Bitterlings are brood parasitic fish which complete their early development in the internal gill spaces of freshwater mussels. Bitterling embryos have wing-like yolk sac extensions that help prevent them from being expelled from the gills by the water flow. The ability to resist expulsion may be helped by the consistent ‘head-down’ position that all embryos adopt in the gills. The mechanism behind this positioning is unknown. We hypothesise here that it might lie in a process of unknown function, specific to bitterlings. That process is blastokinesis — the rotation of the embryo on the yolk ball before hatching. We used time-lapse imaging, histology, X-ray tomography, and expression profiling of the genes fgf8a, krt8, msx3 and ctslb by in situ hybridization in the pre-hatching and hatching stages of the rosy bitterling (Rhodeus ocellatus). We find that blastokinesis is a gastrulation process that has been ventralized by the shape of the yolk mass. Furthermore, we show that bitterlings, unlike other teleosts, hatch mechanically without hatching enzymes, and we provide evidence that this is mediated instead by the apical tubercles on the yolk sac extension. Finally, our data suggest that blastokinesis is functional, because it represents the mechanism behind the optimal, ‘head-down’ positioning of the embryo. Our study provides an example of how selection pressures can lead to a suite of dramatic and coordinated modifications of early development.

Rhodeus ocellatus embryos

Rhodeus ocellatus embryos of synchronized developmental age were collected at the Institute of Vertebrate Biology in Brno, Czech Republic, following in vitro fertilization (IVF). Embryos of various developmental stages (SI Appendix, Table S1) were fixed in 4% paraformaldehyde, dehydrated step-wise in methanol and stored in 100% methanol at -20°C. The developmental staging of the embryos was based on Yi et al. Two or three replicates were used for each stage.

Danio rerio embryos

Embryos of the zebrafish Danio rerio (AB/TL line) were collected in the fish facility of the Institute of Biology Leiden. The eggs were fertilized by 1:1 spawning (single crossing) at the beginning of the light period (14 h light, 10 h dark). The fertilized eggs were collected and incubated in egg water (containing 60 μg/ml “Instant Ocean” sea salts) at 28.5 °C. After collection, the embryos were immediately fixated in 4% paraformaldehyde. Fixed embryos were dehydrated step-wise in methanol and stored in 100% methanol at -20 °C. The developmental stages were determined according to Kimmel et al. (37). For each stage (SI Appendix, Table S1), ten replicates were used.

Primer design

Zebrafish (Danio rerio) is relatively closely phylogenetically related to the rosy bitterling (the same family, Cyprinidae). We BLAST searched the zebrafish genes of our interest (fgf8a, msx3, krt8 and ctslb) since the entire zebrafish genome is known and aligned it to other species in the Cyprinidae (e.g., Carassius auratus and Cyprinus carpio). We searched for conserved regions. After the gene of interest was found, forward and reverse primers of approximately 20 base pairs were designed in the conserved regions that would provide fragments of at least 600 bp with a preferred melting temperature between 55°C and 60°C and a favourable CG percentage (around 30-40%). These primers were ordered from Sigma Aldrich and used to amplify the target gene from cDNA from reverse transcribed form Rhodeus ocellatus total RNA (see SI Appendix, Table S2 for primer sequences and accession number of target genes). These PCR products were cloned in the TOPO-TA PCRII Vector (Invitrogen) and transformed into E. coli bacteria.

Probe synthesis

The probe templates were then obtained using PCR with M13-pUC primers and used for RNA polymerase suited to the sequence (T7/sp6, SI Appendix, Fig. S3). Afterwards these fragments were run through a 1% agarose gel to verify whether the fragment length was correct. The sequence of the template was determined by Sanger sequencing performed at Baseclear (Leiden) to verify that the probes contained the correct gene, and the product was compared using a BLAST with the family Cyprinidae. After this procedure, the probes were purified and made ready for use.

Whole-mount in situ hybridization

Whole-mount in situ hybridization (WISH) was performed as described for Danio rerio(38), with a few specific modification for Rhodeus ocellatus. Specifically, the Proteinase K (10 µg/ml) digestion time for R. ocellatus is longer than for D. rerio. For R. ocellatus embryo at blastula and gastrula stages, the digestion time was 1 min; for early somitogenesis stages, 5 min; for late somit

Micro-computed tomography

Micro-computed tomography analysis was carried out at Naturalis Biodiversity Center (Leiden, the Netherlands) using a Xradia 520 Versa 3-D X-ray microscope (Zeiss) based on described methods(8), and visualized with Avizo software (Avizo 9.5, Thermo Scientific™).

Histology

Sample were embedded in Technovit 7100 for routine histology followed by hameatoxylin and eosin, periodic acid Schiff, methenamine silver and oil red O staining. The section thickness was 0.5-0.7 µm. Samples were embedded in epon for toluidine blue staining and semi-thin sectioned with a glass knife. For scanning electron microscopy, we used critical-point drying, and scanned the samples with a JEOL SEM 7600. Ultra-thin sections were prepared for transmission electron microscopy and examined in a JEOL 1400 transmission electron microscope.

Time-lapse recording

R. ocellatus embryos at the desired developmental stage were mounted in glass view chamber. Stereo microscope (Nikon SMZ1500) was used for recording and the focal level has been adjusted manually to keep the embryo in focus. Photographs were captured with a Nikon DS-Fi1-L2 camera. Time-lapse images were assembled into video recordings using Fiji (39). To quantitatively describe the movement of embryos during blastokinesis, we used the Manual Tracking plugin developed by F. Cordelières (http://rsbweb.nih.gov/ij/plugins/track/track.html) for Fiji. The ventral and dorsal margins of the blastoderm, and the head region and tail region of embryo were labeled and their trajectories during the blastokinesis movement were tracked. The distance and velocity information of each trajectory were then imported into Excel for statistical analysis.

Velocimetric estimation of overall morphogenetic movements was performed by particle image velocimetry (PIV) analysis using the PIVlab software package (40). The size of the vectors is proportional with the velocity of tissue movements. The analysis spanned four periods: (1) Epiboly period until the yolk plug closure. (2) Front-flip period where the embryo's head region moved from the animal pole (12 o'clock position) to the vegetal pole (9 o'clock position). (3) Elongate period with continued head migration towards the vegetal pole (7 o'clock position) and tail relocation from 3 o'clock to 12 o'clock. (4) Rostral protrude period concluding at the head's arrival at the 6 o'clock position before hatching. The deformation modes of each period were described by computing the temporal mean value of the velocity field. The resulting plots were imported to Adobe Illustrator to label the collective direction of vectors. Frame-by-frame PIV analysis was assembled into video (SI Appendix, Movie S3-S5).

Files can be accessed in Image J.