Skip to content

Advertisement

  • Research
  • Open Access

Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus

EvoDevo201910:2

https://doi.org/10.1186/s13227-019-0115-8

  • Received: 5 December 2018
  • Accepted: 30 January 2019
  • Published:

Abstract

Background

The sea urchin is a basal deuterostome that is more closely related to vertebrates than many organisms traditionally used to study neurogenesis. This phylogenetic position means that the sea urchin can provide insights into the evolution of the nervous system by helping resolve which developmental processes are deuterostome innovations, which are innovations in other clades, and which are ancestral. However, the nervous system of echinoderms is one of the least understood of all major metazoan phyla. To gain insights into echinoderm neurogenesis, spatial and temporal gene expression data are essential. Then, functional data will enable the building of a detailed gene regulatory network for neurogenesis in the sea urchin that can be compared across metazoans to resolve questions about how nervous systems evolved.

Results

Here, we analyze spatiotemporal gene expression during sea urchin neurogenesis for genes that have been shown to be neurogenic in one or more species. We report the expression of 21 genes expressed in areas of neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned). Among those 21 gene expression patterns, we report expression of 11 transcription factors and 2 axon guidance genes, each expressed in discrete domains in the neuroectoderm or in the endoderm. Most of these genes are expressed in and around the ciliary band. Some including the transcription factors Lv-mbx, Lv-dmrt, Lv-islet, and Lv-atbf1, the nuclear protein Lv-prohibitin, and the guidance molecule Lv-semaa are expressed in the endoderm where they are presumably involved in neurogenesis in the gut.

Conclusions

This study builds a foundation to study how neurons are specified and evolved by analyzing spatial and temporal gene expression during neurogenesis in a basal deuterostome. With these expression patterns, we will be able to understand what genes are required for neural development in the sea urchin. These data can be used as a starting point to (1) build a spatial gene regulatory network for sea urchin neurogenesis, (2) identify how subtypes of neurons are specified, (3) perform comparative studies with the sea urchin, protostome, and vertebrate organisms.

Keywords

  • Neurogenesis
  • Neural specification
  • Sea urchin
  • Gene regulatory state

Background

The formation of a nervous system is a key innovation in evolution that allows for an organism to integrate sensory information and interact with its environment through motor output. Neurogenesis is a tightly controlled process that occurs during embryonic development in both indirect (animals with larval stages) and direct developing organisms. Comparisons of genomic sequences across the animal kingdom indicate that metazoan nervous systems largely employ a conserved set of genes during embryonic development [1]. Studying those genes in a number of organisms, especially those with simplified nervous systems, has the promise of revealing not only how nervous systems develop, but also how they evolved. Over the past decade, much has been learned about the origins of the vertebrate nervous system, and information has accumulated for neurogenesis of other deuterostomes, including the sea urchin. Nevertheless, much remains to be learned about how complex nervous systems develop.

Echinoderms are a basal deuterostome group characterized by pentaradial symmetry. Unlike the adults, echinoderm embryos are bilaterally symmetric. The nervous system of the bilaterally symmetric sea urchin embryo is composed of 3 major regions. The first is the apical organ, which is a collection of 2–6 serotonergic neurons as well as several other neural cell types found in the anterior end of the embryo [24]. The apical organ is considered to be the embryonic central nervous system and the processing center of the embryo [5]. The second region is the ciliary band, an ectodermal band of ciliated cells and neurons thought to aid in swimming and sensory behaviors [68]. Associated with the ciliary band is a collection of neurons known as the postoral neurons which lie just outside the ciliary band and connect axons to the ciliary band axonal tract [9]. Together, the ciliary band and postoral neurons are considered to be part of the peripheral nervous system of the embryo [5, 9]. The third region of the embryonic nervous system is in the endoderm where several neurons within the gut, as well as in the developing mouth and anus, aid in swallowing and movement of food through the digestive tract. Neurons in the gut have been shown to be specified directly within the endoderm [10].

Over the last 20 years, much effort has been put into creating a developmental gene regulatory network (GRN) for sea urchin development. A big advance in that research occurred with publication of the first sea urchin genome in 2006, and the availability of the genomic information spurred deeper insights into many properties of the embryo [11]. During the annotation phase of the genome project, a large number of dedicated neural molecules were identified [12], and over the last several years, efforts have begun to build GRN states for the sea urchin [2, 5, 13, 14]. However, only a limited number of these neural molecules have been spatiotemporally and functionally characterized. Fewer than 20 specifiers have been localized to neural precursors and assembled into GRN models of the more than 70 transcription factors identified in the genome (based on sequence orthologs of transcription factors identified as neural in other systems) [2, 5, 1215].

Expanding the sea urchin neurogenic GRN can be beneficial for many reasons. The first is the evolutionary insights that can be gained. The phylogenetic position of sea urchins as a basal deuterostome has already shown, with a limited dataset, that sea urchin neurogenesis resembles vertebrate neurogenesis, more than protostome neurogenesis [2]. This suggests that deuterostome neurogenesis is quite conserved. An examination of how sea urchin neurogenesis occurs can be extremely informative, particularly in instances where neurogenic GRNs differ between deuterostomes and protostomes. Another compelling reason for exploring sea urchin neurogenesis is from a developmental perspective; the simplicity and speed of neurogenesis in the sea urchin embryo, along with an ability to identify and place GRN components efficiently, allow for a thorough dissection of which genes are used and how they are used to build a deuterostome nervous system.

