Sister cell fates were similar regardless of whether they divided before or after cisplatin administration and did not arise from proximity-related factors, suggesting fate determination early in a cell's lifetime. Based on these findings, we developed a theoretical model explaining how the observed correlation structure can arise from oscillatory mechanisms underlying cell fate control. Our model recapitulated the data only with very specific oscillation periods that fit measured circadian rhythms, thereby suggesting an important role of the circadian clock in controlling cellular fates.
Abstract The origin of lineage correlations among single cells and the extent of heterogeneity in their intermitotic times IMT and apoptosis times AT remain incompletely understood. In organisms where germline specification takes place early during development, the PGCs are segregated before gastrulation Extavour and Akam, This also appears to be the case in P. Our and previous observations suggest these cells make only pPGCs Ackermann et al. In particular, the ectodermal and mesodermal cells that constitute the SAZ of the juvenile worm persistently express the GMP genes such as vasa , piwi or nanos Gazave et al.
It is currently not known whether the GMP is shared between these apparently different sets of cells because the program is crucial for multipotency, or whether the GMP expression in SAZ, and growth zone cells in general, reflects a persistent ability of these cells to produce germline cells. A similar situation has been described in planarians, where both the neoblast cells the planarian stem cells and the germ cells express piwi Handberg-Thorsager, ; Nakagawa et al. In planarians, the neoblasts give rise to the germ cells Morgan, ; Newmark et al.
Only further investigation involving genetic cell tracing or cell ablation will establish whether P. However, the lineage that generates the pPGCs in the slipper snail also gives rise to mesodermal cell types. What role these different segregation and specification patterns play, and whether they have implications in the ability to regenerate germ cells at later stages of development and in adulthood in a given species need to be further investigated.
We have established, for the first time, the early pattern of segmental mesoderm formation in P.
Stem cell fate choice: determined in an instant
We found that during embryogenesis, segmental precursor cell segregation is similar to that in the leech or other clitellate embryogeneses. This is in sharp contrast to the 32 segments formed during leech embryogenesis and to the numerous segments formed in the other non-leech clitellates Anderson, a ; Balavoine, ; Goto et al.
Nevertheless, as in clitellates, pairs of mesodermal precursors called primary blast cells PBCs are sequentially produced in an anterior to posterior progression, and correspond to a mesodermal segment of the larva. The segregation of the series of segmental PBCs in P. These two pairs of anterior head mesoderm precursors may be related to the six pairs of em precursors known in the leech that contribute to endoderm and pharynx musculature Figure 8C Gline et al. We were not able to determine the precise tissue-type contribution of each mesodermal PBC because our lineage analysis ends at the beginning of late trochophore stage, before cell differentiation takes place.
Additional imaging that extends further in development suggests that segmental blocks of mesodermal cells contributed mostly to a single hemisegment Figure 4—figure supplement 3 , Figure 4—video However, we cannot exclude the possibility that each clonal block will eventually contribute complementary sets of different tissue types to two or more contiguous segments in the juveniles, as they do in the leech.
Future studies will also determine whether these precursors give rise to a few neuronal cells as in the clitellates , as well as other tissues such as the gut endoderm, and possibly pygidium the non-segmental posterior end as it has been previously suggested Starunov et al. Another important difference between P. A simplified annelid phylogeny Weigert and Bleidorn, summarizes the main annelid species investigated for segment formation and teloblasts.
The number of segments appearing during the pelagic larval phase but patterned during embryogenesis and the number of segments added posteriorly after the start of benthic life can vary considerably Balavoine, In leeches, a fixed number of segments is made during direct embryonic development and none added after hatching. In non-leech clitellates such as Tubifex , the embryos make a few tens of segments during embryogenesis and add many more after hatching. Embryonic and post-embryonic teloblasts differ in the way they have been evidenced.
Embryonic teloblasts in the Clitellates Weisblat and Kuo, ; Goto et al. By contrast, post-embryonic teloblasts are inconspicuous cells that are identified only so far by their molecular stem cell signature Gazave et al. No direct observation of post-embryonic teloblast patterns of division is available so far.
No direct evidence for embryonic teloblasts giving birth to post-embryonic teloblasts exists in any species so far to our knowledge.
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The ancestor of annelids presumably had both larval and post-larval segments, and thus it raises the question of the presence of both embryonic and post-embryonic teloblasts in this ancestral annelid, and even more broadly in other spiralians such as chitons a type of segmented mollusk. We also found that after the progenitors of the four mesodermal segments have been produced, the remaining M teloblast cells in P.
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The new tissues that form new segments originate from the SAZ which is a specific ring of small cells presumably teloblast-like within the posterior growth zone, and has mesodermal and ectodermal components Balavoine, Much is still unknown about the exact embryonic origins of the SAZ in annelids. Studies using bright-field microscopy, and DiI injections suggested that the mesodermal SAZ originates from the 4d blastomeres Ackermann et al.
Thus, how these precursors go onto form the ring-like mesodermal SAZ and how they contribute to new tissues in juvenile worms remain to be determined.
