E. scolopes general development and axes denomination
E. scolopes develops by bilateral cleavage, typical for decabrachian cephalopods. Development takes about 21 days at 24 °C water temperature and can be divided into 30 distinct stages, as described by Lee et al. [25] (based on Arnold [30]) and are summarized in Fig. 2a. Cleavage is superficial and leads to a discoblastula. During epibolic gastrulation a thin sheet of cells expands over the yolk, forming the outer yolk sac, while the embryo proper develops at the animal pole of the egg. Shortly before the entire yolk is covered by the yolk sac, organ primordia become visible as epithelial thickenings. These increase in size and complexity until the fully developed paralarva hatches resembling a miniature adult. As usual for cephalopods, the embryonic dorso-ventral (DV) body axis is designated corresponding to the embryo’s orientation along the animal-vegetal axis of the egg. Accordingly, the area of the mouth primordium is regarded as anterior while the area of the anus marks the posterior side of the embryo. During late stage development the body axes tilt by 90° relative to the embryonic axes, so that the original dorso-ventral (DV) axis becomes the antero-posterior (AP) axis (Fig. 2b). This tilted orientation of the animal corresponds to its physiological swim position in the water as an adult.
Regarding the axes of the arms the following terms will be used to describe their orientation: “proximal” will be considered the base of the arm, closest to the animal’s body, “distal” will appropriately refer to the tip of the arm. “Anterior” and “posterior” will correspond to the embryonic anterior (facing the embryonic mouth) - posterior (facing the embryonic funnel) axis. The side of the arm covered in suckers and facing the central adult mouth will be denoted oral while the opposing side will be referred to as aboral (Fig. 2c). The following in depth description of arm bud morphology during embryonic development focuses on the growth and differentiation events of the arm pairs II and IV. The latter develop into the specialized prehensile tentacles, which morphologically set them apart from the rest of the arm pairs. Arm pair II was chosen exemplarily in order to provide continuity of description. The position of the sections through the arms shown in this study are indicated in Fig. 2c.
Appearance of the arm crown and early arm outgrowth
The arm crown is first recognizable at stage 18 as two continuous bands of cells around the equator of the egg (Figs. 2a, 3A). At stage 19, the arm crown separates into five distinct arm fields consisting of condensed layers of epithelial cells on each side of the embryo (Figs. 2a, 3B, Additional file 2), which quickly increase in size during the following stages of development (Figs. 2a, 3C-D). Arm fields II and IV grow out first, followed by III and V, while arm field I extends last and remains the smallest until the animal hatches. At stage 22, the entire embryo starts to contract, which leads to a rearrangement of all organs to a more definitive state [7, 15]. This whole body contraction moves arm pair I closer together and separates the outer yolk from the smaller inner yolk sac (Fig. 2a stage 19 and 22; 2a, 3E). At this stage, axon tracts of the axial nerve cord are visible at the base of all arm buds and connect to form the interbrachial connective (Fig. 3E′, arrowheads). Individual arms are apparent as epithelial bulges, which show uniform cell proliferation (Fig. 3F, G). While arm bud II consists of an inner cell mass, which in the histological sections shows no apparent differentiation or regionalization surrounded by an epithelium (Fig. 3F′), the inner cell mass of arm IV is made up by a dense outer layer of cells with elongated nuclei (region of future musculature), which surrounds a loose inner layer of cells with spherical nuclei (region of future axial nerve cord; Fig. 3G′). In both cases, the epithelium is comprised of multiple cell layers except at the distal end, where a monolayer of epithelial cells covers a slightly pointed tip (Fig. 3F′- F″, G'- G″). Several ciliated cells become apparent on the aboral surface of the epithelium of both arm pairs II and IV (Fig. 3F'''-F'''', 3G'''-3G''''). Even though a neuropil cannot be detected in histological sections, individual patches of nerve fibers projecting from clusters of neurons towards the proximal base of the arm are visible in arm II (Fig. 3F'''-F''''). In contrast, a small central neuropil region of the forming axial nerve cord is detectable in arm IV, where axon tracts terminate diffusely in an epithelial region just before the distal tip of the arm (Fig. 3G', dashed line; 3G'''-3G'''').
