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Equalization of Cleavage Is Not Causally Responsible for Specification of Cell Lineage.

Introduction

Classically studied sea urchin embryos exhibit radial holoblastic cleavage with an unequal fourth cleavage in which smaller micromere cells arise at the vegetal pole. Molecular asymmetries become apparent along the animal-vegetal axis following this asymmetric cleavage (e.g., Wikramanayake et al., 1998; Angerer, 2005; Croce et al., 2011). The micro-meres undergo a second asymmetric cleavage that segregates the lineage, giving rise to all of the primary mesenchyme cells (PMCs) that both make up the larval skeleton and serve as the embryonic organizer from multipotent adult progenitors (including the primordial germ cells) (Yajima and Wessel, 2010; Campanale et al., 2014; Martik and McClay, 2015).

Alteration of this cleavage pattern correlates with altered cell identity, particularly loss of the PMC lineage. For example, perturbation of Cdc42 gene expression or function equalizes cleavage, and the resulting embryos lack skeletogenic cells (Martik, 2015; Sepulveda-Ramirez et al., 2018). It has been hypothesized that differential cell size is directly causal to the phenotype, perhaps by disrupting differential localization of maternal determinants (Sharma and Ettensohn, 2010); but there is no evidence that smaller cell size directly causes micromere cell identity and, if so, by what molecular means.

The addition of a surfactant such as sodium dodecyl sulfate (SDS) during a critical window for asymmetric cleavage delays cleavage until SDS has been washed out. When the embryos recover, subsequent cell divisions are equalized, but normal cleavage planes are maintained (Filosa et al., 1985; Langelan and Whiteley, 1985; Duncan and Whiteley, 2011). This treatment eliminates PMCs; and none of the embryos' cells perform typical PMC cell behaviors, such as cell cycle arrest and ingression, prior to gastrulation in SDS-treated embryos (Tanaka, 1976; Filosa et al., 1985; Langelan and Whiteley, 1985; Sharma and Ettensohn, 2010; Duncan and Whiteley, 2011). Treated embryos show delayed or defective gastrulation and reduced skeletons, each of which can be explained by the dual roles of PMCs as the endomesodermal organizer and the source of all skeletogenic cells in normal embryos.

Molecular phenotypes align with the observed morphological phenotypes. The SDS treatment abrogates localized expression of two PMC marker genes in the planktotrophic sea urchin Lytechinus variegatus (Sharma and Ettensohn, 2010): alx1, which is necessary for PMC commitment to skeletogenic cell fate (Ettensohn, 2003; Ettensohn et al., 2007), and delta, which is necessary for PMCs to induce non-skeletogenic mesoderm in adjacent cells (Sweet et al., 2002). However, the early PMC regulator pmar1 is unaffected, and the molecular mechanism by which these expression patterns are changed is unknown (Sharma and Ettensohn, 2010).

Only planktotrophic euechinoid urchins display the stereotyped asymmetric cell divisions that create PMCs. However, many other echinoderms (e.g., cidaroid urchins, lecithotrophic euechinoids, holothurians, ophiuroids) possess larval skeletal elements. Much of the gene regulatory network for PMC specification appears to operate in the prospective skeletogenic cells of these other echinoderms. Some urchins form variable numbers and variably sized "micromere" cells (particularly cidaroid urchins) (Schroeder, 1981; Wray and McClay, 1988; Yamazaki et al., 2012). Other euechinoids exhibit secondarily derived equal cleavage, in particular, the lecithotrophic urchin Heliocidaris erythrogramma (Raff, 1987; Wray and Raff, 1989); other lecithotrophs, such as the sand dollar Peronella japonica have lost the highly stereotyped cleavage pattern but may still divide asymmetrically (Amemiya and Arakawa, 1996).

A camarodont sea urchin that has lost the asymmetric cleavage, such as H. erythrogramma, offers an opportunity to separate cell size from other developmental differences that other echinoderms cannot, precisely because its equal cleavage represents secondary loss. Heliocidaris erythrogramma is phylogenetically closer to the sea urchin species in which previous surfactant experiments were done (Tanaka, 1976; Filosa et al., 1985; Langelan and Whiteley, 1985; Sharma and Ettensohn, 2010;Duncan and Whiteley, 2011) than a member of an echinoderm class with symmetric cleavage would be; this reduces a potential confounding variable introduced by phylogenetic distance. In addition, H. erythrogramma could be expected to retain other, perhaps still unknown, features of development besides the asymmetric cleavage that may underlie the change in cell specification observed in other euechinoids.

I hypothesized that SDS treatment would not affect specification of the skeletogenic cell lineage in an equally cleaving euechinoid, H. erythrogramma, based on the widespread association between differential size and differential fate reported in the literature. However, I found phenotypes very similar to those in planktotrophic euechinoids. This result suggests that the phenotype induced by SDS treatment in sea urchins is not a result of equalizing cleavage but may share a common cause.

Materials and Methods

Adult Heliocidaris erythrogramma (Valenciennes, 1846) were obtained off of the east coast of Australia at Little Bay, New South Wales (33[degrees]58'S, 151[degrees]14'E), and were maintained in natural seawater at ambient temperature (20-23 [degrees]C). Animals were spawned by intracoelomic injection of 0.5 mol [L.sup.-1] KCl, and gametes were collected in Millipore-filtered seawater (MFSW).

In a pilot experiment titrating the SDS concentration by orders of magnitude, 0.001% w/v was found to be the highest non-lethal concentration. The effective dose of 0.001% w/v SDS was similar to reports in other urchins; 15-30 [micro]g [mL.sup.-1] (= 0.0015%-0.003% w/v) was used in relatively recent papers that treated different species (Sharma and Ettensohn, 2010; Duncan and Whiteley, 2011).

