The small, yolky eggs of O. vulgaris are roughly 2.5 mm long and 1 mm wide. Octopus embryos are described to develop poorly without maternal care (2,33). However, we have found that O. vulgaris embryos can develop without maternal care in artificial oxygenated seawater at continuous strong flow rate and dim light. The standalone system ensured a continuous flow in the tanks resulting in an oblique orientation and soft swirling of the strings, likely mimicking the jet flow the mother normally provides (Fig. 2). The embryos developed highly synchronous within the string and hatched after approximately one month at 19 °C. We provide a summary table with key characteristics of each stage to allow consistent staging of O. vulgaris embryos (Table 1) as well as a comparative table including Arnold and Boletzky stages for easy translation between cephalopods (Table 2). As the developmental stages presented by Naef are based on days of development rather than on morphological characteristics and contain considerable gaps in development, we split some events and added ‘.1’ or ‘.2’ in such cases. For all descriptions presented, the morphological axes of the embryo are used (Fig. 1B). According to these axes, the location of the funnel is posterior, the embryonic mouth anterior, the arm crown ventral and the mantle dorsal.
Cleavage, Gastrulation and Epiboly
The germinal disc is restricted to the animal pole of the egg, at the micropyle side, which is opposite from the stalk. Meroblastic, bilaterally symmetrical cleavage and subsequent formation of the blastodisc (Stage I) takes place over the first 24-48 hrs after fertilization, depending on water temperature. The first three cleavages are incomplete and generate eight equally sized blastomeres in octopods (Fig. 3A-D), which differs from decapods where the two dorso-medial cells are more narrow compared to the ventro-medial cells (2). Further cell proliferation results in the formation of the blastodisc at Stage I (Fig. 3E). At Stage II, formation of the blastula is completed (Fig. 3F), followed by the onset of epiboly at Stage III, characterized by lateral expansion of the blastoderm over the yolk by cell division (Fig. 3G). The blastodisc, which can be found at the very top of the yolk at Stage II starts to grow and expand over the yolk, generating a cap-like structure by Stage IV (Fig. 3H). At Stage V, a quarter of the yolk is covered by the embryonic cap (Fig. 3I). Using bright-field imaging, the embryo looks uniform at this stage. However, using light sheet microscopy and DAPI as a nuclear stain, the embryo proper with its densely packed nuclei can be clearly distinguished from the extraembryonic ectoderm with larger nuclei spaced further apart (Fig. 3I-I’). At Stage VI, the germinal disc covers half of the yolk mass (Fig. 3J-J’). From this stage onwards, the embryo slowly rotates clockwise when observed from the micropyle side of the egg, along its longitudinal axis (Additional file 1 shows a movie of embryo rotation accelerated to 8x original speed at Stage XI) (12,14). By the end of Stage VII.1, the embryo and yolk envelope (extraembryonic) cover 3/4th of the yolk, followed by complete closure at the vegetative pole, ready for the first reversion (Fig. 3K).
Organogenesis and Maturation
At Stage VII.1, the surface of the embryo appears smooth. The first organ primordia can be visualized using DAPI, revealing the prospective arms as patches of dense nuclei close to the yolk envelope (Fig. 3K-K’). The embryo makes its first reversion at the end of Stage VII. This process takes 7 to 36 hrs, depending on the incubation temperature (14), in which the embryo migrates over the yolk from the micropyle to the stalk side of the egg and can be observed in different topologies (Fig. 3M-O). At Stage VII.2, primordia become visible by bright-field microscopy as thickenings and depressions that arise from the surface of the embryo (Fig. 3L). The eye placodes, mantle anlage, arm primordia and mouth are the first distinguishable structures (Fig. 3L’) and become more discernable towards Stage VIII (Fig. 4), when the mantle rim is elevated.
During the next stages of organogenesis, the organ primordia become more prominent and are clearly distinguishable from the yolk, giving rise to an immature embryo at Stage XVII (Fig. 4-7; Additional files 2-13 show movies of embryos imaged with LSFM). At Stage IX, the arm buds are clearly separated from one another, the mantle appears more elevated and first yellow pigmentation of the retina is visible. The yolk sac envelope that contains blood lacuna and a network of muscular elements starts to create peristaltic waves of surface contraction at this stage, establishing blood circulation for the early embryo (Additional file 14 shows yolk contraction at Stage XI) (34). This phenomenon will cease around Stage XVI, when the embryonic heartbeat is well established and when the area of contact between the yolk envelope and the chorion becomes too small (12).
