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. 2023 Oct 21;5(4):455–466. doi: 10.1007/s42995-023-00194-x

Functional evidence that FGFR regulates MAPK signaling in organizer specification in the gastropod mollusk Lottia peitaihoensis

Sujian Tan 1, Pin Huan 1,2, Baozhong Liu 1,2,✉
PMCID: PMC10689715  PMID: 38045550

Abstract

The D-quadrant organizer sets up the dorsal–ventral (DV) axis and regulates mesodermal development of spiralians. Studies have revealed an important role of mitogen-activated protein kinase (MAPK) signaling in organizer function, but the related molecules have not been fully revealed. The association between fibroblast growth factor receptor (FGFR) and MAPK signaling in regulating organizer specification has been established in the annelid Owenia fusiformis. Now, comparable studies in other spiralian phyla are required to decipher whether this organizer-inducing function of FGFR is prevalent in Spiralia. Here, we indicate that treatment with the FGFR inhibitor SU5402 resulted in deficiency of organizer specification in the mollusk Lottia peitaihoensis. Subsequently, the bone morphogenetic protein (BMP) signaling gradient and DV patterning were disrupted, suggesting the roles of FGFR in regulating organizer function. Changes in multiple aspects of organizer function (the morphology of vegetal blastomeres, BMP signaling gradient, expression of DV patterning markers, etc.) indicate that these developmental functions have different sensitivities to FGFR/MAPK signaling. Our results reveal a functional role of FGFR in organizer specification as well as DV patterning of Lottia embryos, which expands our knowledge of spiralian organizers.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42995-023-00194-x.

Keywords: FGF signaling, Organizer specification, BMP signaling gradient, DV patterning, Mollusk

Introduction

The Spiralia is one of the three major bilaterian clades, including mollusks, annelids, nemerteans, and several other marine invertebrate phyla. Many spiralian lineages share an ancient and stereotypic early developmental program known as spiralia cleavage (Henry 2014; Laumer et al. 2015; Martín-Durán and Marlétaz 2020). Cell lineage analyses reveal that there are high levels of conservation in fate maps (Boyer and Jonathan 1998; Boyer et al. 1998; Hejnol et al. 2007; Henry and Martindale 1998, 1999). During early cleavages a particular blastomere forms, called the D-quadrant organizer, which regulates the specification of the dorsal–ventral (DV) axis and major mesodermal organs by specifying the fates of adjacent cells (Gonzales et al. 2007; Henry 2002, 2014; Henry and Martindale 1987; Lambert 2008; Lyons et al. 2012). Interference with organizer function results in the loss of both the DV axis and featured organs, such as the molluskan shell, foot, and eye (Clement 1962; Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2003; Tan et al. 2022; van den Biggelaar and Guerrier 1979).

There are two principal mechanisms by which the D quadrant is established in spiralian embryos: (1) in some species, the first two rounds of cell division are unequal, and one blastomere (D) is larger than the other three. The establishment of D blastomere identity relies on vegetally localized unidentified factors (Henry 1986, 2014; Henry and Martindale 1987; Render 1989; van den Biggelaar 1993; Verdonk and Cather 1973); (2) in other species the first two rounds of cell division are equal, blastomeres cannot be distinguished at the four-cell stage, and the D quadrant is specified by cell–cell interactions between the micromeres in the animal hemisphere and one of the vegetal macromeres at approximately the fifth cleavage (Arnolds et al. 1983; Boring 1989; Gonzales et al. 2007; Henry 2002; Henry et al. 2017; Kühtreiber et al. 1988). Once specified, the D-quadrant blastomere acts as an organizer to determine the DV axis and gives rise to the majority of the mesodermal organs in both unequally and equally cleaving embryos (Gonzales et al. 2007; Henry 2002, 2014; Henry and Martindale 1987; Lambert 2008; Lyons et al. 2012).

