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From spiral cleavage to bilateral symmetry: the developmental cell lineage of the annelid brain

Background During early development, patterns of cell division—embryonic cleavage—accompany the gradual restriction of blastomeres to specific cell fates. In Spiralia, which include annelids, mollusks, and flatworms, “spiral cleavage” produces a highly stereotypic, spiral-like arrangement of blastomeres and swimming trochophore-type larvae with rotational (spiral) symmetry. However, starting at larval stages, spiralian larvae acquire elements of bilateral symmetry, before they metamorphose into fully bilateral juveniles. How this spiral-to-bilateral transition occurs is not known and is especially puzzling for the early differentiating brain and head sensory organs, which emerge directly from the spiral cleavage pattern. Here we present the developmental cell lineage of the Platynereis larval episphere. Results Live-imaging recordings from the zygote to the mid-trochophore stage (~ 30 hpf) of the larval episphere of the marine annelid Platynereis dumerilii reveal highly stereotypical development and an invariant cell lineage of early differentiating cell types. The larval brain and head sensory organs develop from 11 pairs of bilateral founders, each giving rise to identical clones on the right and left body sides. Relating the origin of each bilateral founder pair back to the spiral cleavage pattern, we uncover highly divergent origins: while some founder pairs originate from corresponding cells in the spiralian lineage on each body side, others originate from non-corresponding cells, and yet others derive from a single cell within one quadrant. Integrating lineage and gene expression data for several embryonic and larval stages, we find that the conserved head patterning genes otx and six3 are expressed in bilateral founders representing divergent lineage histories and giving rise to early differentiating cholinergic neurons and head sensory organs, respectively. Conclusions We present the complete developmental cell lineage of the Platynereis larval episphere, and thus the first comprehensive account of the spiral-to-bilateral transition in a developing spiralian. The bilateral symmetry of the head emerges from pairs of bilateral founders, similar to the trunk; however, the head founders are more numerous and show striking left-right asymmetries in lineage behavior that we relate to differential gene expression.


