Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.


| INTRODUCTION
Extant vertebrates are composed of cyclostomes, i.e., hagfish and lamprey, and gnathostomes, i.e., jawed vertebrates (Janvier, 2015). One of the major anatomical differences between them is that only gnathostomes generate bones. Paired appendages are a unique feature of gnathostomes, which are formed during embryogenesis by contributions from the ectoderm, paraxial mesoderm, and lateral plate mesoderm (Cass et al., 2021;Gilbert, 2016;Nakamura et al., 2021). The lateral plate mesoderm is a layered structure that gives rise to a variety of cell types including the heart, smooth muscles, blood, and fin/limb skeletons ( Figure 1a; Prummel et al., 2020). When the lateral plate mesoderm is formed, it is initially connected to the somites (transient mesodermal blocks). However, later in development, it is divided into the somatic mesoderm, which forms the body wall, and the splanchnic mesoderm, which forms the external layer of the digestive tract; the space between these layers is called the coelom (Figure 1a,b). The formation of coelom has been described in chicken and mouse embryos but not in zebrafish embryos (Prummel et al., 2020). This is even visible in shark embryos, implying that the two-layered (somatic and splanchnic) lateral plate mesoderm evolved before the split of the Chondrichthyes and Osteichthyes (Adachi & Kuratani, 2012). Fin/limb fields are defined as specific regions in the body walls of gnathostome embryos that coordinate the formation of paired appendages.
Extant cyclostomes consist of lampreys and hagfish, which diverged from the common ancestor of vertebrates in the early phase of vertebrate evolution (Janvier, 2015). Previous research indicates that despite their derived features, cyclostomes retain several morphological elements critical for understanding the emergence of gnathostomes. It also indicates that comparisons between cyclostomes and gnathostomes can be useful for estimating the early evolution of vertebrates (Janvier, 1996;Kuratani et al., 2018). The lateral plate mesoderm of lamprey embryos expresses regional marker genes similar to those in gnathostomes (e.g., hand1/2; Onai et al., 2017). It is thought to be patterned into an anterior part that forms the heart tube and a more posterior part (Onimaru et al., 2011). Morphologically, the study indicated that the posterior lateral plate mesoderm does not transform into the somatic/splanchnic mesoderm in lamprey embryos, whereas another report (Tulenko et al., 2013) indicated that this transformation occurred at stage 28 when seven pairs of gill openings became visible.
A previous study proposed that sequential changes, including a gain of the somatic mesoderm in the trunk of the ancestral lateral plate mesoderm, enabled the evolution of paired appendages (Onimaru et al., 2011;Tanaka, 2016). In contrast, the latter suggests that the somatic mesoderm is present in lamprey embryos, whereas persistent somatopleure is a gnathostome synapomorphy (Tulenko et al., 2013). This conflict is caused by the technical limitations of the histology of the embryos. In lamprey embryos, yolk granules are abundant, and the space between tissue layers is difficult to observe. Thus, light microscopic analysis (e.g., paraffin sectioning) alone cannot determine whether the lateral plate mesoderm contains two layers, necessitating ultrastructural investigations using transmission microscopy (TEM). Likewise, whether the somatic mesoderm has a mesenchymal identity in lamprey embryos remains unclear, which is the fundamental identity of the somatic mesoderm of gnathostomes. Therefore, in this study, TEM analysis was performed on lamprey embryos to examine these features.

| Collection of adult lampreys and in vitro fertilization
Adults Lethenteron camtschaticum collected from rivers in Hokkaido were reared in laboratory tanks at 12°C. Fertilization was performed as previously described (Onai et al., 2015). Fertilized eggs were washed several times with water and reared in 10% Steinberg's solution until development reached the desired stage.

