Formation of body appendages during caudal regeneration in Platynereis dumerilii: adaptation of conserved molecular toolsets

Across the animal kingdom, body appendages have evolved in various phyla. Morphology of individual appendages has been adapted according to their function. During morphogenesis, the position at which a new appendage is supposed to form is determined and additional body axes, the proximodistal axes, of the new appendages are established. Concurrently dorsoventral and anteroposterior compartments of the primordium are patterned. Despite large morphological differences between body appendages of different phyla, it has been found that homologous or orthologous genes are often involved in similar regulatory processes during body appendage formation. The regulatory gene networks controlling the formation and shaping of body appendages during embryogenesis have so far been studied in detail in dipteran insects and vertebrates only. However, formation and patterning of a vertebrate limb differ in many aspects compared with development of the arthropod leg. Vertebrate limbs are mainly formed by mesodermal tissue [1], whereas arthropod legs are epidermal structures [2]. Two regions, organizing the growth and patterning of the vertebrate limb, have been described before: the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) [3]. A positive feedback interaction between the AER and the ZPA is important for the development of the limb and has no complement mechanism in arthropods [1]. Other differences involve the number of leg elements and joints, the position of skeletal and muscular elements as well as their innervation. All of these differences may, however, be regarded to be apomorphic traits. Therefore, despite shared aspects of some attributes of developmental gene expression, the appendages of vertebrates are independently evolved and non-homologous as structures in an evolutionary sense.

There is a third, morphologically highly diverse group featuring a variety of different body appendages: polychaete annelids. The most prominent body appendages of polychaetes are bifurcated parapodia flanking the segments of the trunk region. In Platynereis dumerilii, these biramous appendages are formed by a dorsal and a ventral branch referred to as noto- and neuropodium. Both bear cirri, the dorsal and ventral cirrus as well as chaetae. The latter project laterally being flanked by spinning glands. Mechanical stability of these lobes is granted by aciculae, stabilizing rods originating in the trunk (Fig. 1). Spatial and temporal expression patterns of several genes known to play important roles in patterning of limb buds in vertebrates and imaginal disks in insects have meanwhile been studied in a variety of representatives of non-dipteran insects and non-insect arthropods [4, 5]. To determine whether these genes are also activated during formation of parapodia and if so to analyze whether these expression patterns show similarities to those observed in other taxa, orthologs of five genes involved in the development of body appendages in arthropods and vertebrates were isolated from the annelid polychaete Platynereis dumerilii: homothorax (Pdu-Meis/hth), extradenticle (Pdu-Pbx1/exd), Distal-less (Pdu-Dll), specific protein 1/buttonhead (Pdu-sp/btd) and decapentaplegic (Pdu-dpp).

Early expression of Distal-less (Dll) in arthropod appendages starts in broad developmental fields with the distal tip in center, but becomes restricted to the distal region during growth of the appendage. The only exception is the proximally located Dll domain present in the insect leg imaginal disk [4, 6]. In contrast to the expression of Dll, in non-insect arthropods homothorax (hth) and extradenticle (exd) are primarily co-expressed and located in proximal regions of the appendage [4, 7–10]. In Drosophila, both proteins seem to be interdependent for stabilization of expression in vivo, but also for nuclear localization of EXD and DNA-binding activity of HTH-EXD [11, 12]. The expression domains of both genes show an interesting aspect. In insects, exd is present in all parts of the appendage anlagen, whereas hth is limited to the proximal region. However, in non-insect arthropods the size of the expression domains of these two genes is reversed. Prpic et al. demonstrated this reversal of spatial expression in the spider Cupiennius salei and described it as an evolutionary shift [8]. Expression of Dachshund (dac) during arthropod development is located between the expression domains of the proximally and distally expressed genes forming a well-conserved intermediate domain [7–9, 13–15] partially overlapping with the Dll domain [6]. For Drosophila, Estella et al. showed that buttonhead (btd) and specific protein 1 (sp1) are required for Dll expression in the future leg primordia [16]. In a later publication, this statement was modified. Btd seems to play a more insignificant role compared with sp1 during early leg development. However, both genes are required in later development [17]. Expression of decapentaplegic (dpp) does not seem to be conserved throughout the arthropods like the previously mentioned genes, and their patterns are by no means similar [4].

The hth homolog Meis disappears from the distal-most parts as the vertebrate limb grows and regresses to proximal regions during elongation of the limb [18, 19]. Interestingly comparable to the situation in insects, co-expression of the hth and exd homologs Meis and Pbx and the formation of heterodimeric complexes of MEIS and PBX [19] was observed in vertebrates. The Dll homologs in vertebrates form the Dlx gene family. These genes were initially found in the ectoderm of the limb bud. Later, Dlx expression is limited to the AER, the region located at a distal rim of the limb anlagen [20]. Sp8 and Sp9 are the vertebrate homologs of the arthropod btd and sp1 genes [17, 21]. At the beginning of the development, Sp8 and Sp9 are expressed in the ectoderm of the limb. Later on, both are found exclusively in the AER. They regulate Fgf8 expression, which in return is required for the expression of Dlx in the distal limb [21]. Dach1 is the vertebrate homolog of dac and is expressed during the initial phase of limb formation in medial and distal parts of the mesenchyme including the AER, but is not expressed in the posterior distal region. During a later stage of limb development, all parts of the mesenchyme and the AER show Dach1 expression [22].