In order to distinguish embryos between Stages IX and XIII, mantle size and the angle relative to the imaginary plane through the eyes, as well as folding of the funnel tube are easily recognizable morphological characteristics (LSFM images in Fig. 4, 5, funnel in Fig. 6). The shape of the funnel is visible through the chorion, but is easier to observe after dechorionation. At Stage IX, the funnel tube rudiments become visible (Fig. 6A) and fuse at the margins by stage X (white arrow Fig. 6B). At Stage XI, the funnel tube rudiments have grown in size and bend towards the midline (Fig. 6C). Then, at the beginning of Stage XII (Stage XII.1), the funnel starts to form a real tube that is fused at the ventral extremity by Stage XII.2 (Fig. 6D-E). But, it is at Stage XIII that the formation of the siphon shaped funnel tube is complete (Fig. 6F). In the subsequent events, the position of the mouth on the anterior side changes (Fig. 7, white arrows on LSFM images). The mouth is situated between the first pair of arms on the anterior side from Stage VIII to XIV and is still open to the outside at Stage XV.1. It will start to internalize, becoming encircled by the anterior arms at Stage XV.2. By Stage XVI, the mouth is covered by the arm crown, waiting to take its final position as soon as the outer yolk is reduced.
As the embryos grow, the shape of the mantle goes from depressed towards the middle at Stage VII.2 to flat and perpendicular to the longitudinal axis at Stage X. At Stage XI, the mantle is elevated on the posterior side and thus tilted and clearly grows in size by Stage XII. At Stages XIII and XIV, the length of the mantle equals and exceeds the length of the head in the dorsoventral axis, respectively (Fig. 5, 7), and at Stage XIV, a heartbeat can be observed at the mantle tip (Additional file 15 shows embryonic heart beat at Stage XVII). From Stage IX to stage XIV, the color of the retina changes from light yellow to dark red/brown. The color of the eye and retina continues to darken during development, until the eye is completely black and covered by an iridescent layer, clearly visible from Stage XIX onwards.
The chromatophore pattern (appearance, color and size of chromatophores) is another convenient characteristic to stage O. vulgaris embryos (Fig. 7, 8, 9). At Stages XV.1 and XV.2, the first chromatophores appear as small yellow dots on the posterior side, next to the funnel and on the mantle, respectively. By Stage XVI, the first chromatophores on the anterior mantle appear. From Stage XVIII.2 onwards, the chromatophores react to changes in light intensity under the microscope (expand under light stimulation and contract in the dark). The ratio of the size of the external yolk sack in relation to the size of the embryo is another measure that can be used for staging (Fig. 7, 8, 9). At Stage XIV, this ratio approximates 1:1 and rapidly decreases to 1:3 at Stage XVI, 1:4 at Stage XVIII.1 to 1:6 at Stage XIX.1. This latter stage is also characterized by the first appearance of ink in the ink sac on the posterior side. The embryo undergoes the second reversion at Stage XIX. We annotate these stages as XIX.1 before and XIX.2 after the second reversion.
At Stages XX.1 and XX.2, the external yolk sack is nearly and completely depleted, respectively (Additional file 16 shows a movie of a Stage XX.2 embryo imaged with LSFM). It has been described that cephalopod embryos are likely slightly sedated in the egg by a tranquillizing factor to prevent premature hatching which can occur at these stages (35). What precisely induces natural hatching is still unknown, but it is easily triggered by several factors, such as mechanical stimuli, photoperiodicity and sudden changes in light levels or temperature (15). We observed that natural hatching starts approximately seven days after the second reversion at 19 °C, but is detrimental to the paralarvae in the tank system under continuous flow. Therefore, seven days post second reversion, we moved the strings from the system to a different tank containing aerated artificial seawater, which induced hatching within minutes.
Assays to evaluate embryonic fitness
Yolk contraction can be observed from Stage IX to Stage XVI under the stereomicroscope and is a valuable readout to evaluate embryonic survival at early organogenesis stages. Furthermore, upon development of the retina, a "saddle" to discoidal shape of the pigmented layer is typical of high-quality embryos. Frowning or folding of the retina points towards poor health. From Stage XIV onwards, a heartbeat can be recognized in the transparent embryos. Occasionally, small crustaceans can be observed on the strings. Generally, these are part of the natural ecosystem of the string and are not impacting embryonic development. Nevertheless, poor rearing conditions (insufficient flow, dissolved oxygen levels and strings floating or sunken) can trigger strings to overgrow with fungi (white thread-like structures or parts turning pale or pink) or get infected by worms. A final readout of state of the art rearing is the hatching of actively swimming paralarvae that display positive phototaxis, reported for most cephalopod hatchlings (9,36,37).