INTRODUCTION
During early development, embryonic cleavage produces blastomeres via a rapid series of cell divisions without significant growth, relying on maternally deposited messengers and proteins. During these processes, the initially broad developmental potential of blastomeres becomes gradually restricted towards distinct cell fates. This can occur via two basic modes, called regulative (conditional) or mosaic (determinate) development. In regulative development, exhibited by vertebrates, cnidarians and sea urchins (Gilbert, 2000), almost all blastomeres share a broad developmental potential and cell fate determination largely depends on local signaling events. In mosaic development, most blastomeres inherit distinct maternal determinants and signaling is assumed to play a minor role. This requires sophisticated in ovo localisation, a stereotypic arrangement of cleaving blastomeres and an invariant cell lineage. Prime examples for mosaic development are the nematodes (Sulston et al., 1983) and ascidians (Nakamura et al., 2012;Nishida, 1987). Recent results however hint at a considerable degree of cellcell signaling in their invariant lineage (Lawrence and Levine, 2006;Lemaire, 2009), which underscores that regulative and mosaic development only differ in the relative contributions of autonomous versus conditional cell fate determination.
Mosaic development with invariant early cell lineage and autonomous cell fate decisions is also characteristic for the Spiralia, or 'Lophotrohozoa', a large assembly of marine invertebrate phyla (Henry, 2014). Their eponymous 'spiral cleavage' produces a highly stereotypic, spiral-like arrangement of blastomeres ( Fig. 1A) (reviewed in (Nielsen, 2004(Nielsen, , 2005: The first two cleavages, perpendicular to each other, subdivide the embryo along the animal-vegetal axis into four blastomeres, representing the four future embryonic 'quadrants' A, B, C and D (Henry, 2014). The subsequent cleavages are asymmetrical, generating quartets of smaller micromeres towards the animal pole and quartets of bigger macromeres towards the vegetal pole. In addition, due to an oblique angle of these divisions, the originating micromere quartets are alternately turned clock-or counterclockwise against the macromere quartet, so that the micromeres come to lie in the furrows between the macromeres (Fig. 1A). This way the characteristic spiral-shaped organization of the embryo is generated. The initial cleavage pattern is identical for each quadrant, so that the whole early embryo shows rotational symmetry around the animal-vegetal axis.
Corresponding cells with similar lineage in the four quadrants are referred to as quadriradial analogues.
In most spiralian phyla, spiral cleavage produces spherical planktonic larvae with characteristic apical ciliary tuft and bands of motile cilia that drive larval swimming. In annelids and molluscs, these are called trochophora larvae (Fig. 1A). The larvae form a simple nervous system that integrates sensory information from photo-, mechano-and chemosensory receptor cells for the control of ciliary locomotion (Jekely et al., 2008;Marlow et al., 2014;Tosches et al., 2014). Its most prominent features are an apical nervous system with an apical organ underlying an apical tuft. The apical nervous system is connected via radial nerves to a ring nerve (Nielsen, 2004(Nielsen, , 2005.
The ring nerve innervates a pronounced circular ciliary band, the prototroch, which subdivides the larva into upper episphere and a lower hyposphere.
During settlement metamorphosis, the larva transforms into an adult body with overt bilateral symmetry (or more or less complex derivatives thereof, see for instance the development of Crepidula (Hejnol et al., 2007;Lyons et al., 2017) and Ilyanassa (Goulding, 2009). The adults of most phyla develop a wormshaped body form as manifest for example in basal mollusks and, most prominently, in flatworms and annelids. In these worms, the former episphere of the larva develops into the head including the paired cerebral ganglia. The hyposphere gives rise to the trunk including the ventral nerve cords (Nielsen, 2004(Nielsen, , 2005. Hence, the most peculiar feature of spiralian development is the transition from rotational (spiral) point symmetry to bilateral symmetry, which has puzzled embryologists for more than a century (e.g., (Wilson, 1892)). How is this spiral-to-bilateral transition accomplished? In the hyposphere, bilateral symmetry is established through the unique behavior of two cells, 2d and 4d (Wilson, 1892), which derive from the D-quadrant. These cells divide once into the left and right bilateral founder cells of the entire trunk, which proliferate in a stem cell-like fashion and give rise to trunk ectoderm and mesoderm, respectively (Fischer and Arendt, 2013;Gline et al., 2011;Lyons et al., 2012). The situation is more complicated in the episphere, where the bilateral symmetry has to emerge from a pre-existing array of spirally arranged micromeres. Here, the spiral-to-bilateral transition may involve a 'rearrangement' of micromere position via complex cellular movements, or start from selected bilateral founders analogous to the trunk founders. The latter solution was favored by E. D. Wilson (Wilson, 1892), who gave an early and detailed account of spiral cleavage in the annelid Nereis. He observed a sudden transition from spiral to bilateral cleavage pattern after the appearance of the prototroch that he attributed to such (yet to be identified) founders.
To decide between these options, we reconstructed the full developmental cell lineage for the marine annelid Platynereis dumerilii from the fertilized egg to the swimming trochophore stage (~30 hpf), by confocal microscopy. We linked early lineages to gene expression using a cellular resolution gene expression atlas for several embryonic stages (compare (Vergara et al., 2017)). This resource is explored here for the episphere spiralto-bilateral transition. Platynereis is especially suited to study this transition, because the larval phase is rather short and the bilateral head and trunk structures are developed in the swimming trochophore, so that the full transition can be observed microscopically. Previous studies in Platynereis and other spiralians had established the bilateral fate of early micromeres by injection of tracer dyes, yet did not resolve their lineage in cellular resolution (Ackermann et al., 2005;Hejnol et al., 2007;Meyer et al., 2010). The timelapse recordings, software tools and lineage analysis presented here generate an unprecedented resource for spiralian biology available so far only for nematode and tunicate model systems.
Our lineage analysis allows tracking the spiral-to-bilateral transition in cellular detail. As postulated by Wilson, we identify bilateral founder cells; yet, we observe a whole array of bilateral founders distributed over the episphere at around 12hrs of development. Some of them, located in the lateral episphere represent quadriradial analogues, that is, they stem from similar (i.e., corresponding) lineage in their respective quadrants. Others, located more medially, stem from dissimilar lineage in their respective quadrants.
Mapping the expression of the conserved bilaterian head patterning genes otx and six3 onto the developmental lineage, we find that lateral otx expression marks the bilateral founders with similar lineage, whereas medial six3 marks those of dissimilar lineage. Moreover, we find that while the otx+ lateral founders show strong proliferation during larval stages and remain mostly undifferentiated at 30hpf, the six3+ medial founders differentiate earlier and give rise, among others, to bilateral pairs of cholinergic neurons in the larval brain. Finally, we find that the apical organ proper does not derive from bilateral founders at all. Instead, it develops from the most medial cells that do not show bilateral symmetry.
We discuss our findings as the result of a secondary overlap of two initially separate phases of development, namely an early mosaic and determinate and a later regulative phase, representing larval and adult development, respectively.

