Transcriptomic analysis of Nodal – and BMP- associated genes during development to the juvenile seastar in Parvulastra exigua (Asterinidae)

Abstract

The molecular mechanisms underlying development of the pentameral body of adult echinoderms are poorly understood but are important to solve with respect to evolution of a unique body plan that contrasts with the bilateral body plan of other deuterostomes. As Nodal and BMP2/4 signalling is involved in axis formation in larvae and development of the echinoderm body plan, we used the developmental transcriptome generated for the asterinid seastar Parvulastra exigua to investigate the temporal expression patterns of Nodal and BMP2/4 genes from the embryo and across metamorphosis to the juvenile. For echinoderms, the Asteroidea represents the basal-type body architecture with a distinct (separated) ray structure. Parvulastra exigua has lecithotrophic development forming the juvenile soon after gastrulation providing ready access to the developing adult stage. We identified 39 genes associated with the Nodal and BMP2/4 network in the P. exigua developmental transcriptome. Clustering analysis of these genes resulted in 6 clusters with similar temporal expression patterns across development. A co-expression analysis revealed genes that have similar expression profiles as Nodal and BMP2/4. These results indicated genes that may have a regulatory relationship in patterning morphogenesis of the juvenile seastar. Developmental RNA-seq analyses of Parvulastra exigua show changes in Nodal and BMP2/4 signalling genes across the metamorphic transition. We provide the foundation for detailed analyses of this cascade in the evolution of the unusual pentameral echinoderm body and its deuterostome affinities.

Introduction

The molecular mechanisms underlying patterning of body axes in development are remarkably conserved across invertebrates and vertebrates, despite the great diversity of animal body plans. In most animals, the major body axes, the anteroposterior, dorsoventral and left-right are readily apparent in a bilateral body organisation, facilitating comparative analysis of axial patterning mechanisms. Within the deuterostomes however, the echinoderms have evolved a radial body plan characterised by 5-fold symmetry with many hypotheses proposed as to how this evolved and relates to the bilateral plan of groups in the chordate line to which they are related (Raff and Popodi, 1996; Morris, 2012; Lacalli, 2014; Arnone et al., 2015; Byrne et al., 2016). Echinoderms have a bilateral body organisation as larvae and the earliest echinoderms in Cambrian fossils were bilateral as adults, indicating that adult radial symmetry is secondarily evolved (Zamora and Smith, 2012; Smith et al., 2013). While the evolutionary origin of this body plan has been the subject of debate, the molecular mechanisms underlying its development are poorly understood. Understanding how pentamery is patterned in development is important in tracing its evolution.

Most molecular studies of echinoderm development have focussed on early embryos and larvae and the gene regulatory networks (GRNs) involved with patterning the primary germ layers and the sea urchin larval skeleton (Peter and Davidson, 2011; Arnone et al., 2015; Cheatle Jarvela et al., 2016; Cary and Hinmann, 2017; Gildor et al., 2017, Gildor et al., 2019). Investigation of molecular and cellular mechanisms underlying adult development requires access to the developing juvenile, which in most species, requires long-term rearing of feeding larvae making it difficult to access the juvenile stage (Wray et al., 2004; Hodin et al., 2019). To address this challenge, rapidly developing species that have evolved non-feeding lecithotrophic larvae and metamorphose soon after gastrulation provide model systems to investigate development of the adult body plan (Raff, 1992; Wray, 1996; Byrne, 2006, Byrne, 2013; Raff and Byrne, 2006). The evolution of lecithotrophy in echinoderms has long been the focus of studies of alternative larval life history modes (Raff, 1992; Wray, 1996; Raff and Byrne, 2006). Recent generation of transcriptomes that incorporate developmental stages through metamorphosis and adult body formation for lecithotrophic species including the sea urchin Heliocidaris erythrogramma and the seastar Parvulastra exigua, provides an important foundation for analysis of the molecular processes underlying development of pentamery (Wygoda et al., 2014; Byrne et al., 2015, Byrne et al., 2018, Byrne et al., 2020; Israel et al., 2016; Koop et al., 2017).