| Transmission electron microscopy and 3D reconstruction of the embryos
Embryos fixed in 2% paraformaldehyde/2.5% glutardialdehyde in PBS (−) were replaced in 0.1 mol L −1 phosphate buffer (PB) (pH 7.4) for 10 min three times. The samples were then fixed in 1% osmium tetroxide in 0.1 mol L −1 PB at 4°C for 2 h. Block staining was performed with 2% uranyl acetate at 4°C for 60 min. Dehydration was performed in 50%-100% EtOH, and the solution was changed twice with propylene oxide for 15 min. Resin embedding was performed using Quetol 812 (Nissin EM). Resin sections with a thickness of 90 nm were prepared using an ultramicrotome. Next, double staining with uranyl acetate and lead citrate was performed, and the sections were observed using an transmission electron microscope (Hitachi H-7650). For the plastic sections, normal toluidine blue staining was performed. The number of samples examined at each stage was as follows: 21 (2), 23 (2), 24 (2), 25 (2), and 27 (2). For 3D reconstruction of the somatic mesoderm at stage 27, TEM images were obtained by sectioning 90 nm thick sections. The images were then analyzed and reconstructed using Dragonfly software (Object Research Systems).

| RESULTS
3.1 | Development of coelomic lateral plate mesoderm in early to mid-pharyngeal lamprey embryos The lateral plate mesoderm was situated lateral to somites. The lateral plate mesoderm first appears as an elongated cell sheet, in F I G U R E 1 A scheme of the lateral plate mesoderm. (a) Generalized mesoderm organization in vertebrates. Along with the anterior/posterior axis, the mesoderm is regionalized into the head/trunk mesoderm. Head mesoderm consists of premandibular, mandibular, hyoid, and peri-otic head mesoderm. The lateral plate mesoderm is situated lateral to somites. There are conflicting views about its position. Some authors regard Cardio-pharyngeal mesoderm as LPM but are considered the head mesoderm by others (Lescroart et al., 2022;Prummel et al., 2020;Schubert et al., 2019). Both viewpoints are considered in the identification of lateral mesoderm. (b) The lateral plate mesoderm consists of somatic and splanchnic mesoderm; the coelom is formed between them. hm; hyoid head mesoderm, mm; mandibular head mesoderm, ov; otic vesicle, pmm; premandibular head mesoderm, pom; peri-otic head mesoderm, s; somite.
F I G U R E 2 Lethenteron camtschaticum; formation of the lateral plate mesoderm from stages 21 to 23. (a) Stage 21 lamprey embryo. White lines indicate sectioned planes. a1 indicates a transverse plastic section of the white Line 1 and a2 indicates the white Line 2 section. (b) Stage 23 embryo. Two transverse sections are indicated as white lines. b1 and b2 panels indicate the sectioned regions in (b). In each panel, the white dotted box is zoomed area that is shown as TEM images in the right panels. (c) A scheme of lateral plate mesoderm formation is examined in the above stages. en; endoderm, lpm; lateral plate mesoderm, nc; notochord, nt; neural tube, s; somite; TEM, transmission electron microscopy. which the ventral tip contains fewer cells than the more dorsal part.
The lateral plate mesoderm is recognized between the ectoderm and endodermal gut. These topological and morphological characteristics were used to identify the lateral plate mesoderm in this study. By stage 21(early pharyngeal stage) of lamprey embryos, the lateral plate mesoderm is located lateral to somites (Tahara, 1988 ;Tahara, 1988). Damas (1944) showed that there was somatic and splanchnic mesoderm lateral to the somites near the B1 section plane at stage 23; however, such an organization was not observed in my observations. In the caudal region, there was a coelom in the lateral plate mesoderm and the number of cells was much higher than that at the cardiac level ( Figure 2b, Line 2).
Transmission electron micrographs showed that the coelom was filled with the extracellular matrix ( Figure 2b