episphere
The brain is almost entirely formed by the offspring of the apical micromeres 1a-1d, collectively referred to as "1m" (Ackermann et al., 2005;Dorresteijn, 1990), easily accessible to life imaging by standard scanning confocal microscopy. To track cell divisions in the apical brain, we injected embryos at different stages post fertilization (1, 2 or 4 cell stage) with H 2 B-dsRed and Lyn-EGFP mRNAs (Haas and Gilmour, 2006), which label chromatin and cell membranes, respectively. Then we recorded time-lapse movies of these apically mounted embryos ( Fig. 1A Taken together, our comparative analysis shows that Platynereis brain development is highly stereotypical at the level of overall cell arrangement and lineage tree topology.

The cell lineage of the first differentiated cells is invariant
Although the stereotypic tree topology and cell positions suggest an invariant cell lineage, we wanted to prove this assumption by investigating whether the same cell types are produced by the same cell lineage in different embryos.
To date, the only differentiated cells for which the cell lineage has been fully described in Platynereis are the primary prototroch cells (Dorresteijn, 1990). In summary, we addressed the cell lineages of 62 differentiated cells in a 30hpf episphere, summarized in ( Figure 2F and Table I). For the vast majority of investigated cell types (exiting the cell cycle before ~15hpf), the cell lineage is strictly conserved among multiple embryos (column "Support" in Table I).
Interestingly, the cell lineage varies substantially in later-born cells (ChATpositive cell Nr 52 exiting the cell cycle at ~28 hpf, cell Nr 50, ~20hpf). Based on the analyzed embryos and available literature we created a consensus lineage tree with the identified cell types ( Supplementary Fig. 2). In summary, our analyses show that the Platynereis larval brain develops via stereotypical cell divisions and that the lineage of early differentiating neuronal cell types is conserved.
Using our atlas, we found that the transcription factors coe, ngn, neuroD, and prox are co-expressed with the neuronal differentiation markers elav and Syt, the cholinergic marker ChAT, and the neuropeptidergic marker Phc2 in the apical organ cells (Nr. 46 and Nr. 53, later serotonergic, in Table I) We first focused on lineages that retained the initial rotational symmetry. In Platynereis, these lineages give rise to early differentiating cells of the prototroch and apical organ. The primary prototroch develops from the two vegetal-most quartets of the first micromeres, that is, 1m-22 and 1m-21, in a strictly radial arrangement (Fig. 3A-B). The blastomeres 1m-12, located slightly more apically, divide twice in a spiral mode (with an exception of 1d-12, see below) ( Fig. 3B). They produce the non-dividing accessory prototroch cells 1m-122 and 1m-1212.
The apical organ develops from the four cells 1m-111 that form a prominent "apical rosette" in early development, characteristic for the spiral cleavage pattern (Dorresteijn, 1990)

