Studying early embryogenesis in the flatworm Maritigrella crozieri indicates a unique modification of the spiral cleavage program in polyclad flatworms

Background Spiral cleavage is a conserved early developmental mode found in several phyla of Lophotrochozoans with highly diverse adult body plans. While the cleavage pattern has clearly been broadly conserved, it has also undergone many modifications in various taxa. The precise mechanisms of how different adaptations have altered the ancestral spiral cleavage pattern is an important ongoing evolutionary question and adequately answering this question requires obtaining a broad developmental knowledge of different spirally cleaving taxa. In flatworms (Platyhelminthes), the spiral cleavage program has been lost or severely modified in most taxa. Polyclad flatworms, however, have retained the pattern up to the 32-cell stage. Here we study early embryogenesis of the cotylean polyclad flatworm Maritigrella crozieri to investigate how closely this species follows the canonical spiral cleavage pattern and to discover any potential deviations from it. Results Using live imaging recordings and 3D reconstructions of embryos, we give a detailed picture of the events that occur during spiral cleavage in M. crozieri. We suggest, contrary to previous observations, that the 4-cell stage is a product of unequal cleavages. We show that that the formation of third and fourth micromere quartets are accompanied by strong blebbing events; blebbing also accompanies the formation of micromere 4d. We find an important deviation from the canonical pattern of cleavages with clear evidence that micromere 4d follows an atypical cleavage pattern, so far exclusively found in polyclad flatworms. Conclusions Our findings highlight that early development in M. crozieri deviates in several important aspects from the canonical spiral cleavage pattern. We suggest that some of our observations extend to polyclad flatworms in general as they have been described in both suborders of the Polycladida, the Cotylea and Acotylea.

has clearly been broadly conserved, it has also undergone many modifications in various taxa. 23 The precise mechanisms of how different adaptations have altered the ancestral spiral 24 cleavage pattern is an important ongoing evolutionary question and adequately answering this 25 question requires obtaining a broad developmental knowledge of different spirally cleaving 26

taxa. 27
In flatworms (Platyhelminthes), the spiral cleavage program has been lost or severely modified 28 in most taxa. Polyclad flatworms, however, have retained the pattern up to the 32-cell stage. 29 Here we study early embryogenesis of the cotylean polyclad flatworm Maritigrella crozieri to 30 investigate how closely this species follows the canonical spiral cleavage pattern and to 31 discover any potential deviations from it. While the adult morphology of the different phyla gives few obvious clues as to their close 52 relationships, it has long been recognised that a subset of lophotrochozoan phyla share 53 striking similarities in the earliest events of their embryology, most notably in the spatial 54 arrangement of early blastomere divisions, a developmental mode known as spiral cleavage 55 (Hejnol, 2010;Henry, 2014;Lambert, 2010). Representative lophotrochozoan phyla with spiral 56 cleavage comprise annelids, molluscs, nemerteans, flatworms, phoronids and entoprocts 57 (Henry, 2014;Lambert, 2010) and recent phylogenetic results show that these spirally 58 cleaving phyla form a clade within the Lophotrochozoa (Marlétaz et al., 2019). The monophyly 59 of the spirally cleaving phyla strongly suggests a single origin of the spiral cleavage mode. 60 The fact that spiral cleavage has been maintained in these animals since they diverged in the 61 early Cambrian, over half a billion years ago, argues that some selection pressure for 62 maintaining spiral cleavage exists. 63 There are several aspects of spiral cleavage that appear to be highly conserved. The first is 64 the spiral pattern itself: Embryos of the eight-cell stage consist of four larger vegetal 65 macromeres, 1Q, and four smaller animally positioned micromeres, 1q, each sitting skewed 66 to one side of their sister macromere, above the macromeres' cleavage furrows. The typical 67 spiral deformations (SD) of macromeres show a helical twist towards one side with respect to 68 the animal-vegetal axis. This is best seen if the embryo is viewed from the animal pole. The 69 resulting spiral shape taken by all four macromeres is either clockwise (dexiotropic) or counter 70 clockwise (laeotropic). In subsequent rounds of division, the larger macromeres again divide 71 unequally and asymmetrically sequentially forming the second and then the third quartets of   Henry, 1986; Henry, 1989; Henry and Martindale, 1987;Render, 1983;Render, 1989). 4d 1 and 4d 2 appear equal and this equality is easily observed in 4d 2 due to its larger size and 295 exposed external position. Both descendants of 4d 1 and 4d 2 (4d 11 and 4d 12 and 4d 21 and 4d 22 ) 296 then undergo another round of roughly meridional cleavages. This is similar to Surface's 297 descriptions in H. inquilina (Surface, 1907).  One observation suggesting blebbing has an important function in polyclad embryogenesis is 358 that when embryos are mounted in high concentrations of agarose (>0.6%) we observed 359 severely abnormal development (n=5/5). We speculate that these defects may be caused by 360 blebbing being hampered by the stiff agarose. Common to all of the blebbing events is that 361 they occur in cells which contain most of the yolk and which undergo asymmetric divisions. 362 Additionally, we show here that they coincide with increased cytoskeletal activity (mitosis). 363 One simple explanation for their occurrence may be that they are the visible manifestation of 364 actomyosin contractions of the cortex during strong cytoskeletal movements involved in 365 asymmetric cleavage in yolk-rich blastomeres. may be a broader pattern within polyclads, including species so far regarded as "equal" 383 cleavers, but requires precise measurements to be carried out in different species. In M. 384 crozieri the question remains as to whether the observed size differences at the 4-cell stage 385 truly reflect an unequal cleavage mechanism, meaning that the D quadrant is already specified 386 by maternal determinants at this early stage. Clearly, we need to know more about the 387 molecular basis of putative maternal determinants and the mechanisms by which they could 388 be sequestered but an early specification of the D quadrant via cytoplasmic localization seems 389 to be supported by previous studies on Hoploplana inquilina (Boyer, 1987;Boyer, 1989). 390

