The making of an octopus arm

Embryonic development and appearance of the arm crown in O. vulgaris

The embryonic development of O. vulgaris including the general arm crown formation was first described by Naef [42] and will be briefly summarized in this paragraph. Like all cephalopods, the O. vulgaris embryo exhibits a direct development, which takes about 35 days at 20°C. Embryos develop by bilateral, meroblastic cleavage in which the first cleavage furrow sets the primary body axis (Figure 1C, stage 2). During epibolic gastrulation, the yolk gets covered by a thin sheet of cells (the outer yolk sac) while the embryo proper is forming at the animal pole of the egg (Figure 1C, stage 11). Shortly thereafter, first organ primordia appear as epithelial thickenings, which in the process of organogenesis increase in size and complexity (Figure 1C, stage 16 - hatchling). The O. vulgaris hatchling is considered a paralarva, which undergoes a pelagic phase before settling to the sea floor.

The arm crown primordium appears right before the end of gastrulation as two bands of cells on each side of the egg’s ‘equator.’ Both bands are situated closer to the posterior part of the embryo, resulting in a larger gap on the anterior side (Figure 1C, stage 16). As the embryo expands over the yolk, the arm crown rudiment becomes more distinct and subdivides into four arm fields on each side of the embryo (Figure 1C, stages 18 to 19) [42].

Embryonic formation and differentiation of the O. vulgaris arm crown

In order to understand the basic dynamics of octopus appendage development, we analyzed the growth, formation, and differentiation of single arms within the arm crown by studying their histology and cell proliferation pattern. We thereby focused on stages right after the arm fields had formed and for continuity of the study analyzed the morphology of either arm II or arm III. The terminology used for describing the arm axes is summarized in Figure 1B. As soon as the arm fields are established, individual arm rudiments can be distinguished as bulges of moderate size at which arm rudiment III appears before rudiments II and IV and arm pair I remains developmentally delayed until stage 19 (Figure 2A). After this initial asynchronous development, all arms show a similar level of maturity during subsequent stages. Early arm rudiments consist of a few layers of undifferentiated cells enclosed by an epithelium (Figure 2A′), both of which are heavily proliferating (Figure 2A″).

In the following course of development, the arm bulges set themselves apart from the original band-like structure and can readily be distinguished by their unique position within the arm crown (Figure 2B). At this stage, the arm buds are made up by an epithelium consisting of cells similar in shape and size, encircling an underlying differentiating cell mass (Figure 2B′). Unlike the epithelium, these underlying cells are composed of several inhomogeneous cell types. In particular, we observed cell nuclei towards the proximal and central parts of the arm buds which are slightly darker, smaller, and rounder (Figure 2B′, open arrowhead). Similar types of cells in these regions were previously described as neuroblast cells migrating from the base and lateral edges of the epithelium into the arm primordium in the O. vulgaris embryo [44]. All cell types continue to proliferate strongly within the now hemispherical arm rudiments (Figure 2B″).

While the arm crown slowly contracts, wedge-shaped tissue starts to grow from the anterior proximal base of arm bud II and posterior proximal base of arm bud III, towards the anterior and posterior part of the eye (Figure 2C, arrows), which later envelopes the eyes to form the primary eyelid [28]. Within the arm bud, the previously seemingly undifferentiated cell mass starts to clearly organize into distinct tissue layers at stage 23 (Figure 2C′). Underneath the epithelium, we observed a loose layer of darker stained and larger cells adjacent to an area of densely packed elongated cells within the areas of the future dermis and muscle mass, respectively. A central mass of small and rounded cells is making up the future axial nerve cord. In addition to an organization into distinct cell layers, we noted a dramatic change of shape from a hemispherical arm bud to an elongated arm. Cell proliferation is more pronounced in the lateral epithelium and at the distal tip of the arm bud in an area underneath the epithelium (Figure 2C″).

As the embryo increases in complexity, the four arm pairs begin to encircle the originally anterior situated mouth (Figure 2D). At this stage, we could clearly distinguish between the epithelium, dermis, muscle layer, and axial nerve cord (Figure 2D′). Several cells have submersed under the epithelium and differentiated into the basal cells of what will become the Kölliker’s organs. These tegumentary structures are a larval feature unique to most incirrate octopods, which assist in hatching and facilitate post-hatching swimming behavior [51] (Figure 2D′, open arrowhead). Underneath the epithelium, the thick, loose dermis is taking up a large part of the proximal, aboral fraction of the arm adjacent to which the dense muscle layer becomes evident. The axial nerve cord consists mostly of round cell bodies; however, at this stage, a ganglionic structure is not visible yet. Proliferation is most pronounced in the lateral and proximal parts of the epithelium, the muscle layer, and the cell bodies within the axial nerve cord (Figure 2D″).