An array of bilateral founder cells
We next focused on those lineages, for which a transition from the initial rotational to bilateral symmetry was manifested. Previous reports from Nereis (Wilson, 1982) and Platynereis (Dorresteijn, 1990;Pruitt et al., 2014) identified the first divisions with bilateral symmetry between 7 and 12 hpf, yet, could not track the progeny of these cells at subsequent stages. Using our tracked lineage, we interrogated these cell divisions with regard to their clonal progeny. We defined 'bilateral founders as cells that would i) have a bilateral counterpart (in position); ii) produce bilaterally symmetrical clonal progeny with similar lineage topology; and iii) appear at roughly the same developmental time point. To our surprise, we identified not only few, but a whole array of pairs of bilateral founders situated on the right and left sides of the Platynereis episphere (Fig. 4A). These appear as early as 6 hpf and continue to arise until 18 hpf. The entirety of paired bilateral founders and their bilaterally symmetrical progeny is visible in (Fig. 4A). How do these arise?
The first 'bilateral' divisions (i.e., divisions with a bilaterally rather than rotationally symmetrical orientation of spindle poles) take place after less than 6 hpf (Fig. 4B). Specifically, the 1m-112 cells cleave into two bilaterally positioned daughter cells (1m-1121 and 1m-1122). At 8 hpf, these cells continue to divide in a bilaterally symmetrical manner (Fig. 4C) About half of the bilateral founders -the ones located more laterally -are generated in perfect bilateral symmetry, as mirror images on the right and left body sides. This is reflected by a bilaterally symmetrical arrangement of the resulting lateral clones (with dark blue color in Fig. 4D). All descendent lineages show full bilateral symmetry, as is apparent from the equivalent lineage history of right and left counterpart clones (Fig. 4E). Counter-intuitively, the other half of the bilateral founders -the ones located more mediallyarise in a very asymmetric manner, which is reflected by the dissimilar outlines of the medial clones on the left and right body sides (with dissimilar colors in Fig. 4D). For each pair of bilateral founders generated by these sublineages (Fig. 4F), the lineage history of the left and right founder is very different. They originate by asymmetric divisions at different branches of the lineage tree and in some cases even differ in the lineage depth. As an example, the offspring of 1c-1121211 and 1d-1121111b (lineage depth difference 1, lineages diverged 4 cell cycles ago) produce a bilaterally symmetric domain (light green clones in Fig. 4F).
Finally, we noted a peculiar difference in how the four initial quadrants 1a, 1b, 1c, 1d contributed to the multiple pairs of bilateral founders. In most cases, founder pairs that were produced from the 1c quadrant on the left side came from the 1d quadrant on the right side, and those produced from the 1b quadrant on the left side came from the 1a quadrant on the right side. In few rare cases, however, pairs of bilateral founders came from the 1a versus 1c quadrants, or resulted from a single quadrant (Fig. 4G-H). The most striking examples are represented by the bilateral clones 1a-1121211 and 1a-1121121 (light and dark blue clone in Fig. 4G) and small clones 1b-12111aa and 1b-121121b (dark green in Fig. 4H) that originate from single quadrants.
In summary, the overall contributions of quadrants to bilateral progeny and the processes by which bilateral symmetry is reached are rather complex, forming a "patchwork" of processes schematized in Fig. 4I.