Analysis of transient gene expression in forming segments of Platynereis dumerilii during normal growth by segment addition from the posterior growth zone region is very difficult. Normal segment formation is neither synchronized within the Platynereis culture nor is it predictable. Even animals of exact same age show great variations in size and length and also in growth rates. In addition, segment formation during non-regenerative growth is extremely fast, activating the genes participating in this process only transiently and in an extremely short time frame (minutes to hours compared with days during caudal regeneration). Therefore, formation of posterior structures and activation of genes is studied during caudal regeneration. The favorable conditions of rapid segment growth and body appendage morphogenesis during caudal regeneration allow a thorough and continuous survey of expression patterns.

The development of body appendages during normal development and during the regeneration process does not necessarily require activation of the same subset of genes. Normal development of Platynereis dumerilii is well described by Fisher et al. [26]. The development of the egg starts with spiral cleavage and leads to a first larval stage—the trochophore larva. This trochophora and the subsequent metatrochophore larva possess the typical prototroch used for locomotion; the trunk already possesses the anlagen of the first setigerous segments—three pairs of so-called setal sacs [27, 28] that can be regarded as the imaginal disks of the parapodia. During larval development, these anlagen shift outward and form protruding parapodial buds and setae. Soon afterward, the typical three-segmented young worm—the nectochaete stage—starts first active crawling movements utilizing these parapodia. These early larval segments thus develop simultaneously and are referred to as deutomeres, whereas all subsequent segments develop iteratively from a posterior growth zone region and are called tritomeres. Platynereis dumerilii is capable of caudal regeneration [23, 29], i.e., the loss of a posterior region of the trunk can be regenerated completely. During regeneration, a blastema is formed at the posterior end. While some of the newly formed tissues derive from blastemal cells, others grow into the regenerating end from the posterior stump. The nervous system is built by an outgrowth of the remaining nerve cord in the remaining segments in the stump [25, 30]. Wattez-Combaz demonstrated that the outgrowing nervous system is the driving force for dorsoventral morphogenesis within the regenerating segments [31]. The developmental processes of parapodial morphogenesis in deutomeres, tritomeres as well regenerating segments thus vary, and our results clearly show this for the expression of a typical subset of genes that are also involved in body appendage formation in normogenesis of Platynereis, other annelids and arthropods.

Pdu-Dll expression in young parapodial buds of polychaetes shows similarities to the expression of Dll in appendage primordia during early development of arthropods and vertebrates. The subsequent relocation of the expression domain in distal direction as seen in these taxa [32] can also be observed in the polychaete annelid Platynereis dumerilii. Two of three main expression domains of Pdu-Dll are shifting distally during the ongoing morphogenesis of the segment, these domains persisting at the bases of the dorsal and ventral cirrus, respectively. No expression, however, is found in the distal-most part of the cirri. In arthropods and vertebrates, the Dll expression domain generally extends into the distal tips of appendages in all developmental stages [4]. The third expression domain of Pdu-Dll remains in a proximal position. These results correspond to the work of Winchell et al. on the polychaete Neanthes [33]. During parapodial growth, Pdu-Dll seems to be expressed in an atypical way not explicitly indicating a role in distal patterning of the appendage.

Sp1 and btd in arthropods as well as the vertebrate orthologs sp8 and sp9 are both required for Dll expression in the arthropod and vertebrate leg primordia, respectively. Therefore, expression of these genes can be detected primarily in distal regions of the developing appendage [16, 17, 21]. In Platynereis dumerilii, expression of Pdu-sp/btd seems to be related to Pdu-Dll expression. At first, in young segments Pdu-sp/btd expression is found in the proximal part of the parapodia like Pdu-Dll. During ongoing segment morphogenesis, a shift in distal direction comparable to the shift of Pdu-Dll expression can be observed. The weaker expression and the final position of Pdu-sp/btd after the shift differ from Pdu-Dll expression in the notopodium. This could be explained by the function of sp/btd activating Dll. In older segments, the expression of Pdu-Dll is terminated and likewise Pdu-sp/btd is downregulated. In the neuropodium, no shift in distal direction was observed. The neuropodium shows a persisting proximal expression domain of Pdu-Dll and expression of Pdu-sp/btd at the same position. In addition, expression of Pdu-sp/btd is not downregulated. We thus show that spatial expression of both genes exhibits some striking similarities during development of the appendage. The interaction between Pdu-sp/btd and Pdu-Dll and a resulting role in patterning of the parapodia, however, can only be assumed. Additional experiments (e.g., knockout) could clarify whether Pdu-sp/btd has the same function as in arthropods and vertebrates or is involved in a completely different developmental process.