Over the next stages of development, the morphology of the arm crown matures considerably. The individual arms become connected by a velar web, which surrounds the proximal base of the arm (Figure 2E, arrowheads). Within the epithelium, the basal cells of the Kölliker’s organs have multiplied and formed into cup-shaped invaginations secreting spine-like setae (Figure 2E′). The dermis underneath the epithelium has developed into a loose, fibrous layer. A thin layer of longitudinal and transverse muscle fibers borders a dense layer of differentiating cells, which envelopes the axial nerve cord. These differentiating cells were previously described by Kier [52] as nerve cell bodies; however, cells within this area in the tentacle of the cuttlefish have been considered as differentiating muscle cells (myocytes) by Grimaldi et al. [53]. Given the position of these cells within the arm, we believe that they may constitute a combination of both. Proliferation is now mostly restricted to the distal tip of the arm, to the newly forming longitudinal muscle layer and the suckers (Figure 2E″).

At hatching stage, the mouth comes to lie in the center of the four pairs of seemingly homonomous arms (data not shown) while the velar webs further increase in complexity (Figure 2F, arrowhead). The aboral side of the arm is entirely covered in Kölliker’s organs, which start to break through the epithelium. Underneath the dermis, the layers of longitudinal muscle fibers have increased and transverse muscle fibers are more abundant. The amount of cells surrounding the axial nerve cord as well as those within the neuropil is reduced. Furthermore, the axial nerve cord has formed into three ganglionic regions, corresponding to the three suckers on the oral side of the arm. In terms of overall shape, we noticed that the distal end of the arm becomes drawn-out into a pointed tip, typical for adult animals. This area was termed the growth zone of the arm (gza) or flagellum by Naef [42]. Cell proliferation is mostly restricted to this area, to the muscle layer right beneath the dermis and the suckers (Figure 2F′). Interestingly, the overall complexity decreases from the proximal base towards the distal tip of the arm with the tip remaining in a state of development comparable to stage 26 (Additional file 3).

Arm bud outgrowth and elongation

During the early stages of arm development (stages 19 to 23), we observed a drastic elongation of the spherical arm bulge along its proximal-distal axis. Since axes elongations are often accompanied by epithelial cell dynamics in embryonic development [54-58], we monitored the shape and orientation of cells stained for F-actin during early arm formation. Rows of elongated, epithelial cells appear at the proximal base of the arms and are extending obliquely into the arm buds from stage 19 until stage 23 (Figure 3A,B,C; Additional files 4, 5, and 6). At stage 24 (Figure 3D; Additional file 7), only remnants of these elongated cells remain (dashed lines).

To understand whether an elongation of epithelial cells can indeed contribute to the elongation process of the early arm buds, we disrupted actin polymerization within the cytoskeleton using Cyt D. We performed treatments with two concentrations of Cyt D (1 or 5 μM) for the duration of early arm outgrowth and elongation (stages 19 to 23). Embryos raised in control medium form anatomically normal and elongated arms (n = 35, 100%; Figure 3E). The basement membrane distinctly separates the epithelium from the underlying cell mass (Figure 3F, arrow) and the surface epithelium includes elongated cells (Figure 3G,H). In contrast, the arms of embryos treated with 1 μM Cyt D fail to fully elongate and appear malformed (n = 69, 100%; Figure 3I). In particular, the arms’ lengths, widths, and thicknesses are reduced (n = 7; ANOVA, P < 0.05; Additional file 8A) and the basement membranes are highly disorganized (Figure 3J). However, the average cell number in these arms does not significantly differ from the cell number in the control arms (n = 6; ANOVA, P = 0.164; Additional file 8B). Furthermore, epithelial cells on the oral surface of the arms are not elongated (Figure 3K,L). Cell shape measurements show a clear reduction in the length to width ratio with a decrease in length of otherwise elongated epithelial cells (n = 40; ANOVA, P < 0.05; Additional file 8C). Arm buds of embryos treated with 5 μM Cyt D fail to grow out and remain in a developmental state comparable to stage 19 embryos (n = 34, 94%; Figure 3M). The arm bulges consist of a thin epithelium separated by a discontinuous basement membrane from the few underlying cell layers (Figure 3N). Arm length, width, and thickness in these arms is significantly reduced when compared to control embryos (n = 14; ANOVA, P < 0.05; Additional file 8A) as well as the number of cells (n = 7; ANOVA, P < 0.05; Additional file 8B). Cells on the surface epithelium are not elongated but round and do not show clear cell boundaries (Figure 3O,P). Cell shape measurements show a clear reduction in the length to width ratio with a decrease in length in these embryos (n = 40; ANOVA, P < 0.05; Additional file 8C).