The conserved apical six3, otx and nk21 domains show different lineage behavior
A number of recent studies revealed a conserved role of the homeodomain transcription factors six3, otx and nk21 in the specification of the apical region (Marlow et al., 2014;Steinmetz et al., 2010;Tessmar-Raible et al., 2007). In general, six3 is expressed most apically, surrounded by a ring of otx expression. Nk21 is expressed in the ventral apical region, overlapping with six3 and otx. Taking advantage of our cellular atlas, we have identified the cells expressing six3, otx and nk21 at 12hpf and compared the clonal progeny of these cells at later developmental time points to the actual expression domains (Fig. 5A-D). This comparison reveals that, while the six3 and otx domains are initially mutually exclusive, six3 expression later spreads into the otx clonal descendants (compare Fig. 5A and Fig. 5D at 24 hpf). In contrast, otx expression is turned off in clonal descendants from 20hpf onwards. Nk21 expression is less dynamic and largely remains expressed in clonal descendants (Fig. 5D).
We set out to characterize the six3, otx, and nk21 further with regard to lineage behavior. At 6 hpf, otx is expressed in the 1m-12 primary trochoblast cells (Supplementary fig. 5), which later give rise to the accessory prototroch.
At 12hpf, the cells expressing otx match the 1m-1122 descendants with few exceptions ( Fig. 5C and E), thus including the bilateral founders that produce the set of strictly bilateral clones (compare Fig. 4C). This means, the otx domain develops from specific quartets of micromeres, in line with a possible specification by maternal determinants. In addition, the dorsal otx region is most proliferative among episphere cells in that it shows the highest lineage depth and the shortest cell cycle length (Fig. 5G-H). Except for the prototroch and accessory prototroch cells, it produces no differentiated cells until 22hpf (whereas the ventral cells 1ab-1122 give rise to the gland cells, Table 1).
Cells in this territory differentiate much later, such as the adult eyes (Arendt et al., 2002).
In contrast, at 12hpf six3 matches the 1m-1121 quartet ( Fig. 5A and Fig. 4B), suggesting maternal specification. Yet, the bilateral founders emanating from these cells do not represent quadriradial homologs (and thus are unlikely to be specified maternally). On average, the six3 cells divide less, and generate several differentiated cells at 22hpf, including the crescent cell (Nr 40 in Table   I

DISCUSSION
Here we reconstructed the full lineage of the larval episphere in the marine annelid Platynereis dumerilii, from spiral cleavage to fully bilateral larval stages, including individual lineages for 62 differentiated cells that make up the larval body. Overall, our data confirm earlier observations that the development of spirally cleaving embryos is highly stereotypic, and extend these observations to early larval stages. Consistent with this, we find that the cell lineage of early differentiating cells is highly invariant.
To relate the Platynereis lineage to embryonic and larval stages, we constructed a gene expression atlas for several embryonic and larval stages, for 23 genes with known roles in developmental specification and cellular differentiation. Taking advantage of this multi-stage expression atlas, we have explored how gene expression and early cellular differentiation relate to the transition from rotational to bilateral symmetry.
Lineage tracking, concomitant with the mapping of gene expression and identification of differentiated cells in the consensus lineage tree, is part of ongoing efforts (Achim et al., 2018;Achim et al., 2015;Tomer et al., 2010;Vergara et al., 2017) to resolve and understand Platynereis development at single cell level. The comparison of these resources to similar pioneering efforts in other developmental models (e.g. (Du et al., 2015;Santella et al., 2016;Stach and Anselmi, 2015;Tassy et al., 2010), will be especially rewarding for understanding the evolution of development at cell type level.