Although spiralian D-quadrant organizers have been extensively studied at the cellular level, knowledge regarding the molecular pathways regulating their specification is still limited (Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2001, 2003). MAPK signaling has been revealed to play essential roles in organizer function. This signaling is activated in the organizer and in some species investigated, mediates organizer function by regulating downstream BMP signaling (Lambert and Nagy 2003; Lambert et al. 2016; Tan et al. 2022). Treatment with a specific inhibitor (U0126) can cause phenotypes resembling those induced after removal of the organizer, including abolished DV axis and reduced mesodermal structures (Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2003; Pfeifer et al. 2014; Seudre et al. 2022; Tan et al. 2022). However, given the various molecules that can activate MAPK signaling, it is necessary to further determine the activator of MAPK. In representative deuterostomes and ecdysozoans (within the protostomes), MAPK is one of the fibroblast growth factors (FGF) signaling targets and mediates the role of FGF signaling in regulating the dorsal fates and mesodermal development (Dorey and Amaya 2010; Fan et al. 2018; Kadam et al. 2009; McFann et al. 2022). Studies in two spiralian species have also shown that FGF signaling is involved in mesodermal induction (Andrikou and Hejnol 2021). Therefore, it is reasonable to propose that FGF may act as an upstream regulator driving the function of MAPK signaling in organizer specification and subsequently regulating mesodermal development (Fig. 1A, B). Recently, a pioneering report supported this hypothesis in the annelid O. fusiformis, revealing an FGF receptor (FGFR) as the upstream regulator of organizer-related MAPK signaling in a spiralian for the first time (Seudre et al. 2022). However, given the wide involvement of MAPK signaling in spiralian organizer specification (Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2001, 2003; Vellutini et al. 2017), a key question remains regarding whether this regulatory mechanism is prevalent in spiralian lineages.

Fig. 1.

Fig. 1

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The molecular and cellular mechanisms of spiralian organizer specification and function. A In major animal clades, FGF signaling plays a crucial role in DV patterning and mesoderm development by regulating the MAPK pathway. In mollusks, the common role of MAPK signaling in 3D blastomere (organizer) specification and subsequent DV patterning and mesodermal development indicates a potential role of FGF signaling in organizer specification. The ellipses indicate that there are no available data at the corresponding regulatory node. The diagrams of representative animals are derived from PhyloPic (http://phylopic.org) and Wikipedia (https://www.wikipedia.org) licensed under CC BY 3.0. B A hypothesis regarding relationships between the organizer specification and FGF signaling in an equal-cleaving mollusk. C A schematic diagram depicting the flow of the FGF signaling pathway, in which the targeting node of the selective inhibitor SU5402 is highlighted. The diagram has been modified from (Seudre et al. 2022)

The Mollusca is a representative phylum of Spiralia and is an ideal system in which to address the above question (Fig. 1A). Recently, we revealed the role of MAPK signaling in mediating organizer function by modulating BMP signaling in the gastropod mollusk Lottia peitaihoensis (formerly L. goshimai) (Tan et al. 2022). Here, we investigated the role of FGFR in the development of L. peitaihoensis using the specific inhibitor SU5402. Our results revealed the importance of FGFR in organizer specification. These findings suggest a conserved role of FGFR in specifying spiralian organizers and provide insights into the evolution of organizer identity mechanisms in Spiralia.

Results

Identification of the FGFR gene in L. peitaihoensis

We searched for genes showing sequence similarities with FGFR genes from the developmental transcriptome of L. peitaihoensis, and one candidate was retrieved. Subsequent phylogenetic analysis confirmed that the gene was orthologous to FGFR genes, and thus it was designated lpe-fgfr (GenBank accession OP758846) (Fig. 2A). We next analyzed the expression patterns of lpe-fgfr at the 60-cell stage, when the organizer specification was completed. The gene was expressed in the whole embryo at this stage, despite relatively faint staining (Fig. 2B).

Fig. 2.

Fig. 2

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L. peitaihoensis FGFR gene and mRNA expression at the 60-cell stage. A Maximum likelihood tree of lpe-fgfr. B Whole mount in situ hybridization for lpe-fgfr at the 60-cell stage. Panels a and b show the animal and vegetal views with the 3B side to the left, respectively. The bar represents 50 μm. C Multiple protein alignments of FGFR orthologs from various species

