fgfr (D), myo (E), and babo (F) in diapause and HCl‐treated eggs. Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Gene expression levels relative to rp49 were normalized to the mean of Day 0 diapause eggs. Each data point represents the mean ± SD (n = 4). Squares indicate diapause eggs, and circles indicate HCl‐treated eggs. Asterisks indicate significant differences between diapause and HCl‐treated eggs from the same day (by Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4A showed the temporal expression patterns of NT in diapause and HCl‐treated eggs. In diapause eggs, NT expression remained low during the first 9 days post‐oviposition, whereas HCl‐treated eggs exhibited a pronounced expression peak on Day 6. Temporal changes in spz1–5 gene expression were shown in Figure 4B–F. The expression levels of spz2 and spz4 exhibited pronounced peaks on Day 6 in HCl‐treated eggs, whereas low levels were observed in diapause eggs. The expression of spz5 peaked between Days 7 and 8 in HCl‐treated eggs but remained very low in diapause eggs. In contrast, spz1 expression did not differ significantly between diapause and HCl‐treated eggs. Conversely, spz3 expression was relatively higher during the first 2 days post‐oviposition in diapause eggs than in HCl‐treated eggs.

Figure 4.

Figure 4

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Changes in expression levels of NT (A), spz1 (B), spz2 (C), spz3 (D), spz4 (E), and spz5 (F) in diapause and HCl‐treated eggs. Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Gene expression levels relative to rp49 were normalized to the mean of Day 0 diapause eggs. Each data point represents the mean ± SD (n = 4). Squares indicate diapause eggs, and circles indicate HCl‐treated eggs. Asterisks indicate significant differences between diapause and HCl‐treated eggs from the same day (by Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001).

3.4. Changes in Expression of vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spz Family Genes in Non‐Diapause Eggs and Chilled Eggs

The above results showed differential expression patterns of vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spz family genes (spz2, spz4, and spz5) between diapause and HCl‐treated eggs, with significantly higher expression observed during embryonic development in HCl‐treated eggs. To further confirm that these temporal changes are related to embryonic development, non‐diapause eggs and eggs whose diapause was terminated by chilling (chilled eggs) were examined. As shown in Figure 5, non‐diapause eggs exhibited expression patterns similar to those observed in HCl‐treated eggs for vn, Jeb/Alk, Fgf/Fgfr, myo, NT, and spz family (spz2, spz4, and spz5) genes. Similarly, chilled eggs showed increased expression during embryonic development, comparable to that observed in HCl‐treated and non‐diapause eggs (Figure 6).

Figure 5.

Figure 5

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Changes in expression levels of vn (A), Jeb (B), Alk (C), Fgf (D), Fgfr (E), myo (F), NT (G), spz2 (H), spz4 (I), and spz5 (J) in non‐diapause eggs. Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Gene expression levels relative to rp49 were normalized to the mean of Day 0 non‐diapause eggs. Each data point represents the mean ± SD (n = 4). Different letters above the bars indicate significant differences (ANOVA followed by Tukey's multiple comparison test).

Figure 6.

Figure 6

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Changes in expression levels of vn (A), Jeb (B), Alk (C), Fgf (D), Fgfr (E), myo (F), NT (G), spz2 (H), spz4 (I), and spz5 (J) in eggs whose diapause was terminated by chilling (chilled eggs). Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Gene expression levels relative to rp49 were normalized to the mean of Day 0 after transfer to 25°C. Each data point represents the mean ± SD (n = 4). Different letters above the bars indicate significant differences (ANOVA followed by Tukey's multiple comparison test).

3.5. Changes in Expression of Pointed and hkb Genes in Diapause, HCl‐Treated, and Non‐Diapause Eggs

Figure 7A,B presented a comparison of pointed and hkb gene expression between diapause and HCl‐treated eggs. HCl‐treated eggs displayed a more pronounced and broader peak in pointed and hkb expression. In diapause eggs, an increase in pointed expression was detected on Day 1 after oviposition; however, between Days 2 and 9, the levels gradually decreased. Diapause eggs showed very low levels of hkb expression. The changes in pointed and hkb gene expression were also examined in non‐diapause eggs. As shown in Figure 7C,D, non‐diapause eggs exhibited temporal changes in pointed and hkb expression similar to those in HCl‐treated eggs.