The transcription factors shown to be necessary for neurogenesis in the sea urchin include soxC and brn1/2/4, which are expressed broadly in the nervous system and are required for neurons throughout the embryo [5, 13]. Furthermore, some transcription factors are expressed only by specific types of neural progenitors in the sea urchin and are required for the development of subsets of neurons in the nervous system. Neurogenin is required for specification of cholinergic neurons in the ciliary band, orthopedia is required for specification of postoral neurons, and achaetescute and zfhx1 have been shown to be required for specification or differentiation of serotonergic neurons in the apical organ, respectively [2, 13, 15]. Additionally, soxb1, insm, and sip1 are expressed during the initial proneural specification period in the sea urchin [14]. Perturbations of these transcription factors led to the construction of preliminary gene regulatory networks for neurogenesis. Inhibition of signaling pathways such as Nodal, FGF, Delta/Notch and signaling molecules such as Wnt ligands has also contributed to the knowledge of inputs into nervous system development in the sea urchin [2, 5, 14, 15]. Before GRNs can be assembled to reveal detailed neurogenic specification sequences, and allow for evolutionary comparisons, many additional neural transcription factors and signaling molecules must be characterized. Thus, identification of additional genes involved in neurogenesis and their spatiotemporal expression profiles is a necessary beginning point toward that goal. With a vastly expanded group of involved genes, one will then be able to ask questions about patterning: Is neural development similar to vertebrates? Are the same genes used in similar ways as flies and vertebrates? Which neural processes and phenomena are vertebrate or deuterostome innovations, and which evolved earlier? What are the innovations (if any) made within the echinoderm lineage to get their unique organization of the nervous system?

Here we report the spatial and temporal expression patterns of 21 genes from blastula through pluteus stage in the sea urchin Lytechinus variegatus that are expressed in areas of the nervous system and have been reported to be involved in neurogenesis in other model systems. These patterns of gene expression provide a template for future perturbation studies to determine whether they are involved in, or essential for, proper neural patterning and behavior in the sea urchin and ultimately for understanding how neural GRNs are established in development. However, these patterns of gene expression provide more than a simple list of molecular players for sea urchin neurogenesis. The data presented here reveal the incredibly dynamic nature of gene expression required for neurogenesis to occur. Some of these expression patterns are complex and show the initial expression in a non-neural area of the embryo early, then turn off, and later express in areas of the nervous system. Others come on later in developmental time, with their expression remaining in a single area of the nervous system. Still others are expressed in multiple neurogenic areas previously shown to have unique regulatory states [2, 14].

Taken together, the data presented here reveal that even simple embryos, such as the sea urchin, must possess complex gene regulatory networks to build a functioning nervous system. We believe that the sea urchin embryo—with its known developmental gene regulatory network, molecular tractability, and simple nervous system—can be a developmental model for understanding of how a nervous system is built. Indeed, the sea urchin is unique in that it is one of the few organisms that have the potential for revealing gene regulatory networks, beginning from the initial maternal inputs through zygotic transcription, that lead to a functioning nervous system. To this end, the data shown here are a critical and necessary step to bring the sea urchin to the forefront of neural development research.

Results

Identification of genes expressed in the embryonic sea urchin nervous system

As part of an effort to identify components of the neurogenic GRN in the sea urchin, a molecular cloning and an in situ hybridization screening were performed. To choose candidate genes for this screening, we queried the published Stronglyocentrotus purpuratus developmental transcriptome (www.echinobase.org) and focused on annotated sea urchin orthologs of genes expressed during embryonic development that belong to the functional classification of neurogenesis [16]. For each candidate chosen, coding sequences were isolated from Lytechinus variegatus cDNA and spatiotemporal expression was analyzed by in situ hybridization from blastula stage through pluteus larval stage. These time points were chosen so that the in situ screening covered hatched blastula stage, the onset of earliest neural specification, through pluteus stage, when much of the nervous system has been patterned and larvae have begun to use their simple nervous system. Within this time frame, embryos were fixed every 2 h in development and candidate genes were examined so that dynamically changing expression of these genes and their spatial distribution could be detected.

Transcription factors expressed in the apical organ

Members of the Early Growth Factor Response (Egr) family are zinc finger transcription factors and include the vertebrate gene krox20 which is involved in the development of the hindbrain and neural crest [17, 18]. Lv-egr is detected very faintly at 10–12 h postfertilization (hpf) in the anterior two-thirds of the embryo (brackets in Fig. 1a, b). This expression is then diminished (Fig. 1c–g), and Lv-egr is expressed in cells of the apical organ starting at 24 hpf (Fig. 1h–l). In many pluteus-stage embryos, Lv-egr is strongly expressed in a single cell, while 1–4 other cells faintly express Lv-egr in the apical organ. The beginning of the specification sequence of neural progenitors in the apical organ, which is marked by expression of soxC and delta, has been shown to begin at blastula stages [2, 5]. Since expression of Lv-egr in the apical organ does not begin until 24 hpf (early pluteus stage), which is several hours after the onset of delta and soxc, this suggests that Lv-egr is most likely involved in differentiation or survival of neurons there rather than in specification of progenitors.
Fig. 1
Fig. 1

Transcription factors expressed in the apical organ. al Lv-egr is expressed in the anterior two-thirds of the embryo from 10 to 12 hpf (hours post fertilization) (brackets in ab) and then comes on in cells in the apical organ starting at 24 hpf (h). mx Lv-hey is expressed in scattered cells of the vegetal plate from 10 to 12 hpf (arrowheads in n, o). Inset image of o shows posterior view of vegetal plate. One to two cells express Lv-hey in the elongating archenteron at 16 hpf (arrowhead in p). Then, at 24 hpf, Lv-hey is expressed in 2–4 cells in the apical organ (tx). Lv-hey is also expressed in scattered mesodermal cells at 30 hpf onward (arrowhead in W) and in 1–2 cells in the gut endoderm (arrowhead in x). a′l′ Lv-elk is expressed lightly in skeletogenic mesodermal cells from 10 to 18 hpf (a′e′) (arrows). From 20 to 22 hpf, Lv-elk is expressed in the non-skeletogenic mesoderm (brackets in f′, g′). At 24 hpf, Lv-elk turns off in the non-skeletogenic mesoderm and turns back on in a subset of skeletogenic mesoderm (arrow in H′). At 28 hpf, Lv-elk is expressed in 1–2 cells of the apical organ (arrowheads in j′l′). Scale bars = 50 μm