Genes associated with Nodal and bone morphogenetic protein (BMP) signalling are integral to axis formation in patterning animal development (Bier and De Robertis, 2015). These signalling pathways have been a focus in comparing the molecular mechanisms patterning body axes across the Metazoa and in identifying potential homologies (Shen, 2007; Bier and De Robertis, 2015). In sea urchins and seastars the role of the Nodal-BMP cascade in larval axis formation is well documented (Smith et al., 2008; Raff and Smith, 2009; Duboc et al., 2010; Angerer et al., 2011; Luo and Su, 2012; Bessodes et al., 2012; Molina et al., 2013; Yankura et al., 2013; Su, 2014; Sasaki and Kominami, 2017; Fresques and Wessel, 2018). For H. erythrogramma, temporal and spatial expression patterns revealed that genes of the Nodal-BMP cascade are also expressed during metamorphosis and juvenile development (Wygoda et al., 2014; Byrne et al., 2015, Byrne et al., 2018; Koop et al., 2017). BMP2/4 and several downstream genes are located in the five hydrocoele lobes, the first morphological expression of pentamery, and core of the echinoderm body plan (Koop et al., 2017). This indicates a role for Nodal-and BMP signalling in patterning body plan development. The 5-fold expression of Nodal and BMP2/4 signalling genes in the hydrocoel as well as pentameral expression of genes of the Drosophila retinal GRN indicates that pentamery may have evolved through duplication of the ancestral single anterior-posterior axis (Byrne et al., 2007; Koop et al., 2017), as hypothesised (Raff and Popodi, 1996; Morris, 2012; Byrne et al., 2016).

As genes associated with Nodal and BMP2/4 signalling appear to be involved in patterning development of pentamery in sea urchins, we investigated the expression of these genes in development to the juvenile seastar, Parvulastra exigua. For the Echinodermata, the asteroid model represents the basal-type body architecture with a distinct (separated) ray structure and are likely to be a better system to understand evolution of pentamery than the highly modified sea urchin body, which has cohesion between the rays and other derived features including the most radical echinoderm metamorphosis forming the juvenile almost entirely from axial elements (skeleton associated with the water vascular system) (David and Mooi, 1998; Byrne et al., 2016). Parvulastra exigua releases its large eggs (360 μm diameter) in benthic masses that adhere to the substratum with their sticky jelly coat, a hypertrophied extracellular matrix that attaches the embryos to the substrate (Cerra and Byrne, 1995). The larval feeding program has been deleted as the blastopore closes soon after gastrulation. Parvulastra exigua has holobenthic development and hatch as larvae that maintain a tenacious attachment to the substratum (Fig. 1). The larvae have the tripod morphology of benthic seastar larvae with a hypertrophied attachment complex. Metamorphosis ensues as the larval body degenerates and the juvenile tube feet take over the role of attachment over approximately 2 weeks. Evolution of development in P. exigua has been the focus of studies of evolution of benthic larvae in asteroids (Byrne, 1995, Byrne, 2006, Byrne, 2013).

We used the de novo developmental transcriptome generated for Parvulastra exigua, from the embryo through metamorphosis, incorporating 6 stages from gastrula (1-day post fertilization - dpf) to the definitive juvenile (21 dpf) to determine the expression profiles of genes associated with Nodal and BMP2/4 signalling. A recent investigation of global variation in gene expression in P. exigua showed that marked changes occur during metamorphosis and gene ontology analysis revealed dynamic changes in gene expression through the transition to pentamery (Byrne et al., 2020). Many of the terms enriched in late metamorphosis included signalling genes (Byrne et al., 2020). In situ hybridisation shows that axial patterning genes (e.g. hox4, eng, otx, eya, pax6), including genes associated with Nodal and BMP2/4 signalling, are expressed in coelomic, neural and sensory structures (Byrne et al., 2005, Byrne et al., 2020; Cisternas and Byrne, 2009). Thus, as for sea urchins, gene expression in development of the pentameral seastar shows 5-fold expression including in structures core to the adult body plan (Byrne et al., 2005, Byrne et al., 2020; Cisternas and Byrne, 2009).

We identified 39 genes associated with the Nodal and BMP2/4 network in the P. exigua transcriptome and determined their temporal expression patterns and profiles through metamorphosis to the pentameral juvenile. Our results revealed complex changes in gene expression through development and that groups of genes change their expression in concert. The expression profiles point to genes likely to be involved in metamorphosis and juvenile development. Our results were compared with those from a similar study of Nodal and BMP2/4 associated genes in sea urchin juvenile development (Byrne et al., 2015).