Larval phenotypes after FGFR inhibition

We treated the embryos with SU5402, a selective inhibitor of FGFR phosphorylation, from the 16- to 64-cell stage to examine the requirement of FGFR activation in organizer specification and function (Fig. 3A). The peptide encoded by lpe-fgfr contained conserved residues for interaction with SU5402 (Fig. 2C). If the specification of the L. peitaihoensis organizer is indeed regulated by FGF signaling, inhibiting FGFR activation should cause a deficiency in the development of featured organs. Our findings support this prediction: treatment with 24 μmol/L SU5402 influenced the development of the foot, shell, and eyes in 48-hpf veligers. In the control group, the shell of veliger larva completely covered the larval trunk, and the foot comprised a distinguishable propodium and metapodium (Fig. 3B). SU5402 treatment resulted in a range of abnormal larvae that fell into three categories depending on the severity of the phenotype: (1) In 52 cases (58.43%, n = 89) they exhibited a mild phenotype, the feet developed, but showed abnormal morphology (Fig. 3D). Specifically, the larval feet tissue could be roughly recognized as protruding tissue mass adjacent to the prototroch (velum), but a consistent pattern could not be recognized, and the propodium and metapodium were undistinguishable. Regular mineralized lines in larval shells comparable to those in the control group were also observed, reflecting generally uninfluenced biomineralization (Fig. 3D). (2) For the embryos showing moderate phenotype (25 cases, 28.1%), the foot and shell were reduced. The propodium and metapodium were indistinguishable, and the shell lost regular mineralized lines (Fig. 3D’). (3) In 12 cases (13.5%) disorganized larvae occurred that possessed only partial or even undiscernible feet and shells (Fig. 3D’’). The development of larval eyes was also influenced. We found that 38 of 89 manipulated larvae had no more than one eye, half (45 cases) of the larvae had two eyes, and extra eyes were observed in six cases (Fig. 3C–G). A chi-square test revealed a statistically significant difference (P < 0.05) in the proportions of larvae with varying phenotypes between the groups with and without SU5402 treatment. These finding revealed that short-term treatment with SU5402 during the course of organizer specification caused serious developmental deficiency of organizer-induced organs, similar to phenotypes with the loss of organizer function (Koop et al. 2007; Lambert and Nagy 2001), indicating that FGFR signaling plays roles in organizer function.

Fig. 3.

Fig. 3

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Larval phenotypes after SU5402 treatment. A Diagram illustrating SU5402 inhibitor treatment. The asterisk indicates the presumptive organizer specification. B–E The general larval morphology (lateral views with anterior to the top) and eyes of manipulated larvae in the upper and lower panels, respectively. The control larva has a well-formed shell with regular mineralized lines, and the foot comprises of the propodium (pp) and metapodium (mp). pt, prototroch. The bars represent 50 μm. F A statistical summary of the manipulated larvae. G The ratios of manipulated larvae with different phenotypes and eye numbers (*P < 0.05, Chi-square test)

Effects of FGFR inhibition on organizer specification and BMP signaling gradient in early embryos

We next investigated how SU5402 treatment affected organizer functions. In L. peitaihoensis, when the organizer (3D) is specified, the blastomere as well as the adjacent ones showed characteristic morphological changes, and BMP signaling is modulated to form a gradient throughout the embryos (Tan et al. 2022). We thus used DAPI, phalloidin and anti-phosphorylated Smad1/5/8 (pSmad1/5/8) antibody staining to explore organizer specification and the change of BMP signaling in the manipulated embryos.

In the control group, the organizer could be identified based on the characteristic four-cell arrangement pattern (3D-2d22-3c2-3d2) at the vegetal pole of the 60-cell embryo (Fig. 4Ab-Ae). Specifically, the more centered macromere (3D, the presumptive organizer) was surrounded by three small blastomeres, 2d22, 3c2, and 3d2 (Fig. 4Ab, Ae). This morphological characteristic could not be observed on 3B and adjacent blastomeres, indicating an asymmetric cell arrangement pattern along the 3B–3D axis. SU5402 treatment changed the characteristic cell arrangement pattern, and no asymmetry associated with the specification of the organizer was detected, i.e., the four macromeres (3M) as well as adjacent 3m2 blastomeres were symmetrical (Fig. 4Ab’–Ae’, despite slight asymmetry between 3M blastomeres). These results indicated that FGF signaling influences organizer specification.

Fig. 4.

