Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii

Abstract

Development of sexual characters and generation of gametes are tightly coupled with growth. Platynereis dumerilii is a marine annelid that has been used to study germline development and gametogenesis. P. dumerilii has germ cell clusters found across the body in the juvenile worms, and the clusters eventually form the gametes. Like other segmented worms, P. dumerilii grows by adding new segments at its posterior end. The number of segments reflect the growth state of the worms and therefore is a useful and measurable growth state metric to study the growth-reproduction crosstalk. To understand how growth correlates with progression of gametogenesis, we investigated germline development across several developmental stages. We discovered a distinct transition period when worms increase the number of germline clusters at a particular segment number threshold. Additionally, we found that keeping worms short in segment number, by manipulating environmental conditions or via amputations, supported a segment number threshold requirement for germline development. Finally, we asked if these clusters in P. dumerilii play a role in regeneration (as similar free-roaming cells are observed in Hydra and planarian regeneration) and found that the clusters were not required for regeneration in P. dumerilii, suggesting a strictly germline nature. Overall, these molecular analyses suggest a previously unidentified developmental transition dependent on the growth state of juvenile P. dumerilii leading to substantially increased germline expansion.

Keywords: annelida; critical size; developmental transition; gametogenesis; sexual reproduction.

Figure 1

Schematics of gametogenesis in Platynereis dumerilii. (a) 3‐segmented larval P. dumerilii has 4 PGCs (4 blue circles) located next to the growth zone (blue stripe to the right of 4 PGCs) at the tail. (b) By the time the larva grows into 5 segments, 4 PGCs migrate anteriorly (indicated by blue arrow in A). (c) In juvenile worms these cells proliferate and form a large cluster in the anterior segments 5‐6. (d) Many small clusters that later appear in posterior trunk segments. These are thought to come from the large anterior cluster in segments 5‐6. The correlation between the total segment number and gonial cluster formation has not been resolved. (d′) Example of a gonial cluster in the trunk segments showing vasa mRNA via in situ hybridization. (e) In pre‐mature worms, later stages of gametogenesis have been better documented. These are the stages when maturing sperm and oocytes can be observed without requiring any molecular markers because they are easy to distinguish based on cell morphology even by simple light microscopy. S: segment. mRNA, messenger RNA

Figure 2

Gametogenesis in the immature juvenile and pre‐mature P. dumerilii. (a) In worms between 10 and 19 segments in length, vasa positive cells can be found towards the anterior of the worm, just behind the jaws. (a′) Close‐up of the anterior cluster in (a). (b) More vasa positive cells appear in a ring‐like structure to encircle the esophagus as the worm grows. (b′) Close‐up of the anterior cluster in (b). (b″) another example of the ring‐like anterior cluster in a different sample. (c) Vasa expression continues to accumulate in this region in worms 30–39 segments long. (c′) Close‐up of the anterior cluster in (c). (c″) vasa expression in the posterior growth zone. (d) Schematic of a worm longer than 40 segments with vasa+ gonial clusters (in blue) populating the trunk segments. (e) Clusters of vasa+ cells begin to appear throughout the posterior body cavity of the worm once it has reached 35–40 segments in length. (e–e″) Close‐up views of the anterior cluster (e′) and trunk clusters (e″). (f) Clusters are more numerous in larger worms, and they start forming pockets (arrowheads in f″) that are regularly present in most segments. (f′,f″) Close‐ups of anterior cluster (f′) and trunk clusters (f″). (g) vasa signal becomes localized in the anterior parapodia of some worms, and become completely absent in other regions. (g′) Close‐up of parapodia showing localized expression in a sample similar to the one in (g). (h,h″) In some of these longer worms, immature oocytes are already present (arrowhead in h, and close‐ups of an oocyte in h′ and h″). These oocytes are not yet fully mature, as evidenced by their diameter (much less than 160 µm, which is the diameter of a mature oocyte)

Figure 3

vasa expression in the mature female and males. (a–b′) Sexually mature females. Traditional whole mount in situ hybridization (WISH) shows vasa expression surrounding the nuclei of oocytes (a′ and b′). (c–c′) Fully mature oocyte processed for hybridization chain reaction (HCR) showing vasa mRNA expression (yellow) and nuclear stain DAPI (cyan) in the perinuclear region. Box in (c) indicates the magnified region in (c′). nl: nucleus, od: oil droplet. (d–d′) Sexually mature male shows no expression of vasa. However, depending on the level of maturation, some males still showed small patches of vasa positive regions (f–g′). (e–e‴) Spermatozoa in a fully mature male processed for HCR show no vasa signal. (e and e″) vasa in yellow and DAPI in cyan, merged. (e′ and e‴) vasa signal shown alone. The acrosome and axial rod can be noted in these magnified views. a: acrosome, ar: axial rod. (e″) and (e‴) are magnified views from (e) and (e)″. (f–g′) Additional samples that were mature males but had some patchy expression of vasa