Elongation along the PD axis
The subsequent stage is characterized by an elongation of all arms along their PD axes (Fig. 4A), and an increase of cilia on the arms’ aboral surfaces (Fig. 4A′). Since in octopus the arms’ elongation is driven by a concomitant elongation of epithelial cells [22], we compared cell shapes in the epithelium of arms II and IV at stages 21 and 23. At stage 21 epithelial cells on the aboral surface of arm II are oriented at an angle towards the corresponding margins of the arm (Additional file 3A), while at stage 23 elongated rows of cells can be observed, which align in a central region along the arm’s PD axis (Additional file 3A′). In contrast, elongated cells at the proximal base of arm IV are already lined up in central rows along the PD axis at stage 21 (Additional file 3B). At stage 23, most cells in the epithelium of arm bud IV are elongated and oriented along the PD axis (Additional file 3B′).
Except for a central region, cell proliferation at stage 23 is still rather uniform in both arms II and IV (Fig. 4B, C). The central region constitutes the forming neuropil of the future axial nerve cord, which is surrounded by a denser layer of the forming muscle cells with elongated nuclei (Fig. 4B′, C′). In both arms the rudimentary musculature at this stage consists of sporadic individual longitudinal and transverse muscle fibers (Fig. 4B'', C''). Furthermore, the neuropil region in both arms extends almost along the entire length of the arm primordium (Fig. 4B''', C''', B'''', C'''').
Tissue differentiation
By stage 25 the central region of the arm crown becomes more restricted, which moves arm pair I closer to each other and towards the mouth. Accordingly, all other arms attain their final position relative to each other and acquire a unique shape and length (Fig. 5A; compare Fig. 4A-A'). During this process the entire arm crown shifts to the anterior region of the head to eventually surround the mouth [7, 9]. Arm pair I remains the shortest, followed by arm pair V, which develops a wider base and grows at an oblique angle relative to the remaining arm pairs. Both arm pairs II and III are rather similar in shape at this stage. Arm pair IV is easily distinguished by its slender shape and its rapid increase in length. Furthermore, the ciliation on the aboral side of all arm pairs becomes localized to arm – specific regions (Fig. 5A′). In particular, the ciliation of arm pairs I, II and V is concentrated to the posterior part of the arms while ciliation of arms III-IV shows a more scattered pattern with a slightly higher density of cilia on the arm’s anterior side.
At stage 25, most cell proliferation becomes localized to the epithelium, a region adjacent to the epithelium, in a central region and the suckers in arm II. Few proliferating cells can also be observed in central regions of the arm (Fig. 5B). The dorsal epithelium in arm bud II consists mostly of large, ovate cells, characterized by a basal nucleus, interjected by interstitial cells. Small, non-secretory, cuboidal cells cover the distal tip, as described in Singley [31] (Fig. 5B′, Additional file 4A). Underneath the epithelium, the layers of longitudinal muscle fibers become more prominent and are partly intertwined with the transverse muscle fibers (Fig. 5B'-B''). The central neuropil region is increasing in size and surrounded by a dense layer of cells with rounded cell nuclei, which Kier [16] identified as neuronal cell bodies (Fig. 5B'). Axon tracts from the axial nerve cord reach the distal tip of the arm (Fig. 5B'''-B''''). In contrast to arm II, cell proliferation becomes most strongly localized to the epithelium, the cell layers adjacent to the distal epithelium and the suckers of arm IV at stage 25. Fewer cells at this stage proliferate in the proximal regions of cells adjacent to the epithelium and in central regions of the arm (Fig. 5C). Furthermore, a single layer of non-secretory cells makes up the epithelium of arm IV (Fig. 5C', Additional file 4B). Underneath the epithelium, longitudinal muscle fibers organized in thick muscle bundles become obvious and are equally intertwined by transverse muscle fibers (Fig. 5C'-5C''). The neuropil area is less prominent than in arm II but is equally surrounded by a dense layer of cells with spherical nuclei (Fig. 5C'). According to Grimaldi et al. [32], these cell bodies surrounding the neuropil constitute differentiating myocytes in the tentacle (arm IV) of the cuttlefish. Here, we consider them as a mixture of differentiating neuronal and muscular cells. The axonal tracts of the axial nerve cord reach throughout the entire length of the arm as well. In addition, intramuscular nerve cords appear on the oral side of arm IV, while the first connective fibers start to project from the axial nerve cord towards the periphery (Fig. 5C'''-C''''). In general, tissue differentiation occurs in a gradual process from the proximal base towards the distal tip in both arms II and IV.