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This study in P. In the leeches, the totality of a species-specific number of segments are made during embryogenesis through the activity of large embryonic teloblasts. In contrast, in most marine annelids, the majority of segments are made during post-embryonic juvenile development, through the activity of inconspicuous posterior stem cells whose characteristics are still largely unknown. Earlier works in P.
Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, CAS
We establish in this article that the mesodermal teloblasts which are very similar to the leech are indeed at play in the P. We also show that these embryonic teloblasts are most likely at the origin of the teloblast-like posterior stem cells of the juvenile post-embryonic teloblasts , thus possibly establishing a cellular continuity in segment formation in P. In addition, it will be useful to determine if embryonic ectodermal teloblasts exist in P. The large teloblasts of Clitellate embryos would have evolved in the context of an acceleration of development, most or all segment formation happening in the embryonic stage Balavoine, Meanwhile, parallels among all spiralians remain to be determined.
It is legitimate to wonder whether embryonic teloblasts are present in mollusks having a form of mesodermal segmentation such as chitons or aplacophorans Scherholz et al. Future studies using high-resolution live imaging and genetic lineage-tracing methods on these exciting but understudied spiralian groups, as well as additional studies in the annelids will help answer the open questions surrounding teloblasts and their involvement in the evolution of different body plans.
Platynereis dumerilii embryos were obtained from lab cultures at the Institut Jacques Monod. Cultures are reared based on previous protocols Dorresteijn et al. Embryos and larvae are staged after Fischer et al. We provide a table with calculated developmental times and stages corresponding to each time point Table 1. Using an alignment of P. To express constructs in P. For preparing the embryos for microinjections, fertilized embryos were dejellied and their cuticle was softened via enzymatic degradation.
For microinjections, an agarose injection platform made of 1. Louis, MO, P, 0. Embryos were injected at one-cell stage, two-cell stage into CD half , and four-cell stage into D quadrant. A A Zeiss inverted scope with a gliding stage, coupled with the Eppendorf Femtojet microinjector and Eppendorf Transferman micromanipulation system were used for injections. The agarose platform was placed into a small petri dish lid and covered with filtered sea water before the embryos were transferred into the dish.
In B and C, the steps of general experimental outline for live imaging samples at different stages are listed. For all mounts, we used glass-bottom dishes MatTek Corporation 35 mm dish, P35G-0—10 C , as this method enabled keeping embryos and larvae in sea water which could be easily renewed if desired. As a result, samples were healthy after mounting during extended periods of imaging, and even after imaging they continued to grow in the agarose.
Mounting was carried out on a warm plate to keep low-melting agarose from solidifying while allowing enough time for rotating the samples to the desired position using an eyelash tool Figure 10—figure supplement 1. Injected embryos and larvae can be checked for normal development based on a number of criteria observed at different stages of development. We picked healthy samples based on these criteria, as well as the samples with optimal fluorescence signal strength which varies based on the amount of mRNA injected , and appropriate angle for mesoderm imaging after mounting.
Depending on the developmental stage to be imaged, samples had to be mounted before or after the ciliary band formation and the start of swimming movements. Mounting embryos or larvae with functional cilia will result in the specimens rotating in their agarose spherical lodge. For stages after the cilia form, immobilization requires deciliation at the time of mounting. We thus carried out imaging for both pre-hatching embryonic and pre-larval and post-hatching larval stages using two different techniques:.
We observed that specimens mounted in low-melting agarose before 15 hpf usually around 5—6 hpf usually will not move even after the ciliary bands develop, because the growing cilia are impaired by the agarose covering. These specimens were simply placed onto the glass coverslip, excess sea water was removed, and low-melting agarose was added to cover the sample. Embryos and larvae can then be mounted in low-melting agarose in the next ten minutes before the cilia start to grow back. These DMSO-treated larvae recover completely and grow without abnormalities.
Mounting injected samples during larval stages may be desirable when a specific and precise orientation of the sample is required starting at a specific stage. Embryos were injected at one-, two-, or four-cell stage with HistoneH2A-mCherry and mVenus-cdt1 aa solution mix, and were raised until 12 hpf. These experiments were done according to previously-published protocols Asadulina et al. For EdU assays, embryos or larvae were incubated in EdU incubation conditions same as above for the desired time period. Then EdU solution was washed by transferring the samples several times through sea water.
EdU reaction was developed on the final day of immunohistochemistry protocol. For live imaging, a Zeiss LSM confocal scope was used. In the samples coinjected with both nuclear and membrane constructs, we took advantage of mVenus YFP being nuclear and EGFP being strictly localized to the cell membrane, and imaged the two fluorophores together, using single laser for excitation.
This allowed quick simultaneous acquisition of all three fluorophores decreasing phototoxicity due to laser exposure. Fluorescent protein production from the injected mRNA started becoming detectable around 5—6 hr after injections, thus imaging could not be carried out earlier. For the samples imaged for cell lineage analysis Samples A, B, C in Figure 5—figure supplement 1 , 1. EdU assays, and immunostaining stacks were analyzed and exported using Fiji release of ImageJ Schindelin et al.