To test whether the observed epithelial cell changes during this particular developmental window (stages 19 to 23) are a crucial factor for the elongation of the arm, Cyt D-treated embryos were washed in normal sea water and allowed to develop until control embryos were at stage 25 (Figure 3Q,R,S). Embryos initially treated with 5 μM Cyt D do not survive past stage 24 (n = 33, 100%). Embryos initially treated with 1 μM Cyt D remain shorter in overall size (n = 52, 100%; Figure 3T). Furthermore, even though the arms continue to differentiate, they fail to elongate (Figure 3U). Measurements of the arms’ dimensions in the treated versus control group confirm this observation showing a tendency of the treated arms to be shorter but thicker (n = 11; t test, P < 0.05; Additional file 8D). The surface epithelium at this stage does not include elongated cells in the control embryos (Figure 3S) and elongated cells are equally absent from the surface epithelium of the initially treated embryos (Figure 3V). Altogether, these results suggest that actin polymerization is crucial for the elongation of epithelial cells and the concomitant elongation of the embryonic arm during a specific period of arm formation.

Formation of the embryonic arm musculature

The O. vulgaris adult arm consists of a complex arrangement of muscle layers (Figure 1A), which are composed of large, mononucleated muscle cells with oblique striation (Additional file 9A). To understand how this network is established during embryonic development, we examined the first appearance of muscle cells, formation of muscle fibers, and their subsequent arrangement into distinct muscle layers within the developing arm buds.

We first analyzed the morphology of O. vulgaris embryonic muscle cells in cell culture so as to readily identify these cells in the context of the developing tissue. We then followed the appearance and subsequent organization of embryonic muscle cells into different muscle layers by staining embryonic arms for F-actin (Figure 4A,B,C,D,E). Embryonic muscle cells from late developmental stages are significantly smaller when compared to adult muscle cells and the striation is not apparent yet (Additional file 9B). We first observed these cells at stage 24, right after the arms had elongated. At this stage, muscle cells are located adjacent to the epithelium in the area of the future longitudinal muscle fibers and in a deeper tissue layer in the area of the transverse muscle fibers (Figure 4D, arrows and arrowheads) surrounding the future axial nerve cord (dotted line). Both muscle layers become more prominent at stage 26 (Figure 4E). Interestingly, muscle fibers in the distal tip within the growth zone of the arm are not organized into distinct layers (Figure 4E, dashed line). Right before hatching, the arm’s musculature consists of a thin longitudinal and an intertwined, sparse transverse muscle layer (Additional file 9C).

Muscle-specific genes and phylogenetic analysis

During invertebrate and vertebrate muscle development, muscle precursor cells (myoblast cells) determined to differentiate into mature muscle cells (myocytes) commonly initiate the expression of muscle genes including Myosin heavy chain, Actin, and Tropomyosin [59-63]. These muscle genes are often activated at different times of muscle formation, dependent on muscle fiber type and location. For instance, in the gastropod Haliotis rufescens, Tropomyosin mRNA accumulates up to 7 days prior to myofibril assembly [64]. In order to examine where early myocytes originate from and how they differentiate into mature myofibers, we therefore cloned and studied the gene expression patterns of the well-conserved muscle genes Myosin heavy chain, Actin, and Tropomyosin.

Full-length gene orthologs of O. vulgaris Actin (Ov-Actin) and Ov-Tm were previously characterized by Ochiai et al. [65] and Motoyama et al. [66], respectively. Both Ov-Actin and Ov-Tm were isolated from the O. vulgaris arm musculature. While the deduced Ov-Actin amino acid sequence shows strong sequence identity to other invertebrate and vertebrate actins, Ov-Tm is highly cephalopod specific. We further identified two fragments of the Ov-Mhc gene, the deduced amino acid sequence of which shows strong sequence similarities to the head and a tail region of known cephalopod Myosin heavy chain gene orthologues (Additional file 10). A maximum likelihood (BS) analysis supports a specific assignment of the Ov-Mhc gene to a distinct clade of MyosinII subfamily orthologs from representatives of cnidaria, lophotrochozoa, ecdysozoa, and chordata with high posterior probabilities (Additional file 11).