Tissue end-differentiation
From stage 27 to hatching the arm crown differentiates into its final adult-like form. During this time, arm pair III becomes slightly longer than arm pair II and forms a velar web on its posterior side through which it becomes connected to arm pair V (Fig. 6A-A'). Ciliation on the aboral side of the arms further intensifies and remains restricted to the posterior region of arms I and II, while it now covers the entirety of arms III-V (Fig. 6A').
The phase of tissue end-differentiation in arm II is characterized by almost an adult-like maturity (Fig. 6B) and a confinement of cell proliferation to the distal tip (Fig. 6 B'). First chromatophores are formed underneath the epithelium within the dermis of arm II, while an additional superficial-longitudinal muscle layer appears adjacent to the dermis (Fig. 6B). Distinct layers of longitudinal, oblique, and transverse muscle fibers enclose an area of undifferentiated cells adjacent to the neuropil of the axial nerve cord (Fig. 6B, C-C'). The latter is almost devoid of cell bodies and is now comprised of series of ganglia, each of which corresponds to a sucker on the oral side of the arm (Fig. 6B). Connective fibers link the axial nerve cord to the suckers as well as the intramuscular nerve cords within the growing muscle mass. The latter are regularly connected by anastomoses (Fig. 6D-D', E-E'). Similar to arm II, tissue maturity is highly advanced in arm IV and cell proliferation is restricted to the distal portion of arm IV at this stage (Fig. 6F-F'). Furthermore, a superficial and circular muscle layer have formed adjacent to the epithelium in addition to the longitudinal and transverse muscle layer (Fig. 6F, G-G'). However, as opposed to arm II, the axial nerve cord is not organized into a series of ganglia, but consists of a tube-shaped neuropil, which is also almost devoid of cell bodies (Fig. 6F, H). While connective fibers and anastomoses are connecting intramuscular nerve cords to the axial nerve cord and to each other throughout the entire length of arm IV (Fig. 6H, I), an increase in complexity similar to the arm II can only be observed at the very distal tip on the level of the suckers (Fig. 6H', I').
Formation of the suckers
E. scolopes exhibits four rows of typical decabrachian suckers on the arm’s oral surface used for prey handling and egg deposition in the female squid, and more than 32 lines of suckers on the tentacular clubs, which are mostly used for prey capture [33]. Suckers are asymmetrical, stalked, and divided into an infundibulum (attachment face) and an acetabulum (sucker chamber) [34].
During embryonic development suckers appear as rounded papillae on the distal rim of the arm’s oral surface and new suckers are added in this area throughout the embryo’s development. On arm II this mechanism produces suckers in a constant manner in which suckers are added one at the time, increase in size, and form a double, triple, and finally quadruple row while the arm extends along its PD axis (Figs. 7a and 8). Conversely, in arm IV multiple suckers are formed simultaneously but do not organize into well-defined rows (Fig. 7b).
Early sucker primordia consist of a mesodermal cell mass surrounded by a simple epithelium (Fig. 7c-h). Starting at stage 25 the largest suckers of both arm II and IV show first signs of differentiation at which suckers on arm II are generally larger than those on arm IV (Fig. 7e, h). At stage 26, a short stalk can clearly be distinguished from the ovate future cylinder, which contains the primordial acetabulum and an infundibulum in both arms II and IV (Fig. 7i, l). Within only a few days of development, by stage 28, the suckers on arm II and IV have matured considerably and show first structural differences (Fig. 7j, m). Suckers on both arms consist of a muscular stalk with a constricted end, which attaches to the cylinder containing the acetabulum. While the extrinsic musculature of suckers on arm IV does not show any specializations yet, the constriction of suckers on arm II consists of a defined layer of extrinsic circular muscle fibers. Unlike the suckers on arm II, the acetabulum of the suckers on arm IV show a well-formed sphincter muscle separating the acetabular roof from the rest of the structure. Both sucker types are connected to the axial nerve cord by a connective nerve fiber, which divides into several acetabular nerve fibers at this stage. Shortly before hatching the cylinder of suckers on arm II consists mostly of circular muscle fibers and does not completely envelope the acetabulum consisting of circular and meridional muscle fibers. The infundibulum is rather small and a dense network of nerves appears at its rim (Fig. 7k). Conversely, the cylinder of the suckers on arm IV is mostly made up of meridional muscle layers and is completely surrounded by the acetabulum. The sphincter muscle at the base of the acetabulum becomes even more apparent and a dense network of nerves innervates the rim of the infundibulum’s broad opening, similar to what is observed in suckers of arm II (Fig. 7n).