Fig. 4

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The states of organizer specification and pSmad1/5/8 staining under SU5402 treatment at the 60-cell stage. A DAPI staining and phalloidin labeling in control and SU5402 treated embryos. Panels Ab-Ae show the characteristic four-cell arrangement patterns in the vegetal view, that is, a 3D macromere (presumptive organizer) closely surrounded by three small blastomeres, 2d22, 3c2, and 3d2. In Ab and Ad, the very different 3q22 blastomeres (3a22, 3d22, 3c2, and 3d2) are highlighted by letters with different colors. After SU5402 treatment, the arrangement pattern of four macromeres (3M) and adjacent blastomeres (3m2) were symmetrical (Ab’-Ae’). Note that one macromere is slightly different from the other three, with a pronounced nuclear envelope (Ac’, white asterisk). B The state of pSmad1/5/8 staining under SU5402 treatment. Although asymmetric cell arrangement patterns at the vegetal pole were eliminated (Bb and Bb’), the manipulated embryos appear to still retain a reduced pSmad1/5/8 gradient (Bc-Be, Bc’-Be’). Bf shows the quantified difference in pSmad1/5/8 activities at the animal pole between SU5402-treatment and the control group (P < 0.05). In the treated group, at a more relaxed alpha level (α = 0.1), there was a statistically significant difference in pSmad1/5/8 activities on two opposite sides ‘‘3B’’ and ‘‘3D’’, and therefore, ‘‘3B’’ and ‘‘3D’’ indicate the presumptive 3B-3D axis based on the residual pSmad1/5/8 gradient (Bc’). The bar represents 50 μm

Then, nuclear pSmad1/5/8 staining was performed to explore the effects of SU5402 treatment on the BMP signaling gradient. For control embryos, this pSmad1/5/8 gradient crossed the 3D and 3B blastomeres. The cells adjacent to the organizer (3D side) showed strong pSmad1/5/8 staining, while only weak activities were detected in the cells distal to the organizer (Fig. 4Bc, Bd). After treated with SU5402, the pSmad1/5/8 activation pattern showed obvious changes. Although it seems that a signaling gradient was still observed, this gradient was greatly reduced at the animal pole (Fig. 4Bc’, Bd’). To support this, pSmad1/5/8 activities were quantified by normalization to background intensities (see “Materials and methods”). The results indicated no significant differences in pSmad1/5/8 activities on two opposite sides of the manipulated embryos, and further showed that SU5402 treatments caused a significant downregulation of pSmad1/5/8 activities on one side (the presumptive “3D” side), while those of the opposite (“3B”) side did not change evidently (Fig. 4Bf, P < 0.05). At the vegetal pole, the pSmad1/5/8 staining was only detectable on the “3D” side, similar to that of the control (Fig. 4Bb’, Bd’).

Taken together, these findings show that SU5402 treatment eliminated the characteristic morphological changes that occurred in the vegetal blastomeres with regard to organizer specification and greatly influenced downstream BMP signaling.

The effect of FGFR inhibition on DV patterning

The spiralian D-quadrant organizer determines the establishment of the DV axis. The absence of molluskan organizers can cause the loss of the DV axis and radialized development (Henry et al. 2006; Koop et al. 2007; Kühtreiber et al. 1988; Tan et al. 2022). In L. peitaihoensis, the marker genes expressed in dorsal-type (gata2/3 and hox1), ventral-type (soxb) and blastoporal (foxa and brachyury) tissues all showed radial expression after inhibiting organizer specification, reflecting the lack of DV axis in the manipulated embryo (Tan et al. 2022). Here we examined the role of FGFR signaling in the patterning of the DV axis of L. peitaihoensis by investigating the expression patterns of those marker genes. When treated with SU5402, the expression polarity of gata2/3 along the DV axis was eliminated and exhibited a radial pattern (Fig. 5A’, B’). Although soxb and foxa expression along the DV axis retained weak asymmetry (Fig. 5G, G’), it showed a radial trend (Fig. 5h, h’), comparable to that after organizer inhibition (Tan et al. 2022). These results indicate that FGFR signaling regulates DV patterning in L. peitaihoensis.

Fig. 5.