Hey (hairy/enhancer of split related with YRPW motif) proteins are basic helix–loop–helix transcription factors that are targets of Notch signaling. In vertebrates, hey genes have many different effects on the developing nervous system including, but not limited to: positively regulating maintenance of neural precursors, antagonism of some neural subtypes, and promotion of neural differentiation [1922]. The Drosophila hey otholog is expressed by differentiating neurons and is involved in asymmetric division of ganglion mother cells [20]. Lv-hey is first expressed at mesenchyme blastula stages (12–14 hpf) in a salt and pepper ring of cells in the vegetal plate (Fig. 1m, arrowheads in Fig. 1n–o). This expression in the vegetal plate is most likely mesodermal. As gastrulation begins, Lv-hey is expressed lightly in 2 cells on either side of the archenteron (Fig. 1p arrowhead). Lv-hey then cannot be detected by in situ hybridization until 24 hpf when it turns on in 2–4 cells in the apical organ (Fig. 1t). Lv-hey remains in 2–4 cells in the apical organ and by 30 hpf is also expressed in 1–2 cells at the base of the larval arms, which are likely mesodermal (arrowhead in Fig. 1w). At this time point, some embryos have Lv-hey expression in several cells in the gut, but that is not consistent in all embryos (arrowhead in Fig. 1x). The late onset of expression in the apical organ suggests that, like Lv-egr, Lv-hey is likely not involved specification of neural progenitors. Rather, it is possible that Lv-hey is used in the maintenance of neural precursors or in the differentiation of neurons, like the role in of hey genes in vertebrate models.

Elk, an Ets family transcription factor, has been shown be expressed in the rodent brain [23, 24]. Knockdown studies have shown that Elk-1 effects transcription of several genes in the mouse brain and is considered a pro-differentiation factor there [25]. In the sea urchin S. purpuratus, elk has been shown to suppress apoptosis and promote cell proliferation in the early embryo [26]. Lv-elk is expressed at 10–18 hpf in the skeletogenic mesoderm (Fig. 1a′–e′, arrows). From 20 to 22 hpf, Lv-elk is expressed faintly in the non-skeletogenic mesoderm but not in the skeletogenic mesoderm (Fig. 1f′–g′, brackets). Beginning in some embryos at 22 hpf and in all embryos by 24 hpf, Lv-elk is back on in a subset of skeletogenic mesoderm near the tips of the arms and is not detected in the non-skeletogenic mesoderm (Fig. 1h′, arrow). Later, starting in some embryos at 26 hpf and through 32 hpf, Lv-elk is expressed faintly in 1–2 cells of the apical organ (Fig. 1i′–l′, arrowheads in j′-l′). The late timing of expression of Lv-elk in the apical organ at pluteus stages suggests that it could have a role in the differentiation of neurons there, similar to its role in the rodent brain. At 32 hpf, many embryos do not show expression of Lv-elk in the apical organ.

Transcription factors expressed in or near the ciliary band

AP-2 is a transcription factor that in Drosophila is expressed in the developing central nervous system and optic lobes. AP-2 null embryos have brain defects [2729]. In vertebrates, AP-2 is expressed in neural crest cells and is required for neural crest induction, differentiation, and survival [3032]. In the sea urchin, Lv-ap2 is expressed starting at 24 hpf in the oral ectoderm in an area encased by the ciliary band (Fig. 2a–l, bracket in h), similar in pattern to expression of Lv-netrin (Fig. 4h–l). This region of the oral ectoderm in the sea urchin embryo has neurogenic potential, and SoxC-expressing neural progenitors are found there at the time Lv-ap2 is expressed [5]. Perturbations of Lv-ap2 will be necessary to determine what role Lv-ap2 might be having in this region.
Fig. 2
Fig. 2

Expression of transcription factors in or near the ciliary band. al Lv-ap2 is expressed starting at 24 hpf in the oral ectoderm near the ciliary band (bracket in h) where it remains through 32 hpf. mx Lv-ese is expressed in the non-skeletogenic mesoderm from 10 to 22 hpf. At 22 hpf, Lv-ese is expressed in the lateral ciliary band (shown in bracket in t, inset image shows lateral perspective). It remains in cells of the lateral ciliary band and also turns on in the postoral neurons (shown in inset images in vx). a′l′ Lv-scratch is expressed beginning at 24 hpf in cells scattered in the ciliary band. Cells expressing Lv-scratch increase in number through 32 hpf. m′x′ Lv-prox is expressed in the non-skeletogenic mesoderm until 32 hpf when it is also expressed throughout the ciliary band (arrow heads in x′). Scale bars = 50 μm

Ese is a transcription factor in the Ets family, members of which play roles in neurogenesis in Drosophila [33]. Expression of ese has been previously reported in a related sea urchin species in the non-skeletogenic mesoderm [34], which is also shown by in situ here in Lytechinus (Fig. 2m–s). However, at 22 hpf, Lv-ese is also expressed in a salt and pepper pattern of cells in the lateral ciliary band (Fig. 2s). By 26 hpf, Lv-ese expression remains in the lateral ciliary band and extends to the postoral neurons (Fig. 2u–x, bracket in T, inset image in T shows a lateral view). It is possible that Lv-ese is involved in either specification or differentiation of neural cells in the sea urchin, though it is unclear why Lv-ese is expressed in only the lateral sides of the ciliary band, and not scattered throughout the entire ciliary band.

Scratch, a member of the snail family of transcription factors, is a pan-neuronal marker in Drosophila and is largely confined to the central nervous system of vertebrates [35, 36]. In Drosophila, scratch promotes neuronal fate as scratch null flies show a significant loss of neurons, while ectopic scratch expression leads to additional neurons in the embryo [35]. In mice, scratch is expressed largely by postmitotic neurons [36]. In the sea urchin, Lv-scratch is not detected by in situ hybridization until 24 hpf when it begins in scattered cells throughout the ciliary band in some embryos (Fig. 2a′–h′). As time proceeds, more Lv-scratch expressing cells are found throughout the ciliary band (Fig. 2i′–l′). The salt and pepper pattern of expression in the ciliary band at pluteus stages is similar to that of the proneural transcription factor neurogenin [2]. The restricted and relatively late expression pattern suggests that Lv-scratch has a function more similar to that in vertebrates.