Highly complex transition from rotational to bilateral symmetry
Our analysis of the full lineage until 30 hpf has allowed an in-depth investigation of the transition from the embryonic spiral cleavage pattern with rotational symmetry to the bilateral symmetry of the swimming trochophora larval stage. As anticipated by Wilson (Wilson 1892), we find that the bilaterally symmetrical parts of the larval body emerge from so-called bilateral founders. However, the generation of these founders from within the spiral cleavage pattern is surprisingly diverse (Fig. 4I), in that the initial quartets of micromeres produce bilateral founders in very different ways.
First, and most intuitively, bilateral founders emerge from two cells of similar (corresponding) lineage in different quadrants, located on the future left and right body sides. This straightforward strategy applies to about half of the bilateral founders (1m-1122) located more laterally in the larval episphere, However, we detected an unexpected difference in how left-right opposing quadrants contribute to these founders. While, in most cases, the left and right cell of a given founder pair stem from the 1a versus 1b, or 1d versus 1c quadrants, a small portion of the episphere bears an A|C bilateral symmetry established by the descendants of the non-adjacent A and C quadrants (blue regions in Fig. 4I). Remarkably, while the A|C bilateral symmetry is less frequent in Platynereis and in other annelids such as Capitella (Meyer et al., 2010), it has shown to be predominant in the mollusks Ilyanassa and Crepidula (Chan and Lambert, 2014;Hejnol et al., 2007).
As a second strategy, we discovered sets of bilateral founders that emerge from two cells of dissimilar (non-corresponding) lineage in left-right opposing quadrants (green regions in Fig. 4I), involving non-bilateral cell divisions at non-related positions within the lineage tree topology (Fig. 4F-H). Even more intriguing, we also observed "single quadrant bilateral symmetry", where two symmetric clones originate both from the same quadrant (brown regions in Fig. 4I). These findings contradict the initial assumptions (Wilson, 1892) that, as observed for the 2d and 4d somatic descendants in the larval hyposphere, strictly bilaterally symmetric divisions should establish the bilaterally symmetric portions of the larval body.

Geometrical constraints may explain the different lineage behavior of bilateral clones
Simple geometrical rules may explain the strikingly different lineage behavior of bilateral clones, as illustrated in (Fig. 6). In a four-fold radially (or rotationally) symmetric arrangements (color coded in Fig. 6B), the diagonals represent the only areas naturally exhibiting bilateral symmetry. These could therefore adopt bilateral behavior automatically, without any need for rearrangement or re-specification. Indeed, our analysis of the rotational-to-bilateral symmetry transition revealed that the clones positioned on the diagonals are the first ones to adopt bilateral behavior (Fig. 6A). In other regions of the spiral cleavage pattern, cells of corresponding lineage in leftright opposing quadrants do not automatically come to lie in mirror-image positions. Instead, in the more medial regions between left and right diagonals, left-right mirror image coordinates are of dissimilar origin within opposing quadrants. We propose that this is reflected by highly asymmetric lineage origin of the bilateral founder cells in these regions.
Bilateral symmetry can be also achieved by expanding the areas at the interface between dorsal and ventral quadrants (purple triangles and arrow in  Fig. 4D). With the above-mentioned assumption of these geometrical constraints it is easy to envision the difficulty in adopting bilateral symmetry within the midline region observed during episphere development. Hence, the axis of bilateral symmetry itself is mostly devoid of cells adopting bilateral behavior and only forms small bilateral clones that emerge at later larval stages.

A signaling centre in the plane of bilateral symmetry?
Taken together, these observations suggest that there is no obvious relationship between the embryonic cell lineage of the spiral cleavage and the future bilateral symmetry of the larval body. Rather, the common theme of all cells adopting bilateral behavior appears to be their symmetric position with regard to the bilateral axis. This suggests involvement of a signaling source positioned in the plane of the bilateral symmetry axis. An obvious candidate for the signaling center is the cell 2d and its descendants, positioned in the anterior part of the dorsal hyposphere on the axis of the bilateral symmetry. These cells are well known for their organizing potential of Platynereis trunk (Pfeifer et al., 2014) and the deletion of the 2d cell in Capitella leads to loss of bilateral symmetry in the head (Amiel et al., 2013).
Interestingly, the regulative potential of the D quadrant does not seem to be limited to the C|D-A|B bilateral symmetry, but might also contribute in establishing the A|C bilateral symmetry, as demonstrated by its involvement in specification of the A and C quadrant-derived eyes in Ilyanassa (Sweet, 1998).