Fig. 5

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The expression patterns of marker genes at 6 h postfertilization (hpf) after SU5402 treatment. Lateral views (animal to the top) are provided in the left panel, and vegetal views are shown in the right panels. A-D The genes expressed in the dorsal-type tissues. E, F The expression of ventral-type markers. G-J The expression patterns of blastoporal genes. Panels h and h’ show the clearest gene expression pattern of foxa enclosed by white dashed lines, which could be observed from the view indicated by red triangles in G and G’. The diagrams are derived from the expression patterns of marker genes shown in A–J and A’–J’. K–N The expression of bmp2/4 and chordin. The white crosses in G, I and G’ indicate endo/mesodermal staining, and the white asterisks in E, E’, K, K’, M and M’ indicate the staining in the pretrochal region, which is not discussed here. The diagrams show the body plans derived from the expression patterns of marker genes shown in other panels. pt, prototroch. The bars represent 50 μm

Furthermore, we also found that some marker genes were not evidently affected by SU5402 treatment. The expression patterns of hox1 (another dorsal marker) and brachyury (a blastoporal marker) in treated embryos showed evident asymmetrical patterns, which were nearly the same as those of control larvae (Fig. 5D’, J’), although the locations were slightly distorted (Fig. 5C’, I’).

We also explored the expression of the BMP signaling components, bmp2/4 and chordin, which mediate organizer signaling in DV patterning. The results revealed that the expression of bmp2/4 showed a bilateral symmetry pattern perpendicular to DV axis (Fig. 5L’), indicating the loss of the DV polarity which was comparable to the radial expression of gata2/3. However, chordin was not seriously affected (Fig. 5M, M’), seemingly resembling the expression of the blastoporal gene brachyury.

Similar phenotypes after incomplete inhibition of MAPK signaling

SU5402 treatment resulted in a range of developmental defects from mild abnormalities to severe disorders related to organizer function, both in larval morphology and gene expression patterns. However, these deficiencies were not as severe as those observed when treating the embryos with the MAPK inhibitor U0126 (Tan et al. 2022). Together, these results seemed to indicate that organizer activity was not completely inhibited by SU5402 treatment. As we continued to increase the concentration of SU5402, the inhibitor showed strong toxicity to larval development. To further illustrate the effects of SU5402 on the organizer activity and function, we performed an alternative experiment using the MAPK signaling inhibitor U0126 using varied concentrations (a high dose (75 μmol/L) as previously used (Tan et al. 2022), and a low dose (25 μmol/L)) (Figs. 6, 7).

Fig. 6.

Fig. 6

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Larval phenotypes after low-dose and high-dose U0126 treatment. Larval morphology and eyes in manipulated individuals. In each group, the general larval morphology is shown in the upper panels (lateral views with anterior to the top), and larval eyes are shown in the lower panels (anterior views). Multiple individuals are shown since the larval morphology showed various degrees of heterogeneity. The bars represent 50 μm

Fig. 7.

Fig. 7

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The statistical summary of larval phenotypes (A) and the organizer states and pSmad1/5/8 activities B under low-dose and high-dose U0126 treatment. A Statistical summary of larval phenotypes. There is significance between treatment conditions if the letters with asterisks differ (P < 0.05, Chi-square test). B The state of cell arrangement patterns of manipulated embryos at the vegetal pole after U0126 treatment (Ba-Bc), and the distribution of pSmad1/5/8 activity at the animal pole after U0126 treatment (Bd-Bf). The bars represent 50 μm

The high-dose U0126 treatments led to the same results that we previously reported (Tan et al. 2022). The development of organizer-induced organs in all embryos (n = 52) was severely disordered (Figs. 6C, 7A), and 61.5% (32/52) of individuals had extra eyes (Figs. 6D, 7A). However, when the U0126 concentration was reduced to 25 μmol/L, the individuals (n = 154) showed phenotypes similar to those under SU5402 treatment (Figs. 3, 6). Approximately 40% (62/154) of treated larvae showed mild phenotype with recognizable shells and feet (Figs. 6E, 7A), and moderate phenotype with obviously reduced feet and shells was observed in 27.3% (42 cases) of individuals (Figs. 6E’, 7A), while the other 50 cases (32.5%) showed severe phenotypes (Figs. 6E’’, 7A). For eye development, we found that 94 cases of the manipulated embryos (n = 154) had two eyes, and in 57 cases, external eyes were observed (Figs. 6F, 7A). Similar to the results in SU5402 treatment, we found the BMP signaling gradient was retained in the low-dose U0126 treatment group, while it was generally eliminated by high-dose U0126 treatment (Fig. 7B; Supplementary Fig. S2). The high similarity between the phenotypes caused by SU5402 and low-dose U0126 treatment supported the idea that the phenotypes in the 24 μmol/L SU5402 treatment were caused by incomplete blocking of organizer activity.

