Elongation During Segmentation Shows Axial Variability, Low Mitotic Rates, and Synchronized Cell Cycle Domains in the Crustacean, Thamnocephalus Platyurus Savvas J

Constantinou et al. EvoDevo (2020) 11:1 https://doi.org/10.1186/s13227-020-0147-0 EvoDevo RESEARCH Open Access Elongation during segmentation shows axial variability, low mitotic rates, and synchronized cell cycle domains in the crustacean, Thamnocephalus platyurus Savvas J. Constantinou1,4, Nicole Duan1,5, Lisa M. Nagy2, Ariel D. Chipman3 and Terri A. Williams1* Abstract Background: Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented. Results: Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphomet- ric measurements of the growth zone and of newly formed segments suggest tagma-specifc features of segment generation. Using methods for detecting two diferent phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation. Conclusions: Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries. Keywords: Arthropod, Segmentation, Growth zone, Mitosis, Wnt, EdU Background simultaneously, through progressive subdivision of the Arthropods are the most diverse phylum on earth, and embryo [1]. By contrast, the vast majority of arthropods much of that diversity derives from the variability in their add their segments sequentially, from a posterior region segmented body plan. Te developmental mechanisms termed the “growth zone”. Tese species elongate while that produce segments have been extensively studied in adding segments, thus posing fundamental questions the model organism, Drosophila. But Drosophila is atypi- that do not apply to the model system Drosophila: How cal among arthropods because it establishes segments does elongation occur in the posterior? How are elongation and segmentation integrated [2]. While some mechanisms of elongation are known (e.g., teloblastic growth *Correspondence: [email protected] 1 Biology Department, Trinity College, Hartford, CT, USA in malacostracan crustaceans [3]), surprisingly little is Full list of author information is available at the end of the article known about the range of cell behaviors (e.g., cell division © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Constantinou et al. EvoDevo (2020) 11:1 Page 2 of 18 or cell movement) responsible for elongation throughout and an undiferentiated trunk. Sequential segment addi- arthropods. tion and progressive diferentiation gradually produce Because most species elongate signifcantly during seg- the adult morphology of eleven limb-bearing thoracic mentation, classical concepts of posterior growth gener- segments and eight abdominal segments, the frst two of ally invoke mitosis, either in posterior stem cells or in a which are fused to form the genital region [5, 30–32]. Te vaguely defned posterior region of proliferation [4–8]. highly anamorphic development of Tamnocephalus, as Cell movement has also been assumed to play a role in well as their phylogenetic position, makes them an inter- elongation in cases where embryonic shape changes dra- esting comparison to other arthropods and we have pre- matically [7–10]—and is documented in the four beetle, viously shown that there are numerous Wnts expressed Tribolium castaneum [11–13]. Te current descriptive in the posterior during segmentation [35]. In addition, data suggest a large degree of variability in how sequen- Notch signaling, a known feature of posterior pattern- tially segmenting arthropod embryos grow (reviewed in ing in some arthropods also slows segment addition in [7, 14, 15]). Tat variability has led to the suggestion of Tamnocephalus [37]. replacing the term “growth zone” with “segment addi- Here, we examine in detail the morphometric changes tion zone” (e.g., [16, 17]) or “undiferentiated zone” [15] and cell behaviors associated with segment addition in as possible alternatives. Because the relative contribution Tamnocephalus. We demonstrate that segments from of various cell processes—division, size or shape change, the third thoracic segment arise at a constant rate. We movement—to embryo elongation have only recently characterize the growth zone and newest added segment begun to be quantitatively and systematically examined, during segment addition using morphometric measures. it is challenging to fnd an appropriate catch-all term for Changes in these measures occur at tagma boundaries. all arthropods. Despite expectations for mitosis to drive elongation, we In contrast to our lack of understanding of cellular demonstrate that mitosis in the growth zone is relatively mechanisms of elongation, the models of the gene regu- rare; it contributes to elongation, but at lower rates than latory networks that pattern segments in sequentially anticipated. Tese results corroborate those of Free- segmenting arthropods are being tested more broadly man [33], who counted cells and mitoses in the trunk of (reviewed in [14, 18–21]). In the posterior growth zone, the frst three instars of Artemia larvae and found more Wnt signaling activates the transcription factor caudal mitoses near the anterior than posterior trunk region. (cad), which, through downstream genes, progressively Examination of cells undergoing DNA synthesis reveals subdivides the anterior growth zone and eventually discrete domains of apparently synchronized cells in the specifes new segments [19, 22]. In some systems, poste- anterior growth zone and newest segment. In Tamno- rior Wnt signaling is also thought to keep posterior cells cephalus, boundaries of cell cycling domains correlate in a pluripotent state, presumably dividing as needed precisely with Wnt and cad expression in the growth and thus fueling elongation [22–25]. To fully under- zone, suggesting direct regulation of these behaviors by stand segmental patterning and interpret function via the segmentation gene regulatory network. knock-down/knock-out studies, we need a more detailed understanding of the cellular mechanisms underlying Results elongation and growth [14]. Segment addition and morphogenesis occur progressively Our collaborating labs analyzed the changes in the in Thamnocephalus larvae growth zone during segmentation in three pancrus- Tamnocephalus hatches with three diferentiated larval taceans to compare between species: including two head appendages (frst antennae, second antennae and insects, the beetle, Tribolium castaneum [12], and the mandibles, [34]). In addition, the frst and second max- milkweed bug, Oncopeltus fasciatus [25]; and the crusta- illae and on average three thoracic segments are already cean described here, Tamnocephalus platyurus. Tam- specifed, as determined by the expression of a monoclo- nocephalus, commonly named fairy shrimp, belong to nal antibody (En4F11) that recognizes the segment polar- the same order as the brine shrimp, Artemia. Both are ity protein, Engrailed (En). As larvae grow, segments are branchiopod crustaceans, a taxon more closely related added gradually from the posterior growth zone (Fig. 1), to insects than are malacostracan crustaceans (e.g., with expression of En at the anterior of the growth zone Parhyale hawaiensis [26, 27]). Tamnocephalus live in indicating specifcation of a new segment. Segments temporary freshwater ponds [28] and their life cycle mature gradually, so the trunk typically shows the pro- includes desiccation-resistant encysted eggs (giving rise gression of segmental development: segment pattern- to commercially available cysts, primarily for toxicol- ing, segment morphogenesis, and limb morphogenesis

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