Embryos were treated beginning at the 4- to 8-cell stage, about 2 hours post-fertilization (hpf), at 22 [degrees]C, for 1 hour. Heliocidaris erythrogramma embryos exhibit greater within-batch asynchrony than most commonly used sea urchins. Embryos were washed with three changes of MFSW and finally were transferred into a fresh culture dish to minimize SDS carryover. Control embryos were treated with an equal volume of sterile water and were subjected to mock washout.

Embryos were reared to 56 hpf, when vestigial larval and juvenile skeletons are biomineralized and hence visible by light microscopy without additional processing. Larvae were fixed overnight (~16 h) at 4 [degrees]C in 4% paraformaldehyde (Sigma 158127; Sigma-Aldrich, St. Louis, MO) + 20 mmol [L.sup.-1] EPPS (4-(2-Hydroxyethyl)-l-piperazinepropanesulfonic acid) (Sigma El 894), washed 3 times in pasteurized MFSW, dehydrated stepwise into 100% methanol, and stored at -20 [degrees]C in nonstick tubes. Embryos were washed with 100% ethanol, then cleared and mounted in 2:1 (v/v) benzyl benzoate: benzyl alcohol. Differential interference contrast (DIC) micrographs were taken on an Olympus BX60 upright microscope with an Olympus DP73 camera.

Embryos were scored by hand, and data were analyzed in R (R Foundation for Statistical Computing, Vienna). Boxplots were generated in R, using the R package ggplot2 (Wickham, 2016).

Results

SDS-treated Heliocidaris erythrogramma embryos exhibit a range of phenotypes, from those that are largely normal to those that lack morphological evidence of most mesoderm derivatives. Severely affected larvae lack red pigment cells and a juvenile rudiment (Fig. 1A, B). Most treated embryos have a greatly reduced or absent biomineralized skeleton (Fig. 1C, D). The variation in phenotypes may result from exactly which cell cycle the individual embryo would have undergone during treatment, because H. erythrogramma embryos exhibit more intra-replicate variation in their timing than more commonly studied euechinoid sea urchins. Overall, treatment with SDS during the second to fifth cleavage cycles significantly reduces the number of skeletogenic cells and non-significantly reduces the number of pigment cells in H. erythrogramma. The adult rudiment, induced by an interaction of ectoderm and non-mesenchymal mesoderm, is rarely absent (Fig. 1E).

Discussion

While a molecular mechanism for the SDS phenotype remains unknown, these data suggest that equalization of cleavage per se does not cause the SDS phenotype. Intriguingly, the second asymmetric cleavage specifying multipotent adult progenitors is reported to recover in such embryos (Langelan and Whiteley, 1985), so only one of two asymmetric cleavages is lost. SDS treatment also has been shown to disrupt asymmetrical localization of maternal determinants in a spiralian by irreversibly inhibiting polar lobe formation (Render, 1990). Polar lobe formation is sensitive to disruptions of the actin and tubulin cytoskeletons, as well as the plasma membrane. Moreover, most non-echinoid echinoderm embryos cleave equally, but many, nevertheless, form a larval skeleton. If members of these other echinoderm classes also respond similarly to SDS treatment, it would suggest a shared underlying mechanism for the earliest steps in specification of these cells. Thus, the SDS phenotype is not specific to urchins, nor is asymmetric cleavage specifically diagnostic of echinoderm larval skeleton.

The correspondence of cell size to PMC number was apparently confirmed by 2,4-dinitrophenol (DNP) treatment (Kominami and Takaichi, 1998). All cells below a size threshold develop into the PMC phenotype when their number is increased by DNP. However, DNP works similarly to SDS by temporarily preventing cleavage; thus, when allowed to cleave again, the embryos undergo an unequal cleavage at the third, rather than the fourth, cell cycle. This causes micro-meres to be born both early, relative to cleavage cycle, and larger than normal. These micromeres subsequently proliferate more than in control embryos. Thus, the possibility remains that localization of crucial determinants is on a molecular clock independent of cell size.

Because no molecular mechanism is known for the SDS phenotype, its utility is limited. For example, SDS treatment of yeast alters genome-wide gene expression and appears to affect many different organelles and cellular processes (Sirisattha et al., 2004). Thus, it remains possible that the effects of SDS represent not a specific phenotype but a recovery from a cytotoxic assault.

However, these results cast doubt on a widespread assumption about an otherwise deeply studied cell determination event. Future work should attempt to dissect the complex relationship between the sea urchin unequal cleavage and its accompanying cell specification event, considering that they may have a shared molecular cause rather than a direct effect of cell size on identity. Given this result, hypotheses such as nuclear-to-cytoplasmic ratio are less likely to explain micro-mere specification than cytoskeletal behaviors or localization of membrane-bound determinants.

Acknowledgments

Maria Byrne, Gregory A. Wray, and David R. McClay provided laboratory space, animals, reagents, and feedback. This work was funded by Australian Research Council grant DP120102849, National Science Foundation IOS-1457305, and National Institutes of Health ROl HD14483 and PO1 HD37105.

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ALLISON EDGAR

Department of Biology, Duke University, Durham, North Carolina, and School of Medical Science and Bosch Institute, Department of Anatomy and Histology, University of Sydney, Sydney, New South Wales, Australia

Received 8 April 2019; Accepted 4 July 2019; Published online 22 November 2019.

Email: ae75@duke.edu.

Abbreviations: DNP, 2,4-dinitrophenol; hpf. hours post-fertilization; MFSW, Millipore-filtered seawater; PMC, primary mesenchyme cells; SDS, sodium dodecyl sulfate.

DOI: 10.1086/705358
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Author:Edgar, Allison
Publication:The Biological Bulletin
Date:Dec 1, 2019
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