Prospero or Prox is a homeodomain transcription factor that in vertebrates and in Drosophila acts in neural stem cells to regulate the switch from self-renewal to differentiation into postmitotic neurons [37, 38]. In a related sea urchin species, prox has been shown to be expressed in non-skeletogenic mesoderm [39, 40]. Lv-prox is expressed similar to previously reported at 10 hpf in the non-skeletogenic cells that undergo an epithelial-to-mesenchymal transition at the tip of the archenteron at 18–20 hpf (Fig. 2m′–r′). Through 30 hpf, Lv-prox remains in mesodermal cells (Fig. 2m′–w′). However, by 32 hpf, Lv-prox is scattered throughout the ciliary band (Fig. 2x′). The late timing of expression in cells that are likely neural suggests that Lv-prox is not involved in early neural specification. Rather, it could be involved in differentiation or in regulating asymmetric divisions of neuroblasts in the sea urchin.

Transcription factors expressed in the developing foregut

Diencephalon/mesencephalon homeobox 1 (mbx1) is expressed in the mouse fore and midbrain regions and is required for proper eye and tectum development in zebrafish [41, 42]. No Mbx ortholog has been found in Drosophila [42]. Lv-mbx1 is expressed in a stripe of expression in the developing gut starting at 16 hpf (Fig. 3d, arrowheads in d, e). Once the archenteron has reached its highest point at late gastrula stage, Lv-mbx1 expression is in the foregut and the ectoderm near the developing mouth through 32 hpf (Fig. 3e–l). Thus, if Lv-mbx1 is neural in the sea urchin, it is mostly likely only used in the development of the neurons surrounding the larval mouth. However, functional perturbations of Lv-mbx are required to determine whether it is playing a role in neurogenesis or if it has a purely endodermal role.
Fig. 3
Fig. 3

Transcription factors expressed in the foregut. al Expression of Lv-mbx begins at 16 hpf in the elongating archenteron (arrowhead in d, e). Lv-mbx is expressed in the foregut from then through 32 hpf and is also expressed at pluteus stages in the ectoderm near the mouth. mx Lv-islet is expressed in the developing foregut from 16 to 32 hpf. Starting at 18 hpf, Lv-islet expression is not just in the foregut but in the stripe of ectoderm near the foregut (q). a′l′ Lv-dmrt is expressed in the foregut from 22 hpf (g′) through 32 hpf. m′x′ Lv-atbf1 is expressed in the ectoderm from 10 to 22 hpf. At 24 hpf, Lv-atbf1 also turns on in the foregut (arrowhead in t′), where it remains through 32 hpf. Scale bars = 50 μm

The LIM homeodomain transcription factor, islet1, is expressed and required for proper the development of motor neurons of the neural tube, neural crest-derived dorsal root ganglia neurons, and sympathetic neurons [4345]. Islet is also required for the development of C. intestinalis palps, which are ectodermal thickenings that give rise to peripheral neurons [46]. Prior to 16 hpf, Lv-islet expression is not detected by in situ in Lytechinus (Fig. 3m–o). Lv-islet is expressed in some, but not all, embryos beginning at 16 hpf in the developing gut (Fig. 3p). By 18 hpf, Lv-islet is expressed in a transverse stripe in the anterior portion of the embryo that spans the oral ectoderm and foregut (Fig. 3q). This stripe of expression remains through 32 hpf (Fig. 3r–x). In some embryos at 32 hpf, 1–5 cells in the apical organ also expressed Lv-islet. It is possible that Lv-islet is involved in neurogenesis surrounding the larval mouth; however, it is unclear what the function of the ectodermal stripe of expression may be. Perturbation assays will be necessary to determine what role, if any, Lv-islet is playing in neurogenesis.

Doublesex- and mab-3-related transcription factors (Dmrt family) are perhaps best studied in the context of sexual development. In vertebrates, C. elegans and flies, Dmrt family members are involved in sex determination and sexual development [47]. However, members of the Dmrt family are also expressed in the central nervous system and placodes of vertebrate embryos, the neural tube of the tunicate C. intestinalis, and have been shown to be required for proper neural development in the X. laevis olfactory system [48, 49]. Lv-dmrt is detected by in situ hybridization beginning at 22 hpf in the foregut, where it remains through pluteus stage (Fig. 3a′–l′). Some embryos begin to express Lv-dmrt in the foregut beginning at 20 hpf. The timing and placement of Lv-dmrt, Lv-mbx, and Lv-islet expression in the foregut endoderm suggest that perhaps this is an area of active neurogenesis that is required for swallowing behaviors in the embryo.

Atbf1, a zinc finger homeobox transcription factor, has been shown to promote differentiation of neurons by inducing cell cycle arrest [50]. Atbf1 has been published as being expressed in the ectoderm of a related sea urchin species, Paracentrotus lividus [51]. Here we show that Lv-atbf1 shows the same pattern of expression in the ectoderm of Lytechinus, but has an additional territory of expression in the foregut. Lv-atbf1 is expressed in the ectoderm from 10 to 18 hpf with a stronger expression in the aboral (non-neural) ectoderm, as reported by Saudemont et al. (Fig. 3m′–q′). At 22 hpf, Lv-atbf1 is also expressed in the ectoderm surrounding blastopore (Fig. 3s′). By 24 hpf, Lv-atbf1 is expressed in the foregut, in a pattern very similar to Lv-dmrt and Lv-mbx1 (Fig. 3t′ arrowhead). Additional sites of expression of Lv-atbf1 in the L. variegatus nervous system include the lateral sides of the ciliary band, apical organ, and the ectodermal ridge where postoral neurons are located (Fig. 3u′-x′).