Figure 4

(a,a′) Example scoring of samples for segment number (blue) and vasa + clusters (green). (a′) Gray arrowheads— background, not scored; Green arrowheads—vasa+ clusters, scored. (b) Scatter plot showing the number of vasa clusters in worms by the number of segments at the time of fixation. Clusters typically begin forming after worms reach 30 segments and become numerous after approximately 40 segments. The estimated conditional median (red solid line) shows that this upward trend begins somewhere between 30 and 40 segments, closer to 35 segments. We also noted that there are no worms that are more than 40 segments in length that have zero vasa clusters in the trunk. The histogram above the scatter plot indicates the distribution of sample size by the number of segments in our dataset; notably, approximately half of the scored samples are below 40 segments and half are above. A 95% confidence interval for the conditional median is also indicated (gray shading between dashed red lines). (c) The same dataset and isotonic regression depicted in (b) but with median number of vasa+ clusters regressed on age. In contrast to segment number, age does not appear to be a strong determining factor for vasa+ cluster formation

Figure 5

(a) Experiment schematic showing the establishment of four new culture boxes from one large high‐density culture at 6 weeks post‐fertilization (t = 0). Two low density (worms per box: n = 20) and two high density cultures (worms per box: n = 100) were created. One of each culture type was fixed at 2 and 4 weeks after the experiment was started. Some samples from the original culture were also fixed at t = 0. (b) Scatterplot showing number of vasa+ clusters and the number of segments per worm (n = 39) fixed at the beginning of the experiment at t = 0 (6 weeks post‐fertilization). All the samples at t = 0 are shorter than 30 segments and have no vasa+ clusters in the trunk segments. (c–c″) Scatterplots showing the number of vasa+ clusters and the number of segments per worm 2 weeks after new low‐ and high‐density cultures were established. (d–d″) Scatterplots showing the number of vasa+ clusters and the number of segments per worm 4 weeks after new low‐ and high‐density cultures were established. Dashed line denotes 30 segment‐length. Overall, irrespective of culture density, worms that reach 30 segments or longer start forming vasa+ clusters in the trunk

Figure 6

(a) Amputation experiment design featuring controls, worms cut after segment 10, and worms cut after segment 20. All groups were fixed at the listed time points. (a′) Examples of “current length” (∑ seg no) versus “total segment number produced” (∑ seg produced) during lifetime. Note that when a worm is amputated after segment 10, it has nevertheless produced 20 segments in its lifetime at the time of amputation. Once this worm regenerates 10 new segments, its current length is 20 segments, but total segment number produced during lifetime is 30. (b) Box plots showing the distribution of the number of vasa+ clusters per sample by time point and experimental condition. Sample size and average total segment number for each group are shown directly under the box plot labels. Note that the group amputated after segment 10 (P10) is delayed in forming numerous vasa+ clusters compared to P20 and control (which are longer in total segment number during the earlier time points). As individuals in group P10 reach 35 segments and longer, they start forming vasa+ clusters (starting Day 25). (b′) Segment number distribution for each group is shown. Magenta line denotes 35 segments, the size around which worms start showing an upward trend in forming vasa+ clusters. Note that group P10 reaches 35 segments at the Day 25 time point. (c,c′) Conditional median regression curves showing vasa+ cluster formation trends in each group, ignoring time, with 95% pointwise confidence intervals given by shaded regions (c′). All three lines show an upward trend around 35 segment‐length, which suggests the worms need to reach this size threshold for forming vasa+ gonial clusters

Figure 7

vasa+ cell clusters are not required for regeneration. (a,a′) and (h,h′) show amputation schematics and the control worms at the start of the experiment (Day 0). (b–g′) Worms amputated after segment 10, fixed at different time points and processed for WISH for vasa. (b′), (c′), and (d′) are close‐ups of the regenerating tail in (b), (c), and (d). (i–n′) Worms amputated after segment 20, fixed at different time points and processed for WISH for vasa. (i′), (j′) and (k′) are close‐ups of the regenerating tail in (i), (j), and (k). Up until 30 dpa time point, neither group has vasa+ clusters in the trunk region other than the anterior cluster in the segments right after the jaw. There is no evidence for vasa+ cell migration towards the blastema. However, all the samples regenerate successfully by forming a regeneration blastema which expresses vasa (b′–d′ and i′–k′). (g′) and (n′) are close‐ups of vasa+ clusters in (g) and (n). Gray bars denote the regenerated region, while white bars denote the original segments. dpa: days post‐amputation