| Formation of somatic mesoderm in the trunk body wall
My histological observations suggest that in lamprey embryos, somatic and splanchnic mesoderm formation is present caudal to the heart; thus, the ancestral condition of the lateral plate mesoderm might not include the absence of the posterior somatic mesoderm (Onimaru et al., 2011). This raises the question whether it is not the absence of the somatic mesoderm, but of other developmental mechanisms that are important for the evolution of the fins/limbs. For example, in fin/limb buds, the epithelial somatic mesoderm transforms into mesenchymal cells and proliferates rapidly to generate buds (Gros & Tabin, 2014). To address this question, later-stage embryos were examined to determine the similarities and differences between lamprey and gnathostome somatic mesoderm  (Figure 4e, 4g, 4l). The coelom-enclosed extracellular matrix (ecm) was similar to that of stages 23 and 24 (Figure 4g).
Several somatic mesodermal cells overlapped and had thin cytoplasmic extensions (Figure 4g, arrowheads). In the caudal region, somites and nephron primordium were formed near the tail bud (Figure 4h, 4i, 4l;Damas, 1944). Several lateral plate mesodermal cells spread ventrally around the nephron primordium (Figure 4h-l). The most ventral somatic mesoderm was located beneath the epidermis with a well-developed basal lamina (Figure 4k). Compared to gnathostome limb/fin buds, the number of somatic mesoderm cells was much smaller in lamprey somatic mesoderm (Figure 4f and 4l; Tulenko et al., 2013), which might be related to the absence of fin buds in lamprey embryos. ). In chicken limb buds, ecm organization is rather poor compared to that in lamprey embryos (Ede et al., 1974). The dermomyotome cells were not arranged in a single layer but rather in a cluster (Figure 5f, pink dotted circle). Moreover, this cell cluster may possess migrating muscle progenitor (MMP)-like properties, resulting in the formation of hypobranchial muscles (HBM; Adachi et al., 2018). Notably, the dermomyotome was no longer visible in the ventral region, but the lateral plate mesoderm extended to the ventral end (Figure 5f[v, vi]). The lateral plate mesodermal cells were aligned horizontally and were densely adjacent to the ventral end (Figure 5f[vi]).