Figure 7.

Figure 7

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Changes in expression levels of pointed (A and C) and hkb (B and D) in diapause and HCl‐treated eggs (A and B) and in non‐diapause eggs (C and D). Egg extracts from each stage were prepared, and gene expression levels were determined by qRT‐PCR. Expression levels of pointed and hkb relative to rp49 were normalized to the mean of Day 0 diapause (A and B) or non‐diapause eggs (C and D). Each data point represents the mean ± SD (n = 4). Squares indicate diapause eggs, and circles indicate HCl‐treated eggs. Asterisks indicate significant differences between diapause and HCl‐treated eggs on the same day (by Student's t‐test, *p < 0.05, **p < 0.01, ***p < 0.001) (A and B). Different letters above the bars indicate significant differences (ANOVA followed by Tukey's multiple comparison test) (C and D).

4. Discussion

The results presented here demonstrate distinct temporal patterns of ERK phosphorylation between diapause and developing eggs in B. mori. Significantly higher levels of phosphorylated ERK were observed between Days 1 and 6 after oviposition in developing eggs (non‐diapause and HCl‐treated eggs), whereas diapause eggs maintained low levels during this period. This result is consistent with previous studies that reported similar changes during the diapause process in B. mori (Fujiwara, Shindome, et al. 2006). Moreover, the present study clearly demonstrated distinct temporal expression patterns of the EGF ligand vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, spz family genes (spz2, spz4, and spz5) between diapause eggs and HCl‐treated developing eggs in Bombyx. In HCl‐treated eggs, relatively high expression levels were observed during the middle and late stages of embryonic development, whereas diapause eggs consistently exhibited low levels. Similarly, non‐diapause and chilled eggs showed temporal expression patterns comparable to those of HCl‐treated eggs. These findings suggest that the expression of vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spz family genes (spz2, spz4, and spz5) is associated with embryonic development. Previous studies have reported changes in EGF signaling during diapause in Caenorhabditis elegans (O'Keeffe and Greenwald 2022) and the correlation of insulin, PTTH, and EGF pathways with chilling‐induced diapause termination in B. mori eggs (Gu and Lin 2024; Gu 2025). The present results extend the analysis to a broader set of ERK‐related upstream regulators, revealing coordinated transcriptional patterns associated with embryonic development (Figure 8).

Figure 8.

Figure 8

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Schematic representation of the current understanding of the signaling network that regulates embryonic development in B. mori. Dashed lines indicate findings supported by the present study. See the text for further details.

In the present study, the dynamic expression patterns of PTTH/torso, EGF (vn, spi, rho, and Egfr), Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spzs (spz1‐5) were examined during embryonic development. The results showed that vn, Jeb/Alk, and Fgf/Fgfr exhibited differential expression patterns between diapause and developing eggs, whereas PTTH/torso and EGF ligands (spi and rho) did not. PTTH is a primary stimulator of ecdysteroidogenesis in the prothoracic glands of larval insects (Pan et al. 2021; Gu 2026). Previous studies have also reported differences in the expression of ecdysteroidogenic enzyme genes and in ecdysteroid levels between diapause and developing eggs (Fujinaga et al. 2020; Gu et al. 2021). The similar temporal expression patterns of the PTTH/torso and EGF ligand genes (spi and rho) between diapause and HCl‐treated eggs suggest that their transcriptional regulation is not responsible for the increased expression of ecdysteroidogenic enzyme genes and ERK phosphorylation levels observed during embryonic development (Fujinaga et al. 2020; Gu et al. 2021). However, a previous study reported that changes in PTTH gene expression during a long chilling period paralleled changes in ERK phosphorylation, suggesting a potential role for PTTH signaling in regulating chilling‐induced diapause termination (Gu 2025). It was also demonstrated that the expression of the EGF ligand genes rho and spi is related to chilling‐induced diapause termination (Gu 2025). These findings suggest that, although ERK activation occurs in both chilling‐induced diapause termination and embryonic development (Fujiwara, Shindome, et al. 2006), the signaling pathways leading to this activation involve distinct upstream ligands. However, the expression of vn showed distinct temporal patterns between diapause and HCl‐treated eggs, with a major peak observed during the middle and later stages of embryonic development in HCl‐treated eggs. Relatively high expression of the Egfr gene was also detected in HCl‐treated eggs. To further assess the correlation between the vn expression and embryonic development, non‐diapause eggs and chilled eggs were examined. Both showed temporal changes similar to those observed in HCl‐treated eggs. This result, together with the higher expression of the vn gene during chilling‐induced diapause termination, supports a potential role for vn in both embryonic development and diapause termination. However, spi and rho expression is related to diapause termination, but not embryonic development, suggesting distinct ligand requirements for chilling‐induced diapause termination and embryonic development. Distinct EGF ligand requirements have previously been reported during Drosophila embryogenesis. In Drosophila, Vn appears to function as the primary ligand for wing identity during early imaginal disc development (Simcox et al. 1996; Yarnitzky et al. 1997; Wang et al. 2000), whereas at later stages, a combination of Spi and Vn is required for vein formation (De Celis 2003). In Tribolium, the results showed distinct requirements for Spi and Vn in the formation of pupal structures such as wings, elytra, and gin traps, suggesting ligand‐specific activation of EGF signaling during metamorphosis (Chafino et al. 2021). Future experiments will examine stage‐specific roles of Spi and Vn during silkworm embryonic development.