Expression of axon guidance molecules

Netrins are secreted laminin-related molecules that have highly conserved roles throughout Metazoa to guide axonal migration [52]. Lv-netrin is expressed in a bilaterally symmetric pattern in the vegetal plate from 12 to 16 hpf (Fig. 4a–d, brackets in b–d). At 18 hpf, vegetal expression of Lv-netrin remains in the vegetal plate and is expressed faintly in the apical organ (Fig. 4e, arrowhead). By 22 hpf, Lv-netrin is expressed in the oral ectoderm in a region not inside the ciliary band itself but bounded by the ciliary band, where it remains through 32 hpf (Fig. 4g–l, arrowhead in G). Axonal tracts have been shown to reside throughout the ciliary band of the sea urchin, but it is unclear what molecules are required for axonal guidance, repulsion, and migration [9]. The expression of Lv-netrin surrounding the ciliary band suggests that it is used in guidance of axons in the sea urchin embryo.
Fig. 4
Fig. 4

Expression of axon guidance molecules. al Lv-netrin is expressed from 12 to 16 hpf in a ring in the vegetal plate (brackets in bd). Lv-netrin turns on briefly in the apical organ (arrowhead in e). By 22 hpf, Lv-netrin is expressed in the edges of the oral ectoderm near the ciliary band. mx Lv-semaa is expressed in a ring in the vegetal plate at 14 hpf (bracket in o). At 16 hpf, it is also expressed in the ventral ectoderm (arrowhead in p) and the apical organ. Through 32 hpf, Lv-semaa is expressed in the apical organ and the hindgut. Scale bars = 50um

Semaphorins are a group of secreted and membrane-bound proteins with roles in axon guidance and repulsion and are highly conserved functionally throughout the animal kingdom [53]. Lv-semaphorina (Lv-semaa) is first expressed at 14 hpf when it is detected in the vegetal plate and in the apical organ in some embryos (Fig. 4m–o, bracket in o). By 16 hpf, Lv-semaa is expressed in the vegetal plate, apical organ domain, and in a band in the ventral ectoderm (Fig. 4p, arrowhead shows ventral ectoderm expression). By 18 hpf, the expression in the ventral band is reduced or off in some embryos and expression remains in the blastopore and apical organ (Fig. 4q, arrowhead shows apical organ expression). Apical and hindgut expression continues through pluteus stage (Fig. 4r–x). It is interesting that Lv-semaa is expressed at blastula stages, since at this point neural progenitors have just begun to be specified and axonal tracts are not detected. The regional specificity of Lv-semaa suggests that different regions of the sea urchin nervous system require different axon guidance molecules. Perhaps a diversity of axon guidance molecules is required for regional specificity and connectivity of the nervous system.

Expression of genes important for neural survival or proliferation

APP or amyloid-β precursor protein is a transmembrane protein shown to be important for the formation and transmission of synapses of neurons in culture and crucial to the pathology of Alzheimer’s disease [54]. Lv-app is expressed in the ectoderm of the anterior two-thirds of the embryo until 16 hpf when its expression is detected lightly in the animal pole domain in some, but not all embryos (Fig. 5a–g, brackets in a–c). At 24 hpf, Lv-app is expressed in the top of the foregut and by 26 hpf it is expressed in the coelomic pouches (Fig. 5h, i, arrowhead in i). At this time point, some embryos begin to express Lv-app in the postoral neurons (arrowhead in inset image of Fig. 5i). At 28 hpf, Lv-app remains faintly in the pouches and is expressed in the postoral neurons (Fig. 5j, arrowheads). From 30 to 32 hpf, Lv-app is expressed in the postoral neurons but is no longer detected in the coelomic pouches (Fig. 5k–l, arrowheads in inset images of k, l show postoral neurons). The late expression of Lv-app in the postoral neurons, several hours after they have been specified, suggests that it is involved in survival, differentiation, or maintenance of these neurons. However, the function of Lv-app in other areas of the embryo, particularly in non-neural tissues such as the mesodermal coelomic pouches, is unclear.
Fig. 5
Fig. 5

Expression of survival and proliferation genes in the nervous system. al Lv-app is expressed in the anterior two-thirds of the embryo from 10 to 14 hpf (brackets in ac). At 24 hpf, Lv-app is expressed in the tip of the archenteron, and by 26 hpf, it is expressed in the coelomic pouches (arrowhead in i) and in the postoral neurons (inset images in il). mx Lv-trk is not expressed until 24 hpf when it turns on in the postoral neurons (arrowhead in t) and remains there through 32 hpf. a′l′ Lv-prohibitin is expressed in the mid- and hindgut (bracket in h′) beginning at 24 hpf through 32 hpf. m′x′ Lv-rasO is expressed in the ectoderm and vegetal plate at 10 hpf. By 14 hpf, Lv-rasO remains in the ectoderm and is in a band of expression surrounding the vegetal plate (arrows in o′, p′). By 20 hpf, Lv-rasO is expressed in one or both lateral sides of ciliary band (arrowhead in r′). By 22 hpf, Lv-rasO expression is on a single side of the lateral ciliary band. Expression then diminishes by 28 hpf. a″l″ Lv-hells is expressed in the ciliary band (arrowhead in h″) as well as the apical organ and hindgut. Scale bars = 50 μm

Neurotrophic receptor tyrosine kinase (TRK) genes are essential for neuron survival in a subset of the vertebrate central and peripheral nervous systems (reviewed in [55]). Lv-trk is first detected in the postoral neurons in some, but not a majority, of embryos by in situ hybridization at 22 hpf (Fig. 5m–t). By 24hpf, Lv-trk is expressed in the postoral neurons where it remains through 32 hpf (Fig. 5t–x, arrowhead in t). This regional expression of Lv-trk in only the postoral neurons suggests that different survival genes are required for different subtypes of neurons in the sea urchin.

Prohibitin is a protein that has been shown to confer neuron survival by reducing the production of free radicals in mitochondria [56]. It has also been shown to play a specification role in Xenopus neural crest [57]. In Lytechinus, expression of Lv-prohibitin is not detected by in situ hybridization from 10 to 16 hpf (Fig. 5a′–d′). Signal begins to accumulate for Lv-prohibitin in the hindgut starting at 18 hpf (Fig. 5e′). Overtime, Lv-prohibitin continues to be expressed in the mid and hindgut (Fig. 5f′–l′, bracket in h′). Neurons of the pyloric and anal sphincters originate in those regions so it is possible that Lv-prohibitin is involved in neurogenesis there, though it is also possible that prohibitin does not perform a neural role in the sea urchin.