| Cell layer composition of the body wall unique to lamprey embryos
The myotome and dermomyotome were differentiated caudal to the heart, and the space between the epidermis and dermomyotome was filled with ecm, which included the basal lamina and the basal side of the epidermis (Figure 6a,b). The ventral distribution of the dermomyotome was considerably less than that in the cardiac region, where the somatic mesoderm encountered the ecm medial to the dermomyotome (Figure 6c[i, ii] and 6g[2]). The somatic and splanchnic mesoderm were separated by a coelom, but they were so close that only a small amount of coelom was visible in several regions (Figure 6c[iii] and 6g[2]). Additionally, there was an opening between the somatic mesoderm and ectoderm (Figure 6c[iv, v]). The lateral plate mesoderm formed multiple layers (Figure 6c[vi]). Cells of the somatic/splanchnic mesoderm displayed thinning cytoplasm at their ends, and serial sections were taken to determine whether these were filopodia, a mesenchymal character. The morphology of the somatic mesoderm was not different in any region, so the projecting portion of the somatic mesoderm corresponding to the Figure 6c-iv region, slightly posterior to the image in Figure 5a section Line 2, was selected for reconstruction. Through a 3D reconstruction of the somatic mesoderm cytoplasmic extension, it was discovered that the length along the Z-axis was significantly longer than that along the XY plane, indicating that the cytoplasmic extension was not filopodia but rather a sheet-like structure (Figure 6d Figure 6. The plastic sections are also depicted (b, c) and the TEM sections (d-f). White or black boxes in TEM images are zoomed regions shown in the right panels for (d, e) or left panels for (f, i-vi). The pink dotted circle in (f) indicates an interface between MMP-like cells and the dermomyotome. bl; basal lamina, dm; dermomyotome, ecm; extracellular matrix, ep; epidermis, im; inner myocardium, k; kidney, lpm; lateral plate mesoderm, m; myotome, MMP; migrating muscle precursor cells, slp; somatic lateral plate mesoderm, splp; splanchnic lateral plate mesoderm; TEM, transmission electron microscopy. a somatic/splanchnic mesoderm that enclosed the coelom (Figure 2). At stage 24, the lateral plate mesoderm encloses the intestinal tract and is connected to both sides (Figure 3). At this stage, unlike in the cardiac to more caudal regions, no coelom was formed rostral to the cardiac region, indicating a region-specific distribution of the somatic/splanchnic mesoderm in lamprey embryos (Figure 3). A similar organization of the somatic/ splanchnic mesoderm was also found at stage 25 (Figure 4). The small coeloms in section Line 2 at stage 25 or section Lines 5 and 6 at stage 24 appear to be related to the formation of the somatic and splanchnic mesoderm at stage 27 (Figures 3q and 4l[6, 7]).
However, the exact mechanisms for the transformation of such a small coelom to rather long coeloms are not clear. The coelom may expand by ventral unzipping of the lateral plate into somatic and splanchnic layers. Alternatively, additional openings appear and merge to form somatic and splanchnic layers. In chick embryos, the initial coelomic opening between the two layers of the lateral plate mesoderm seems to be directly transformed into the somatic and splanchnic mesoderm (Prummel et al., 2020). The small coeloms in the trunk lateral plate mesoderm of lamprey embryos found in this study are unlikely to be a common feature in vertebrates. It would be interesting to determine whether there are any small coeloms found in hagfish embryos in future studies.
At stage 27, the coelomic space between the somatic and splanchnic mesoderm was small, and the cell distribution of the somatic and splanchnic mesoderm was rather complicated ( Figures 5 and 6). The above observations indicate that in lamprey embryos, the lateral plate mesoderm exhibits somatic/splanchnic mesoderm in a region-specific manner. A previous study suggested that the somatic mesoderm is not present in the post-cardiac lateral plate mesoderm (Onimaru et al., 2011). However, another study suggested that the somatic mesoderm is present from stage 28 to later stages (Tulenko et al., 2013). My current study revised these observations in terms of the earlier stage presence of the somatic mesoderm in the post-cardiac lateral plate mesoderm with surprising dynamical organization spatiotemporally. For the organization of somatic/splanchnic mesoderm at stage 27, there were several regions in which the coeloms between the somatic mesoderm and splanchnic mesoderm were narrow, and cells overlapped with each other (Figure 6c). Therefore, future lineage-tracing studies would be useful in determining the origins of such cell populations.