In addition to vn, the current study revealed differential expression of Jeb/Alk and Fgf/Fgfr genes between diapause and HCl‐treated eggs in B. mori. Jeb is the only known ligand for Alk (Englund et al. 2003). Previous studies in Drosophila have shown that Jeb/Alk signaling is critical for several developmental processes, including sparing of nervous system growth during larval nutrient deprivation (Cheng et al. 2011), regulation of body growth under nutrient stress (Okamoto and Nishimura 2015), and neuronal circuit assembly in the developing retina (Bazigou et al. 2007). More recent studies have shown that Jeb/Alk signaling activates the MAPK/ERK pathway to promote ecdysteroidogenesis in prothoracic glands (Pan and O'Connor 2021). Additionally, Jeb/Alk signaling appears to influence longevity, as its inhibition has been shown to extend healthy lifespan (Woodling et al. 2020). In C. elegans, the Alk ortholog SCD‐2 appears to regulate dauer formation by modulating TGF‐β signaling (Reiner et al. 2008). By contrast, elevated cardiac Alk signaling has been associated with a reduced average lifespan and a rapid decline in fitness in young flies (Wolfstetter et al. 2025). This suggests that, beyond its systemic effects on lifespan, Jeb/Alk signaling may also influence age‐dependent organ physiology in a manner similar to insulin signaling (Wessells et al. 2004). In the current study, it was found that Jeb/Alk gene expression increased markedly during mid to late embryonic development in developing eggs (HCl‐treated, non‐diapause, and chilled eggs), whereas expression levels remained very low in diapause eggs, indicating a potential correlation between Jeb/Alk signaling and embryonic development.

Along with Jeb/Alk signaling, FGF signaling plays important roles in numerous developmental and physiological processes, including cell migration, angiogenesis, proliferation, differentiation, and survival (Xie et al. 2020). Studies in Drosophila have shown that FGF signaling plays crucial roles in numerous processes during embryonic development, including regulation of gene expression, changes in cell shape, and cell–cell interactions during mesoderm layer formation, as well as in caudal visceral muscle formation, tracheal morphogenesis, and glial differentiation (Muha and Müller 2013). In the current study, differential expression of Fgf/Fgfr genes between diapause and developing eggs was observed. Expression levels were very low during the first 9 days after oviposition in diapause eggs, indicating a correlation between their expression and embryonic diapause. Although studies in C. elegans have shown that Klotho, a key anti‐aging protein, promotes longevity and stress resistance by modulating FGF and insulin signaling (Paquette et al. 2023), this study is the first to suggest a potential link between FGF signaling and embryonic diapause.