Ras signaling has been extensively studied in the context of neurogenesis. Ras signaling has been shown to be used to select cell fates in the mouse brain [58, 59]. Ras proteins are also involved in specification of glial cells in the embryonic brain [60]. Ras family orphan or Lv-rasO is expressed from 10 to 16 hpf in a ring in the vegetal plate surrounding the future site of the blastopore and diffusely in the ectoderm (Fig. 5m′–p′, arrows in o′ and p′ show blastoporal expression). At 18 hpf, it is expressed in and around the developing blastopore as well as diffusely in the ectoderm (Fig. 5q′). At 20 hpf, Lv-rasO is expressed in one or both lateral sides of the ciliary band (Fig. 5r′, arrowhead). By 22 hpf, Lv-rasO expression is in the ciliary band in a very peculiar expression pattern. In the ciliary band, Lv-rasO is asymmetric- only being expressed in one side of the embryo in the lateral ciliary band (Fig. 5s′). This particular asymmetry within the ciliary band is not seen in any other neural genes in this screening nor in any published neural expression patterns in the sea urchin and the purpose of this asymmetric expression is unclear. Starting at 26 hpf through 32hpf, Lv-raso expression is largely diminished (Fig. 5t′–x′).

Lymphoid-specific helicase isoform 1 (Hells) is expressed in lymphoid precursor cells and has no reported role in neurogenesis [61]. However, Lv-hells is expressed in neural territories in the sea urchin. Lv-hells is expressed lightly from 10 to 16 hpf scattered in the ectoderm (Fig. 5a″–d″). By 18 hpf, Lv-hells is expressed in the ciliary band and blastopore and beginning at 24 hpf in the ciliary band, hindgut/blastopore and foregut (Fig. 5e″–l″, arrowhead in H′ shows ciliary band expression). Members of the helicase family regulate processes such as control of transcription and DNA repair and perhaps are providing the same role in neural territories of the sea urchin [61, 62].

Expression of neurotransmitter-related genes

Vacht is a transporter protein responsible for loading acetylcholine into secretory vesicles in neurons [63, 64]. In a pattern very similar to Lv-chat [2], Lv-vacht is expressed in the postoral neurons. It is not expressed from 10 to 18 hpf (Fig. 6a–e) and begins to be expressed in some embryos at 20 hpf where it remains through 32hpf (Fig. 6g–l, arrowhead in g).
Fig. 6
Fig. 6

Expression of neurotransmitter-related genes. al Lv-vacht is expressed beginning in some embryos at 22 hpf in the postoral neurons (arrowhead in g) where it remains through 32 hpf. mx Lv-drd1 is expressed starting at 24 hpf in the ectoderm near the stomodeum and near the postoral neurons (arrowheads in t), where it remains through 32 hpf. a′l′ Lv-asicl4 is expressed beginning at 24 hpf in the developing apical organ (arrowhead in h′) through 32 hpf. Scale bars = 50 μm

Dopamine receptor D1, or Lv-drd1, is first detected in some embryos beginning at 24 hpf in the anterior ectoderm near the mouth and in a patch in the posterior ciliary band (Fig. 6m–t, arrowheads in t). It remains expressed in those areas faintly until 32 hpf (Fig. 6u–x, arrowheads in inset images show posterior ciliary band expression). It makes sense that expression of Lv-drd1 is found near the mouth and postoral neurons since expression of Lv-th, expressed by dopaminergic neurons, has been reported to be expressed in those regions [2].

Members of the acid sensing ion channel (Asic) family are ion channels found throughout the vertebrate nervous system that help convert chemical signals to electrical current [65]. Asic genes are conserved throughout deuterostomes but are not found in protostome animals [65]. In Lytechinus, Lv-asicl4 shows no expression from 10 to 22 hpf (Fig. 6a′–g′). Expression is detected by in situ beginning at 24 hpf in the apical organ where it remains through 32 hpf (Fig. 6h′–l′, arrowhead in h′). Lv-asicl4 is likely involved in the function of neurons in the apical organ, since its expression begins after the serotonergic neurons have differentiated, as marked by expression of serotonin.

Discussion

The sea urchin embryonic nervous system is subdivided into distinct regulatory states

Over the past several years, we have gained a better understanding of when and where neurons are specified and differentiate in the sea urchin embryo. That provides the opportunity to look at genes associated with neurogenesis elsewhere in the animal kingdom and ask which of these are associated with neural development in the sea urchin. When and where do these genes appear in the sea urchin? Which are exclusively expressed in neural territories and which are expressed in other locations in the embryo? If these genes are neural in the sea urchin, to which regulatory state do they likely belong?

To this end, the in situ hybridization data shown here allow us to begin addressing those questions. The ciliary band, the apical organ, the foregut endoderm, the hindgut endoderm, and the oral ectoderm each harbor neurons in the larva [2, 5, 10, 12, 66, 67]. Most genes in this survey are expressed in one or more of these territories in a manner that suggests a neural association. However, expression within an area of the nervous system alone does not mean that a gene has a neural function. It is entirely possible that some of these genes do not play a role in neurogenesis. However, if a gene has been shown to be neurogenic in multiple organisms of different lineages (including protostome and vertebrate models) and is expressed in developing neural territories in the sea urchin, then it is likely to have a neurogenic role there. This is particularly true of the genes that are expressed in a salt and pepper pattern within the nervous system (such as Lv-scratch, Lv-ese and Lv-prox, Fig. 2), which is a classic characteristic of neural patterning. We believe that all of the gene expression patterns shown here play some role in neurogenesis, be it specification of neurons, axon guidance, or neural survival. Careful co-expression analysis followed by perturbations will be required to determine what role, if any, each of these genes has during neurogenesis in the sea urchin.