Discussion

The role of FGFR signaling in the organizer specification of L. peitaihoensis

The fact that the D-quadrant organizer sets up the DV axis is a crucial characteristic during the development of spiralian embryos (Henry 2014). The current understanding of the molecular mechanisms underlying the process comes only from studies in limited species, which reveal that MAPK signaling determines the D-quadrant organizer specification and mediates its function in regulating DV patterning (Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2001, 2003).

The specification of the organizer can be reflected by activated MAPK signaling (i.e. the dpERK) in various spiralian species. However, the states of MAPK signaling in L. peitaihoensis could not be directly explored by dpERK staining due to technical problems. We therefore inferred the relationship between FGFR and organizer specification based on the changes in cell arrangement at the vegetal pole. The altered cellular morphology and arrangement caused by the failure of organizer specification have been frequently reported. During the stage of organizer specification, embryos treated with specific inhibitors (e.g., U0126, brefeldin A, or monensin) show symmetrical division of 3Q macromeres at the vegetal pole in the gastropods Patella vulgata and L. peitaihoensis, the polyplacophoran Mopalia muscosa, and the scaphopod Antalis entalis (Gonzales et al. 2007; Henry and Perry 2008; Tan et al. 2022), which is similar to the phenotype observed here (Fig. 4A). We also investigated the BMP signaling gradient at 60-cell stage, which was determined by the organizer (Tan et al. 2022). As expected, we found that the pSmad1/5/8 gradient was reduced after treatment with the FGFR inhibitor (Fig. 4Bc’, Bf). Taken together, these consistent phenotypes suggest that FGF signaling regulates organizer specification in L. peitaihoensis. It is reasonable to propose that during the stage of 3D blastomere specification, FGFR activates MAPK signaling and thus determines organizer specification. This, to some extent, answers the open question of which molecule activates MAPK signaling in the molluskan organizer.

The organizer-induced DV patterning of L. peitaihoensis was largely coupled with gastrulation (Tan et al. 2022). As gastrulation proceeded, the blastopore (and related tissues) moved from posterior to ventral, and the expression patterns of marker genes including gata2/3, soxb, foxa and so on, which were generally broad and mostly radial initially, became restricted along the DV axis. After U0126 treatment, the expression of these marker genes showed almost radial patterns, reflecting radialized development after the inhibition of organizer function (Tan et al. 2022). We found that the failure of organizer specification caused by FGFR inhibitor treatment also resulted in radialized development in early embryos (Fig. 5B’, F’, H’), although not all marker genes were expressed in a radialized pattern. These results show a radialized phenotype similar to that reported in embryos of some spiralia species, in which the inhibition of the organizer caused radialized subsequent development, especially at the gastrula stage (Gonzales et al. 2007; Koop et al. 2007). In the gastropod Haliotis asinine, the expression of dorsal shell field markers show a ringed pattern in the perturbed larvae (Koop et al. 2007). In the gastropods P. vulgata and Lymnaea stagnalis, and the polyplacophoran M. muscosa, the inhibition of organizer function causes the embryos to lose the capacity to exhibit bilateral cleavage, and the manipulated larvae showed radialized body plans (Gonzales et al. 2007). Further observations indicated that the effect of FGFR-inhibitor treatment on organizer function was maintained to the larval stage and resulted in a range of phenotypes displaying varying degrees of disturbance to organizer-induced organs, such as foot and shell (Fig. 3D, D’’).

Partial inhibition of organizer function under SU5402 treatment

Although SU5402 treatment interfered with organizer function, the resulting phenotypes were not as severe as those observed when treated with the MAPK inhibitor U0126 (Tan et al. 2022), specifically in the gene expression patterns and larval morphology (Figs. 3D, 5), indicating that this treatment condition could not completely inhibit organizer activity. Due to the nonspecific effects of high concentrations of SU5402 treatment (data not shown), we used treatment with a low dose of U0126 as an alternative to investigate this possibility. This treatment revealed phenotypes similar to those in the SU5404 treatment, in contrast to the severe phenotypes caused by high-dose U0126 treatment (this study and Tan et al. 2022), supporting the prediction that the relatively moderate phenotypes after 24 μmol/L SU5402 treatment was caused by incomplete inhibition of organizer function.