In Drosophila melanogaster, Toll plays central roles in embryonic development—particularly in dorsoventral patterning and early tissue specification—as well as in innate immunity, and is activated by its endogenous ligand, spz (Lemaitre et al. 1996; Lewis et al. 2013). In B. mori, five spz genes (spz1–spz5) showed significant upregulation after infection with Escherichia coli and Staphylococcus aureus. Yeast two‐hybrid assays showed that only spz2 interacted with Toll11 and Toll9‐1, suggesting that activated spz2 triggers AMP expression upon pathogen infection (Yu et al. 2020). In addition, spz3 has been reported to regulate melanization during stripe pattern formation (KonDo et al. 2017). In the present study, spz genes exhibited gene‐specific temporal expression patterns between diapause and developing eggs, with major expression peaks of spz2, spz4, and spz5 observed during the mid to late stages of embryonic development in developing eggs. No significant difference in spz1 expression was detected between diapause and developing eggs, whereas spz3 showed high expression during the early stage of diapause eggs. Studies in D. melanogaster have shown that spz is a secreted protein that contains a cystine‐knot domain structurally related to NT, indicating evolutionary and structural similarity with extracellular signaling proteins involved in protease‐mediated activation processes (Schwer 2001). In the current study, the NT gene showed a pronounced expression peak during the mid‐ to late stages of embryonic development in developing eggs, while expression levels remained very low in diapause eggs. Although studies in D. melanogaster have shown that immune genes, including those in the Toll/spz cascade, are upregulated during reproductive diapause (Kučerová et al. 2016), this study is, to our knowledge, the first to suggest a potential association between embryonic diapause and the differential expression of spz family genes and NT.

Additionally, TGF‐β signaling plays a critical role in embryonic development by regulating cell proliferation, differentiation, apoptosis, and tissue patterning. It also helps control morphogenesis and organ formation through conserved signaling pathways that coordinate cell fate decisions during development (Wu and Hill 2009). In the current study, it was found that the expression of myo/babo, two key components of the TGF‐β signaling pathway, exhibited significantly different temporal changes between diapause and developing eggs. The expression of myo exhibited large fluctuations during early and mid‐embryonic development, with two major peaks observed in developing eggs, whereas expression remained low in diapause eggs. HCl‐treated eggs also exhibited relatively higher expression of babo compared to diapause eggs. Although TGF‐β signaling is downregulated in diapause‐destined pupae of Helicoverpa armigera (Zhang et al. 2022), the present study suggests its potential role in regulating embryonic diapause in B. mori by linking differential gene expression in developing and diapause eggs. In C. elegans, the TGF‐β ligand DAF‐7 and its signaling pathway regulate entry into the dauer, a diapause‐like stage, in response to environmental cues, highlighting a conserved role of TGF‐β signaling in developmental arrest (Murakami et al. 2001).

Although this study revealed significant differences in the expression of vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spz (spz2, spz4, and spz5) genes between diapause and HCl‐treated developing eggs in B. mori, the onset of gene expression during embryonic development varied among these genes. Specifically, myo exhibited the earliest increase in expression (Day 1 after oviposition), followed by Fgf/Fgfr (Day 3), Jeb/Alk (Day 4), and subsequently NT, spz2, spz4, spz5, and vn (Day 5), suggesting a sequential progression of upstream signaling inputs. Based on these temporal patterns, a hypothetical stepwise model is proposed in which early TGF‐β signaling may prime embryonic tissues for development, followed by activation of FGF and Jeb/Alk pathways that regulate cell proliferation and tissue organization, and later engagement of NT, spz, and vn that contribute to differentiation and patterning. Given that these pathways are known to converge on the MAPK/ERK cascade, this sequential activation may collectively contribute to the progressive increase in ERK phosphorylation observed during embryonic development. While this model is based on transcriptional dynamics and does not establish direct regulatory interactions, it provides a framework for how multiple upstream signals may be temporally coordinated to regulate ERK activity during embryogenesis in B. mori. However, the current study investigated only the transcriptional levels of these genes; thus, further studies focusing on protein expression and kinase activity are needed to elucidate their regulatory roles during embryogenesis.