Nevertheless, the data presented here suggest that these territories each have their own unique gene expression profile, and therefore, each represents a distinct regulatory state (Fig. 7, Table 1). Furthermore, gene expression data here suggest that the ciliary band itself can be subdivided further into other regulatory states as suggested by Barsi and colleagues [68]. For example, the transcription factor Lv-ese is expressed in cells only in the lateral sides and posterior ciliary while other transcription factors such as Lv-scratch and Lv-prox are expressed in cells scattered throughout the ciliary band. This suggests neural cells in different regions of the ciliary band express different combinations of transcription factors and perhaps carry out different functions and have different trajectories. At the same time, a number of the genes presented in this study are expressed in patterns that suggest additional territories of expression outside the nervous system.
Fig. 7
Fig. 7

Schematic of genes expressed in different regions of the ectoderm and endoderm at late gastrula and pluteus larva stages. a, b Schematics show 3 ectodermal territories (apical organ, ciliary band, and postoral region) and the list of genes from this study expressed in those regions at two time points (22 hpf and 32 hpf). Expression within a region does not mean that the genes are necessarily co-expressed with one another. c, d Schematics show endodermal territories where genes from this study are expressed at late gastrula and pluteus stages. Expression within a region does not mean that the genes are necessarily co-expressed. A anterior, P posterior

Table 1

Areas and timing of expression

Rows indicate genes, and columns indicate areas of the embryo. White/blank indicates no expression detected in that area. Colors indicate timing of when the gene is expressed in that area. Yellow: early expression (at some point between 10 and 16 hpf). Orange: mid-timing of expression (at some point between 18 and 24 hpf). Red: late expression (at some point between 26 and 32 hpf)

TF transcription factor, Postoral postoral neurons or ectoderm surrounding the postoral neurons, Oral Ecto oral ectoderm, Aboral Ecto aboral ectoderm, Meso. mesodermal cells/tissues, Coel. Pouches coelomic pouches

The data provided here are not intended to be a complete list of molecular players expressed in the embryonic nervous system. It is highly likely that there are many other genes expressed in the sea urchin nervous system but were not identified in this screening because they turn on earlier than 10 hpf or later than 32 hpf. Furthermore, there are likely other genes that are neurogenic in the sea urchin but were not found in this screening because they do not fall under gene ontology (GO) term categories related to nervous system development. While gathering information about regulatory states in an embryo is necessary for building a developmental gene regulatory network, it cannot tell us about interactions between genes and which molecular players are essential for development. What it can do is provide a guide for future experiments designed to determine how neurogenesis occurs in each of the several regulatory regions. It is clear that each region of the embryonic nervous system is distinct in the neural genes expressed so each must be considered separately in terms of specification trajectory, timing, and differentiation.

While understanding a neurogenic GRN for a basal deuterostome will be informative from an evolutionary perspective, it can also allow for comparisons between the embryonic and adult (postmetamorphic) nervous systems. It is believed that the larval nervous system is completely lost during metamorphosis and that the adult nervous system is formed de novo [69]. This leaves the question of whether the same genes are required for the formation of the adult nervous system and whether the same or a modified version of the embryonic GRN is deployed after metamorphosis for the formation of the adult nervous system.

Expression of genes in multiple regions of the embryo

Several genes in this study are expressed in multiple territories in the embryo (Table 1). These include the transcription factors Lv-ese, Lv-prox, Lv-islet, Lv-atbf1, Lv-hey, and Lv-elk as well as the axon guidance gene Lv-semaa. Some of these are expressed in multiple neural territories while others are expressed in neural domains and in mesodermal cells (Table 1). This underscores a limitation of using purely quantitative methods including quantitative PCR and RNAseq alone to build gene regulatory networks. These methods, while extremely sensitive and quantitative, do not give a spatial picture of where in the embryo genes are affected by perturbations. For this, one needs in whole mount expression assays.

Spatiotemporal expression is one of the most valuable components necessary for building GRNs. The spatiotemporal pattern of expression of the genes in this analysis indicates that there are significant differences of expression in the three primary neurogenic territories. This provides a framework for perturbation studies to determine what genes are more upstream in these neural territories and which genes are involved in downstream processes of neural development. The obvious logic of GRNs is that a gene expressed early is far more likely to be nearer to the top of a GRN than a gene that is first expressed later in development. Similarly, genes that are expressed with a close temporal sequence to another gene’s expression may provide hints of direct relationships. Bolouri and Davidson [70] showed for S. purpuratus that it took on average 3 h for the RNA of one gene to be expressed, processed, translated into a functional protein before it could activate the next downstream gene. In Lytechinus, that “step time” is likely shorter since Lytechinus develops in sea water that is about 8–10 °C warmer than for S. purpuratus.

It is possible that genes expressed both in mesodermal tissues and in neurogenic territories are performing similar roles in both regions. Notably, both secondary mesoderm and neurogenesis have been shown to be dependent on Delta/Notch signaling in the sea urchin [2, 10, 15, 7174]. It could be that the genes expressed in both mesoderm and neural territories are dependent on or are regulators of Notch signaling in both tissue types.

Dynamic patterns of expression are also revealed by this analysis. For some genes, expression appears to be broad and later narrows to just a few cells. Expression of other genes begins in one cell at a time in a salt and pepper pattern. Expression of some genes occurs only during a limited timeframe meaning that as a GRN state switches, it must be turned on and then later turned off. The timing of that temporary expression could occur at a specific time for an individual cell or roughly simultaneously for all cells within a territory. Each of these patterns offers early hints toward the role of that protein in establishing a nervous system in the larva.

A major difficulty with this kind of analysis is the inability to track a single cell over time. Since the analysis reports expression of a field of cells in each territory, the analysis quickly becomes complicated if, as is likely, neurogenesis starts with a few cells and then additional cells initiate neurogenesis. Given that likelihood, it is very difficult to establish, a priori, the sequence of gene expression. For that reason, perturbations are absolutely necessary to gain a reasonable understanding of expression sequences and connections in forming a GRN. This analysis thus is a series of snapshots, and one interpretation, lacking other information, is that if a gene is expressed at one stage and then expressed in successive stages, the gene is continuously expressed. However, one should be aware that other possibilities exist, e.g., the gene is transiently activated in neurons which are born at different times during development. Because this analysis did not follow single cells through time, either outcome is possible. Injection of a recombinant BAC for each gene would allow a time lapse expression analysis (via live imaging) and would help resolve the present ambiguity. To produce recombinant BACs to a large number of genes is a cumbersome task but may be important for resolving some of the issues of GRN assembly in future experiments. With all of these caveats, however, it is still extremely useful to obtain a dataset such as that contained in this analysis before proceeding to assemble a GRN reflecting the generation of a nervous system.