Non-bilateral origin of early differentiating cells
The step-wise and complex transition from spiral to bilateral symmetry is also reflected in the different lineage behavior of early differentiating cells, both spatially and temporally. For example, the earliest differentiating prototroch cells have a spiral origin and the equally early-appearing cells of the apical organ are formed by non-symmetrical clones. Earlier work in Platynereis (Dorresteijn and Graffy, 1993)

and early cell dissociation experiments in
Nereis (Costello, 1945) pointed to a high degree of cell-autonomous differentiation for these cells via the inheritance of maternal determinants.
Several studies in mollusks (Damen et al., 1994;Lambert and Nagy, 2002;Rabinowitz and Lambert, 2010) and in Platynereis (Pfeifer et al., 2014) have demonstrated that mRNA segregation into specific blastomeres during the cleavage plays a crucial role in cell autonomous specification.
The spiral and bilateral division patterns co-exist for a certain period, with the first bilateral divisions commencing at ~6 hpf while the last spiral divisions of accessory prototroch cells happen at ~8hpf. Consistently with the notion that the zygotic expression is necessary for the first bilaterally symmetric division in the leech Helobdella (Schmerer et al., 2013), we did not observe any bilateral behavior before the onset of zygotic transcription (Chou et al., 2016).

Six3/6 and Otx specify medial versus lateral bilateral founder cells
Interestingly, the early differentiating cells of the prototroch and the apical organ that are potentially specified via maternal determinants do not express the regional specification transcription factors of the bilateral head regions, Six3/6, Otx and Nk2.1 (Marlow et al., 2014;Tosches and Arendt, 2013). In contrast, six3, otx and nk2.1 expression encompasses all bilateral founders that arise from the 1m-1121 and 1m-1122 micromeres, and thus all lineages of subsequently differentiating cells with bilateral symmetry -at least transitorily. Among these, six3 expression labels the more medially located 1m-1121 founders that are of different lineage in opposing quadrant, whereas otx labels the more lateral bilateral founders that stem from 1m-1122 micromeres, with similar lineages between quadrants. The six3+ founders give rise to a bilateral subset of cholinergic neurons that differentiate early in the larval brain. In contrast, the otx+ lateral founders proliferate heavily during later stages and will differentiate much later, into adult eyes and optic lobes (Arendt et al., 2002). Future integration of our lineage data with single-cell expression data mapped onto the expression atlases constructed for reference embryonic and larval stages, will allow the identification of candidate signals and receptors, as well as the gene regulatory networks establishing bilateral symmetrical behavior and cell fates in each of the founder lineages.

Animals
The larvae of Platynereis dumerilii were obtained from the breeding culture at EMBL Heidelberg.

Injections and time-lapse imaging
The injections of H2A-mCherry and mYFP were performed as described previously . For tracking axonal projections, LifeAct-EGFP mRNA (Benton et al., 2013) was injected into a given blastomere of embryos injected previously at one-cell stage with H2A-mCherry mRNA.
The injected embryos were kept in filtered seawater at 18°C until the desired developmental stage was reached. Selected embryos were then transferred in ~2μl of sea water into 40°C warm 0.8% low-melting agarose (A9414, Sigma-Aldrich), briefly mixed by pipetting up and down and quickly transferred in ~20μl agarose to the microscopy slide with 150 μm spacer on each side (3 layers of adhesive tape Magic™Tape, Scotch®). Before the agarose fully solidified (within ~15 seconds) the embryos were covered by a coverslip and oriented to the apical position for imaging. Seawater was added from the side of the slide to entirely fill the slide chamber. To avoid drying out, the coverslip was sealed using mineral oil. The embryos were imaged using a Zeiss Axio Imager.M1 fluorescent microscope or Leica TCS SPE confocal microscope with 40x oil immersion objective with time resolution 20 min (Movie 3), 15 min (Movie 8, 10 and 11), 6 min (Movie 1) and 12 min (Movie 2).