Varied phenotypes resulting from different degrees of inhibition of organizer activity have also been revealed in several mollusks (Koop et al. 2007; Kozin et al. 2013; Lambert and Nagy 2003; Martindale 1986). In H. asinine, most of trochophores treated with a low concentration of MAPK inhibitor possess normal structures such as the foot and shell, with only 25% of manipulated larvae being severely affected. When the concertation increased, the development of all larvae was severely perturbed (Koop et al. 2007). In Tectura scutum, low concentrations of MAPK inhibitor resulted in only minor developmental deficiencies (head was rotated 90 degrees from the normal relation with the shell), and higher concentrations prevented the specification of ectodermal structures, such as foot and external shell (Lambert and Nagy 2003). Finally, it is notable that although SU5402 has been widely used in the study of FGFR signaling, it also targets VEGFR signaling (Sun et al. 1999). More specific inhibition of FGF signaling (e.g., the application of FGF ligand/FGFR morpholinos) may help support our results.

The potential dose effect of SU5402 treatment, as well as previous reports mentioned above, indicate that the various effects of organizer function have different sensitivities to MAPK signaling. According to our results, the morphology of the vegetal blastomeres, BMP signaling gradient and the expression of particular genes (gata2/3, soxb etc.) seems to be sensitive to the influences of organizer function, while other aspects (the development of organizer-induced organs and hox1/brachyury expression) are more robust. The potential dissociation of different aspects of organizer effects and their divergent sensitivities to influences warrant further investigation.

The evolution of FGFR signaling function in organizer specification

Our results indicate the regulatory mechanisms of FGFR and organizer specification in the mollusk L. peitaihoensis. Recently, the study in the annelid O. fusiformis also showed that FGFR determined organizer specification by regulating MAPK signaling (Seudre et al. 2022). Combined with studies of MAPK signaling in other mollusks and annelids (Henry and Perry 2008; Koop et al. 2007; Lambert and Nagy 2003), our study establishes a possible regulatory network for organizer specification, in which FGFR-induced MAPK signaling determines organizer specification. Moreover, we revealed that BMP2/4 and Chordin mediate MAPK signal activated in the organizer of L. peitaihoensis (Tan et al. 2022). Although chordin has been lost from the genome of many annelids, it has been retrieved from O. fusiformis and showed asymmetric expression in early embryos supporting a role in DV patterning (Martín-Zamora et al. 2023). Together, these results seem to support a common network of organizer function involving FGFR-MAPK-BMP in two major phyla (Mollusca and Annelida), suggesting an ancestral state among spiralians. The various manners of organizer function/DV patterning in several spiralians investigated (the noninvolvement of MAPK signaling or BMP signaling etc.) (Amiel et al. 2013; Kozin et al. 2016; Lanza and Seaver 2020; Pfeifer et al. 2014) may be the result of evolutionary divergence.

Materials and methods

Embryo collection

Adults of L. peitaihoensis (Grabau & S. G. King, 1928) were collected from intertidal rocks in Qingdao, China. This species was identified as a new Lottia species (L. goshimai) (Nakayama et al. 2017), but recently it was recognized to be a junior synonym of L. peitaihoensis (Grabau & S.G. King, 1928) (Zhang and Zhang 2022). Gamete collection and in vitro fertilization were conducted as described previously (Tan et al. 2022). The fertilized eggs were cultured in filtered seawater (FSW) with 100 unit/mL benzylpenicillin and 200 μg/mL streptomycin sulfate in an incubator at 25 °C until the desired embryonic stage. Developmental stages were referred to by hpf except for early developmental stages (e.g., the 60-cell stage).

Genes and orthology assignment

The O. fusiformis FGFR (GenBank accession CAH1782894.1) was used to search against the deduced peptides encoded by the developmental transcriptome of L. peitaihoensis that we developed previously (Huan et al. 2020), to identify the Lottia FGFR homolog peptides with sequence similarities. Phylogenetic analysis was performed using maximum likelihood (ML) methods with the IQ-TREE package (http://www.iqtree.org/). Whole amino acid sequences were used in the phylogenetic analysis.

Inhibitor experiments

Inhibitor experiments were performed in 12-well plates. SU5402 (Selleck; Cat No: S7667) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 60 mmol/L and stored at − 20 °C. Preliminary experiments tested a concentration range from 6 to 90 μmol/L, and 24 μmol/L was selected as the optimal concentration at which there was a stably reproducible phenotype. At the 16-cell stage (~ 1.3 hpf), the SU5402 storage solution was added to FSW at the optimal concentration. In the control group, an equivalent volume of DMSO was added. Embryos were then raised to the 60- to 64-cell stage (approximately 3.5 hpf). After treatment, embryos were transferred to FSW followed by three FSW washes, and then embryos were either collected for downstream analyses or raised until the desired larval stage. The U0126 treatments were conducted as described previously (Tan et al. 2022).