Whole-mount in situ expression patterns

Ov-Mhc expression is first detected in large, spindle-shaped cells (early myocytes) at stages 20 to 21 in the area of the future transverse muscle layer (Figure 4G, arrowheads; Additional file 9D). At stages 22 to 23, the number of cells expressing Ov-Mhc in this region surrounding the future axial nerve cord increases (Figure 4H dotted line, arrowheads) and expression also appears in fewer cells adjacent to the epithelium in the area of the future longitudinal muscle layer (Figure 4, arrows). The Ov-Mhc-positive cells at this stage include cells, which form fibrous extensions (late myocytes). At stages 24 to 25, the Ov-Mhc expression intensifies in both regions and is visible in embryonic muscle fibers (Figure 4I, arrowheads and arrows; Additional file 9E) as well as early myocytes, which form at the tip. At stage 26, we detected Ov-Mhc expression in both the longitudinal and transverse muscle layer, respectively (Figure 4J, arrowheads and arrows; Additional file 9 F) as well as newly forming muscle cells in the growth zone of the arm (Figure 4J, dashed line; Additional file 9G).

We observed strong Ov-Actin expression within the entire inner cell mass of the early arm bulge and elongating arm bud (stages 19 to 23, Figure 4K,L,M). At stages 24 to 25, the expression becomes clearly localized to the muscle fibers of the transverse and longitudinal muscle layers (Figure 4N, arrowheads and arrows). This expression becomes even more pronounced at stage 26 and is mostly localized to maturing muscle layers at this stage (Figure 4O, arrowheads and arrows). We observed a less localized expression in the growth zone of the arm (Figure 4N,O, dashed lines).

At stages 19 to 21, we detected Ov-Tm expression in the cell mass underneath the epithelium (Figure 4P,Q). At stages 22 to 23, a weak Ov-Tm expression becomes localized to the forming longitudinal and transverse muscle fibers (Figure 4R, arrowheads and arrows). The expression intensifies in the following developmental stages and is mostly detectable within the layers of the differentiating longitudinal and transverse muscle layers (Figure 4S,T, arrowheads and arrows). Ov-Tm expression within the growth zone of the arm remains less localized (4S,T, dashed line).

Formation of the sucker

The octopus sucker is an important manipulative and chemosensory structure on the oral side of the arm, allowing the animal to probe its environment and perform fine motor tasks. At hatching stage, the sucker is made up of radial, meridional, and circular muscle fibers and a rudimentary sphincter muscle, surrounded by the extrinsic musculature, which connects the sucker to the arm proper (Figure 5A,B). The nervous system of the sucker at this stage is almost completely formed and consists of a nerve ring surrounding the sucker rim and bundles of nerves connecting the sucker with the sucker ganglion and the nervous system of the arm (Figure 5C).

In order to understand how the musculature of this complex structure is formed, we examined the expression patterns of the aforementioned muscle-specific genes during the embryonic formation of the suckers. Octopus sucker development was previously described by Fioroni [67] and Nolte and Fioroni [68] and is summarized in Figure 5D,E,F. First sucker rudiments appear at stage 23 as small epithelial outgrowths, which include both a mesodermal and an ectodermal component (Figure 5D). These outgrowths soon increase in size (Figure 5E) and develop a primordial acetabulum, which invaginates from the epithelium surrounding the sucker rudiment (Figure 5F, white dotted line).

During sucker development, Ov-Mhc and Ov-Actin are first expressed at stage 25 within the extrinsic acetabulo-brachial musculature (Figure 5G,H,J,K, arrows). Both genes are also expressed in maturing muscle cells of the future acetabulum, which is just about to invaginate (Figure 5H,K, asterisk). At stage 26, all three muscle-specific genes examined (Figure 5G,H,I,J,K,L,M,N,O) are strongly expressed within the extrinsic circular musculature (Figure 5I,L,O, arrows) and the circular muscle of the now invaginating acetabulum (Figure 5I,L,O, asterisk).