Tracking and comparing the cell lineage across multiple embryos
The live-imaging movies were manually tracked using a custom-made tracking macro in ImageJ/FiJI (Schindelin et al., 2012). We used the nuclei count of the episphere in embryos precisely fixed at several time points to calibrate the developmental time in the movies. Due to a high density of nuclei at later stages, we were able to reliably track until about 32hpf. We use the standard spiralian nomenclature of the cells according to (Conklin, 1897).
After 6hpf, even for non-spiral cell divisions we use the index 1 for the more anterior and index 2 for the more posterior daughter cell until around 10hpf.
After 10hpf, we use indices "a" and "b" instead of "1" and "2" and without any added information. To compare the cell lineage across different embryos, we developed a simple algorithm (Supplementary Fig. 1F) automatically identifying corresponding cells in each tracking dataset and highlighting the differences (see also Supplementary materials and methods).

Whole-mount mRNA in situ hybridization
The mRNA in situ hybridization was performed as described in (Tessmar-Raible et al., 2005) with following modifications: For developmental stages earlier than 12hpf, the embryos were washed twice 4 minutes with TCMFSW (Schneider and Bowerman, 2007) prior to fixation. For developmental stages younger than 24hpf, the embryos were acetylated: After the digestion in proteinase K and two washes with freshly prepared 2mg/ml glycine in PTW (1x phospate-buffered saline with 0.1% Tween-20), the embryos were incubated 5 minutes in 1% triethanolamine in PTW, then 3 minutes in 1% triethanolamine with 0.2% acetanhydride followed with 3 minutes of 0.4% acetanhydride in 1% triethanolamine. The prehybridization, hybridization and SSC washes were performed at 63°C. The hybridization mixture: 50% Formamide (Sigma-Aldrich, F9037), 5x SSC pH4.5, 50 μg/ml Heparin (Sigma-Aldrich, H3149), 0.025% Tween-20 (Sigma-Aldrich, P9416), 50 μg/ml Salmon Sperm DNA (Sigma-Aldrich, D9156), 1% SDS. The DIG-labeled antisense mRNA probes: ChAT, Elav (Denes et al., 2007); Syt, TPH, Phc2, Nk2.1, (Tessmar-Raible et al., 2007); VACht (Jekely et al., 2008); Otx (Arendt et al., 2001); Six3/6 (Steinmetz et al., 2010); VGlut (Tomer et al., 2010). S.Q., and Bowerman, B. (2007). beta-Catenin asymmetries after all animal/vegetal-oriented cell divisions in Platynereis dumerilii embryos mediate binary cell-fate specification. Developmental cell 13, 73-86. Stach, T., and Anselmi, C. (2015). High-precision morphology: bifocal 4Dmicroscopy enables the comparison of detailed cell lineages of two chordate species separated for more than 525 million years. BMC Biol 13, 113. Steinmetz, P.R., Urbach, R., Posnien, N., Eriksson, J., Kostyuchenko, R.P., Brena, C., Guy, K., Akam, M., Bucher, G., and Arendt, D. (2010). Six3 demarcates the anterior-most developing brain region in bilaterian animals.   (Dorresteijn et al., 1993). The ventral views are extensively schematized for simplicity. For 66-cell stage a schematic apical view (top) together with a snapshot (bottom) of the time-lapse recording of the developing episphere is shown. The color-coding of the nuclear tracks in the snapshot corresponds to the coloring in the schematic apical view. The dashed blue line represents the border between embryonic quadrants. (B) The overview of time-lapse movies used for the analysis. Nuclei counts of at least three fixed specimens for each stage were used for temporal calibration of the movies. Asterisks mark the movies used to create the consensus lineage tree. (C) The comparison of the cell lineage trees of three larvae up to 30 hpf. The early lineage covered in previous work but not covered by the movies is shown in blue. The corresponding cells/divisions conserved in all three larvae are colored in black. The divisions and cells that are not present in all three larvae are color-coded according to the legend. (D) The snapshots of the z-projection of the life imaging movies showing the differences between the three larvae at different time points. Differences are color-coded as in (C).