Sample fixations were conducted as previously described (Tan et al. 2022). For immunohistochemistry, DAPI staining and gene expression analyses, embryos were fixed with 4% paraformaldehyde (PFA) (1 × PBS, 100mmol/L EDTA, 0.1% Tween-20, pH 7.4) overnight at 4 °C. To investigate larval shell development, the 48-hpf larvae were anesthetized by adding 125 mmol/L magnesium chloride and then fixed in 2.5% glutaraldehyde for 2 h at room temperature. After fixation, the samples were either washed several times with PBST (1 × PBS with 0.1% Tween-20) and stored at 4 °C for whole-mount immunohistochemistry (WMIHC) or dehydrated to 100% methanol and stored at − 20 °C for whole-mount in situ hybridization (WMISH).

Phalloidin staining, WMIHC and WMISH

Phalloidin staining, WMIHC and WMISH were conducted as described previously (Huan et al. 2020; Kurita et al. 2009; Tan et al. 2022). Embryos at the 60–64 cell stage were stained for pSmad1/5/8 using a commercial rabbit monoclonal antibody (Cell Signaling Technology, Cat. No. 13820S). The quantitative analysis of pSmad1/5/8 signals was performed as described previously (Tan et al. 2022). In brief, the pSmad1/5/8 activities of an embryo were estimated based on the relative pSmad1/5/8 signals in blastomeres at the animal pole. Since the pSmad1/5/8 levels showed graded distributions (although this gradient was reduced in treated embryos), these blastomeres were divided into two groups, namely the D-side group and the B-side group. The relative pSmad1/5/8 signals were calculated by normalization against to the background fluorescence intensity. In total, five embryos were measured from each group (Supplementary Fig. S1). For statistical analysis, the average value of relative pSmad1/5/8 levels was calculated for each group of blastomeres. Statistical analyses were performed using ANOVA and a significance threshold of 0.05 was used. The primers used to generate the probes of WMISH have been reported previously (Tan et al. 2022). Two primers, lpe-fgfr-f (5ʹ -ATGAGATACCACTTGATGCAGACTG-3ʹ) and lpe-fgfr-r (5ʹ -CCAATGTTCCAACCAACTGACTGA-3ʹ), were used to generate probes for WMISH. Samples were observed and recorded under a Nikon 80i microscope.

Imaging

The samples were observed and recorded using a Nikon 80i microscope or a ZEISS LSM 710 laser-scanning confocal microscopy system.

Statistical analysis

A chi-square test was performed to test for a statistically significant difference between the treated group and the corresponding control. Larvae were evaluated and sorted into one of several categories depending on phenotypic difference. For shell and foot development, there were four categories: normal, mild, moderate, and severe. For eye numbers there were three categories: fewer than two eyes, two eyes and more than two eyes. To determine differences, we applied an omnibus Chi-square test of homogeneity, followed by post-hoc pairwise comparisons using a z-test of two proportions. Bonferroni correction was applied to account for multiple comparisons.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 367 KB) (366.5KB, docx)

Acknowledgements

The authors thank the two anonymous reviewers for their thoughtful suggestions. This work was supported by the National Natural Science Foundation of China (grant numbers 42206092, 42076123); the Earmarked Fund for China Agriculture Research System (grant number CARS-49); and the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (No. LSKJ202203002).

Author contributions

BL and PH designed the project. ST and PH performed the experiments, analyzed the data and drafted the manuscript. BL contributed to data analyses and critically revised the manuscript. All authors read and approved the final manuscript.

Data availability

The original contributions presented in the study are included in this published article (and its supplementary information files), further inquiries can be directed to the corresponding author.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Animal and human rights statement

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.

Footnotes

Edited by Jiamei Li.

Special Topic: EvoDevo.

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Associated Data

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

Supplementary Materials

Supplementary file1 (DOCX 367 KB) (366.5KB, docx)

Data Availability Statement

The original contributions presented in the study are included in this published article (and its supplementary information files), further inquiries can be directed to the corresponding author.

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