Through the conserved MAPK/ERK signaling cascade, RTK signaling plays a critical role in regulating cell fate determination, differentiation, patterning, proliferation, growth, survival, and lifespan (Lemmon and Schlessinger 2010). In Drosophila, at least 20 genes encoding RTKs have been identified, and studies have shown that different RTKs share many common downstream effectors, with their hierarchical organization preserved across diverse biological contexts (Sopko and Perrimon 2013). Among the genes examined above, vn, Jeb, Fgf, and NT encode RTK ligands (Sopko and Perrimon 2013), along with spz (spz2, spz4, and spz5), which functions through Toll signaling (Yu et al. 2020). It has also been well demonstrated that TGF‐β activates the MAPK/ERK signaling pathway (Mulder 2000; Zhang 2017). Given that expression of the myo, Jeb, Fgf, vn, NT, and spz (spz2, spz4, and spz5) genes increased dramatically in developing eggs, the elevated ERK phosphorylation observed during embryogenesis may result from activation of multiple upstream signaling pathways converging on the MAPK/ERK cascade. In contrast, diapause eggs showed very low ERK phosphorylation, reflecting suppressed signaling during developmental arrest. ERK phosphorylation has been shown to correlate with embryonic development in B. mori (Fujiwara, Shindome, et al. 2006; Fujiwara, Tanaka, et al. 2006); this study proposes potential upstream ligands that regulate ERK activation during embryogenesis and links this regulation to embryonic diapause.

In addition to investigating the potential upstream ligands responsible for ERK phosphorylation, the present study also examined the expression of two downstream ERK target genes, pointed and hkb. The ETS transcription factor pointed is a well‐known nuclear mediator of the MAPK/ERK signaling pathway and is activated through ERK‐dependent phosphorylation, thereby regulating transcriptional responses during development (Brunner et al. 1994; O'Neill et al. 1994). The hkb gene encodes a zinc‐finger transcription factor that plays important roles in early embryonic patterning and terminal structure formation in insects (Bronner and Jäckle 1991; Reuter and Leptin 1994). In the present study, the more pronounced and broader peaks of pointed and hkb expression in HCl‐treated and non‐diapause eggs further confirmed that ERK signaling may be more strongly activated during normal embryonic development. By contrast, the relatively low expression of these genes in diapause eggs indicates that ERK‐mediated transcriptional activity is largely suppressed during diapause. The correlation between pointed expression and chilling‐induced diapause termination has been reported previously (Gu 2025); the present study provides evidence of an association between pointed and hkb expression and embryonic development in B. mori.

In summary, this study revealed clear differences in ERK phosphorylation dynamics and ERK signaling‐related gene expression patterns between diapause and developing eggs in B. mori. Developing eggs exhibited increased expression of vn, Jeb/Alk, Fgf/Fgfr, myo/babo, NT, and spz (spz2, spz4, and spz5) genes, accompanied by markedly elevated ERK phosphorylation during embryogenesis, whereas diapause eggs maintained consistently low levels. The sequential upregulation of these genes during embryonic development suggests stage‐specific roles in regulating embryogenesis. Because many of these genes encode ligands for RTKs that converge on the MAPK/ERK signaling pathway, their increased expression may underlie the enhanced ERK phosphorylation observed in developing embryos. Together, these findings highlight potential upstream regulators of ERK activation and provide new insight into the molecular mechanisms governing embryonic development in B. mori.

While the present study identifies correlations between RTK ligand gene expression and ERK phosphorylation dynamics, it does not provide direct functional evidence that these factors regulate ERK activation. Future studies employing functional approaches, such as gene knockout, overexpression, or pharmacological inhibition, will be necessary to determine whether these candidate ligands directly contribute to ERK phosphorylation and to delineate their specific roles within the proposed signaling framework.

Author Contributions

Shi‐Hong Gu: conceptualization, investigation, funding acquisition, writing – original draft, methodology, validation, visualization, writing – review and editing, formal analysis, project administration, resources, supervision, data curation, software.

Conflicts of Interest

The author declares no conflicts of interest.

Supporting information

Table S1: qRT‐PCR primer sequences.

ARCH-122-e70194-s001.docx (21.2KB, docx)

Acknowledgments

The author gratefully acknowledges the National Science and Technology Council of Taiwan for funding support through Grant NSTC 114‐2311‐B‐178‐001. Appreciation is also extended to the National Museum of Natural Science, Taiwan, for its financial support.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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

Supplementary Materials

Table S1: qRT‐PCR primer sequences.

ARCH-122-e70194-s001.docx (21.2KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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