In Drosophila dpp is involved in the process of determining the anterior–posterior boundary [34, 35], the dorsal and ventral regions of the imaginal disks [36] and the proximodistal axis [6, 35, 37] of the appendage. In other insect arthropods, expression of dpp orthologs is also found in the limb buds. However, this expression is not comparable to the situation in Drosophila and may not have the same function during appendage development [4, 38, 39]. Angelini and Kaufman suggested that a function of dpp in anteroposterior axis formation is conserved, while determination of dorsoventral and proximodistal axis by dpp in other insect arthropods is unlikely [4]. Expression of the polychaete ortholog Pdu-dpp is detectable exclusively in the notopodium, the dorsal part of the parapodium. This is similar to dpp expression in the leg imaginal disk of Drosophila where hedgehog signaling leads to dorsal dpp expression on the anterior side of the anterior–posterior boundary of the disk [34]. This might hint toward a role of Pdu-dpp in defining the anterior–posterior axis of appendages in Platynereis dumerilii. However, expression domains of Pdu-dpp are rather small making it hard to assume they might set clear boundaries in developing parapodia. To confirm or falsify this hypothesis, parapodial expression of other possible anterior–posterior-related genes like wingless and hedgehog orthologs must be investigated in Platynereis in detail.

Hth and exd and their vertebrate orthologs Meis and Pbx are typically expressed in proximal regions of the developing appendages [4, 19]. Spatial expression of these genes is reversed in higher insects compared with the expression domains observed in non-insect arthropods [4, 7–10, 38]. In Platynereis dumerilii, expression of hth and exd is not restricted to the proximal region. Both genes are activated in nearly all regions of the parapodia, excluding the distal part of the neuropodium and the distal tip of the notopodium. Considering the expression in non-insect and insect arthropods, one hypothesis might be an ancestral form of spatial Meis/hth and Pbx1/exd expression with both genes being active all over the appendage primordium. Another possibility for these broad expression domains, however, could be a different function in the developmental process of parapodia compared with that in arthropods or vertebrates. Further functional experiments are needed to reveal the function of hth and exd in formation of annelid appendages.

The question regarding the evolutionary relationship between polychaete parapodia and arthropod limbs has been the topic in several previous publications and lead to an extensive discussion (e.g., [40]). Homology is classically defined as an “historical continuity in which morphological features in related species are similar in pattern or form because they evolved from a corresponding structure in a common ancestor” [41]. Recently, the new definition of “deep homology” has been coined for such cases in which continuity may not directly be obvious (i.e., regulatory mechanisms during development of appendages) [41]. First of all, parapodia and arthropod limbs show no structural similarities except for the basic fact that both develop from ventrolateral buds [42]. Prpic proposed that the early body organization including the limb primordia of the brine shrimp Artemia franciscana is identical to the early development in Platynereis [43–45]. He deduced that both structures are homologous to each other [43]. The primary goal of this study, however, was to determine whether orthologs of genes known to play key roles in patterning of body appendages during development in arthropods and vertebrates are expressed in parapodial primordia during formation of these appendages during caudal regeneration of a polychaete. The second question was whether the patterns observed could be similar to what was shown in arthropods. The morphology of parapodia, however, is rather complex, not only featuring a single proximodistal axis and involving additional regions, as, e.g., dorsal and ventral cirri and several lobes. Expression patterns can thus not easily be compared across phyla. The previously coined “deep homology” hypothesis, however, can not easily be substantiated by results derived from spatial expression data alone. To achieve more clarity, ultimately functional analyses of these genes along with comparisons of the resulting networks are needed to understand their roles during parapodial formation.

The genes analyzed in this study exhibit expression in forming segments including parts of parapodial buds during caudal regeneration of the polychaete Platynereis dumerilii indicating possible roles in formation of the body appendages of annelids. In comparison with data from mostly arthropod clades, however, both similarities and differences of gene expression can be observed. Questions concerning the homology of body appendages across the now obsolete group Articulata, however, could not be answered. The positions at which primordia of segmentally iterated body appendages are formed in arthropods and annelids are not identical. In Platynereis, a segmental compartment exhibits engrailed (en) expression at the anterior segmental boundary and expression of wingless (wg) at the posterior segmental boundary. The parapodial anlagen occupy a mid-segmental position including wnt1/wingless expression at the posterior side of the parapodial base [44]. In insects, transient parasegmental boundaries are established during embryogenesis showing the same distribution of en/wg expression as seen in nereids. However, imaginal disks of insects form at the position of these parasegmental boundaries. Subsequent resegmentation then shifts these anlagen slightly anteriorly.

Representing a toolkit for the patterning of additional body axes, genes have been recruited during evolution to participate in and control the formation of body appendages in general. The high flexibility of this toolkit leads to varying spatial and temporal expression patterns in different body plans and thus differing results across various taxa not necessarily reflecting phylogenetic relationships.