391
Most importantly, we found that the animal-vegetal division of micromere 4d is present in both 392 polyclad suborders, and we suggest this is a conserved pattern across all polyclad flatworms. 393 It would be highly interesting to reinvestigate this cleavage pattern within the Prorhynchida, 394 where the spiral cleavage pattern with quartet formation has also been partly retained but 395 current developmental data are insufficient to conclude whether it follows the pattern as 396 suggested for polyclads in this study. 397 We consider that the exact fate of both daughter cells of micromere 4d must be investigated 398 more thoroughly before we can conclude whether micromere 4d 2 (animally positioned relative 399 to 4d 1 ) indeed represents the mesentoblast or not. Currently, even the fate of 4d 1 , despite its We synthesised mRNAs for microinjections with Ambion's SP6 mMESSAGE mMACHINE kit. 451 The capped mRNAs produced were diluted in nuclease-free water and used for 452 microinjections in order to detect fluorescence signal in early M. crozieri embryos. Nuclei were 453 marked and followed using histone H2A-mCherry (H2A-mCh) and GFP-Histone (H2B-GFP). 454 The plasmids carrying the nuclear marker pCS2-H2B-GFP (GFP-Histone) and pDestTol2pA2- In order to image specimens from 5 angles, which is necessary to perform volume 514 measurements of early blastomeres, sodium azide with 0.1 M PBS containing 0.1% Triton X-515 100 in (PBSTx) was washed off fixed embryos by four washing steps and stained with 1:300 516 Rhodamine Phalloidin (ThermoFisher Scientific R415) for 2-3 h at room temperature or 517 overnight at 4°C. Following several washes of PBST or PBSTx 0.1 μM of the nuclear stain 518 SytoxGreen (Invitrogen), which is difficult to detect at these early stages, was added for 30 519 min and the embryos then rinsed with PBST for another hour. 520 521

Image processing 522
Post-processing of acquired data was performed with the latest version of the freely available 523 imaging software Fiji (Schindelin et al., 2012)

Availability of data and materials 533
The datasets during and/or analysed during the current study available from the corresponding 534 author on reasonable request. 535 536

Competing interests 537
The authors declare that they have no competing interests.

755
The resulting spiral shape taken by all four macromeres has been shown to be either clockwise (dexiotropic) or 756 counter clockwise (laeotropic) among different lophotrochozoans. In the polyclad M. crozieri it is dexiotropic.

757
Notably it has been demonstrated that the mechanism of spiral deformations depends on actin filaments rather