| Body wall environment and the paired appendage evolution
Mesenchymal cells accumulate under the epidermis and generate fin/limb buds during paired appendage development and produce skeletons. Muscles are formed by MMPs, which arise from the ventral tip of the dermomyotome, a lateral somite (Zuniga, 2015).
Since the 19th century, there have been two major hypotheses regarding fin/limb evolution: the gill arch theory (Gegenbaur, 1859) and the fin-fold theory (Balfour, 1881;Thacher, 1877). According to the gill arch theory, the shoulder girdle evolved by modifying a portion of the gill arch skeleton (Gegenbaur, 1859). In contrast, the fin-fold theory predicts that pectoral and pelvic fins evolved from the long elongated ventral fins of ancestral vertebrates (Balfour, 1881;Thacher, 1877). Recent advances in molecular embryology have supported both hypotheses (Diogo, 2020;Sleight & Gillis, 2020;Tulenko et al., 2013). Furthermore, tbx4/5 is important in fin/limb bud formation, and enhancer analysis indicated that its expression in the body wall may have been the most important factor in fin/limb evolution (Adachi et al., 2016;Minguillon et al., 2009). The role of tbx5 was determined to be critical for the epithelial-mesenchymal transition (EMT) of the somatic mesoderm during the early stages of limb development (Gros & Tabin, 2014).
The major developmental innovations in the evolution of paired appendages have been identified as (1) the emergence of MMP in the ventrolateral edge of the dermomyotome to generate fin/limb muscles, and (2) the EMT and extensive proliferation of somatic mesoderm to provide fin/limb skeletal tissues (Brohmann et al., 2000;Gros & Tabin, 2014). Recently, the EMT of lbx1-positive MMPs in shark embryos was confirmed, suggesting conserved mechanisms for MMP EMT (Okamoto et al., 2017). However, a comparison of MMPrelated genes (HGF/MET) in gnathostomes and lamprey embryos indicated that the mechanisms of EMT in vertebrates have diverged (Adachi et al., 2018). This also implies that the cell structure of MMP may vary between vertebrate species. Lamprey HBM is thought to be derived from gnathostome MMP homologs (Adachi et al., 2018).
will be necessary to compare their ultrastructure to that of the gnathostome to clarify the morphology of MMP cells in different vertebrate species. Extracellular matrix structure may be critical for cell migration through the body wall. Furthermore, in stage 27 lamprey embryos, a dense ecm complex was observed (Figures 5f   and 6c). This abundant ecm complex has not been observed in the fin/limb buds of frogs, fish, or chick embryos (Ede et al., 1974;Masselink et al., 2016;Richardson et al., 1998). In chick embryos, a thin collagen layer is located adjacent to the basal lamina on the basal surface of the epidermis, which is significantly less dense than the extracellular matrix in lamprey body walls (Ede et al., 1974). Thus, these findings imply that dermomyotome de-epithelialization and changes in the repertoire of the surrounding extracellular matrix play a role in the evolution of paired appendages. At the molecular level, lamprey MMP-like cells have not been well examined for comparison with gnathostomal MMPs. This information should be added in the future to better understand the fundamental similarities and differences between them.
The somatic mesoderm is located inside the dermomyotome of the body wall (Tulenko et al., 2013; Figure 6g). However, this body wall developmental program is uncommon in vertebrates (Tulenko et al., 2013). In shark embryos, the somatic mesoderm spreads close to the epidermis by enclosing the dermomyotome (Tulenko et al., 2013). The organization is observed in trunk body walls other than the prospective pectoral/pelvic fin regions (Tulenko et al., 2013).
Furthermore, it is essential to understand the regulatory mechanisms of cell proliferation in the lamprey and gnathostome somatic mesoderm. If changes in cell number resulted in changes in the positional relationship of the somatic mesoderm and dermomyotome, then the evolution of the body wall environment that resulted in the fin/limbs would have been straightforward. Moreover, as directional cell activity and physical forces are thought to be important for fin/ limb bud formation, it is critical to understand the evolution of these relationships (Boehm et al., 2010;Lau et al., 2015). However, there are few comparable data on the body wall architecture of hagfish available (Ota et al., 2011). Therefore, research on hagfish embryos is critical.
The potential for mesenchymal identity in lamprey embryonic somatic mesoderm has been a subject of considerable debate (Damas, 1944;Tulenko et al., 2013). This study found that somatic mesodermal cells were lengthy, with sheet-like converging cytoplasmic ends (Figure 6d-f). These findings imply that they are nonmesenchymal in nature. The mesenchymal cells in chick limb buds are flattened and have numerous filopodia (Ede et al., 1974), which connect to other cells, allowing for the long-distance secretion of the SHH protein, a critical factor in the formation of limb buds (Ede et al., 1974;Sanders et al., 2013). These differences demonstrate that the embryonic somatic mesoderm lacks the histological and functional properties of chick embryos. In addition, tbx5 and other key GRNs may have been involved in the evolution of the somatic mesoderm into a type of mesenchyme with filopodia in the body wall.
Furthermore, future studies could shed light on how GRNs such as these are formed.

| CONCLUSIONS
For many years, the evolutionary origin of paired appendages has been studied from the perspective of developmental body plan changes. The somatic mesoderm is thought to be the key embryonic element for evolution. Current findings support the hypothesis that ancestral vertebrates have a body wall with a somatic mesoderm transformed from the lateral plate mesoderm. However, several developmental sequences (e.g., mesenchymal somatic mesoderm and thin extracellular matrix complex near the epidermis) for the gain of

CONFLICT OF INTEREST STATEMENT
The author declares no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.