Conclusions

The results of this in situ hybridization screening provide a map of the regulatory states present in the sea urchin embryo during neurogenesis. The findings presented in this paper will allow for a dissection of the neurogenic gene regulatory network in a basal deuterostome. At this point, we can only speculate about the functions of these genes during development based on findings in other systems. With these data in hand, the next logical step would be to begin using functional perturbation assays to determine the upstream and downstream relationships of these genes and the consequences these genes have on development of the nervous system in the sea urchin. Comparing the results of these functional studies to what is known in other species will provide a looking glass into how evolution shaped neurogenesis in deuterostomes.

Methods

Adult animals and embryo culture

Adult L. variegatus sea urchins were collected from the Duke University Marine Lab (Beaufort, NC, USA) or Pelagic Corp. (Sugarloaf Key, FL, USA). Gametes were harvested by injection of 0.5 M KCl into adult sea urchins. Embryos were cultured at 23 °C in filtered artificial seawater (ASW).

Cloning and in situ hybridization

Cloning of full length or partial coding sequences for all genes in this study was carried out by designing primers against a transcriptome data set and cloned into pGEM T-easy vector (Promega). PCR was carried out with High Fidelity Phusion Master Mix (NEB). Accession numbers are shown in Table 2. Additional candidates that, in our hands, did not produce a discernable expression pattern between 10 and 32 hpf by in situ hybridization are listed in Table 3. Whole mount in situ hybridization (ISH) was performed using RNA probes labeled with Digoxigenin-11-UTP (Roche). Large cultures of embryos were fixed in a time course for analysis. For each gene, two different sets of embryos were analyzed for each time point. Embryos were fixed by gentle rocking overnight at 4 °C in 4% paraformaldehyde made in filtered artificial sea water (FASW), washed with FASW at room temperature, and stored in methanol at − 20 °C. RNA probes were synthesized in vitro and hybridized at 65 °C. Probes were visualized using AP-conjugated anti-DIG antibody (1:1500, Roche [Indianapolis, IN, United States]). Color was developed using NBT/BCIP (Roche). For each antisense probe, a sense probe was also tested and shown to show no expression pattern or background signal only (Additional file 1: Figs. S1–S6).
Table 2

Plasmids deposited in GenBank

Plasmid

Fragment

Accession

Ap2

Partial CDS

MK015667

App

Partial CDS

MH996686

AsicL4

Partial CDS

MH996683

Atbf1

Partial CDS

MK012222

Dmrt

Partial CDS

MK012223

Drd1

Partial CDS

MH996687

Egr

Partial CDS

MK015664

Elk

Partial CDS

MK029662

Hells

Partial CDS

MK015665

Hey

Full CDS

MK012227

Islet

Partial CDS

MK012226

Mbx

Partial CDS

MK012221

Netrin

Partial CDS

MK015666

Prohibitin

Full CDS

MK015662

RasO

Full CDS

MK015663

Scratch

Partial CDS

MK012224

SemaA

Partial CDS

MK012225

TRK

Partial CDS

MK029664

Vacht

Partial CDS

MK029665

“Partial CDS” means for that gene’s in situ riboprobe, a partial region of the coding sequence was used. “Full CDS” means that the in situ riboprobe was synthesized from the entire coding sequence

Table 3

List of candidate genes screened

Gene name

TF?

arnt

 

ato

TF

bace1

 

clock

TF

drd2

 

gad

 

gbx

TF

glia

TF

gnmt2

 

grb2

 

id

TF

homer

 

ldla/gpcr1

 

machrm5

 

mec2

 

nel1l

 

neurotrophin

 

p75ntr

 

pou4/brn3

TF

rhop2

 

semaB

 

sim

TF

These genes produced no discernable expression pattern between 10 and 32 hpf in our hands

TF indicates genes that act as transcription factors

Declarations

Authors’ contributions

LAS and EMM conducted and designed experiments. LAS wrote manuscript and prepared figures. DRM supervised the study and revised/reviewed manuscript and figures. All authors read and approved the final manuscript.

Acknowledgements

We would like to thank members of the McClay laboratory for their insightful comments and support. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (NSF DGF 1106401). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Competing interests

The authors declare no competing interest or financial interests.

Availability of data and materials

All data underlying the current analyses are publicly available or are included in the Additional files.

Ethics approval and consent to participate

Not applicable.

Funding

This work was supported by: National Science Foundation GRFP Grant DGF 1106401 to Leslie A. Slota, National Institute of Child Health and Human Development (NICHD) NIH RO1-HD-14483 to David R McClay, National Institute of Child Health and Human Development (NICHD) NIH PO1-HD-037105 to David R McClay. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biology, Duke University, 124 Science Dr., Box 90338, Durham, NC 27708, USA

References

  1. Hartenstein V, Stollewerk A. The evolution of early neurogenesis. Dev Cell. 2015;32:390–407. https://doi.org/10.1016/j.devcel.2015.02.004.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Slota LA, McClay DR. Identification of neural transcription factors required for the differentiation of three neuronal subtypes in the sea urchin embryo. Dev Biol. 2018;435:138–49. https://doi.org/10.1016/j.ydbio.2017.12.015.View ArticlePubMedGoogle Scholar
  3. Bisgrove BW, Burke RD. Development of serotonergic neurons in embryos of the sea urchin. Strongylocentrotus purpuratus. Dev Growth Differ. 1986;28:569–74. https://doi.org/10.1111/j.1440-169X.1986.00569.x.View ArticleGoogle Scholar
  4. Yaguchi S, Katow H. Expression of tryptophan 5-hydroxylase gene during sea urchin neurogenesis and role of serotonergic nervous system in larval behavior. J Comp Neurol. 2003;466:219–29.View ArticleGoogle Scholar
  5. Garner S, Zysk I, Byrne G, Kramer M, Moller D, Taylor V, et al. Neurogenesis in sea urchin embryos and the diversity of deuterostome neurogenic mechanisms. Development. 2016;143:286–97. https://doi.org/10.1242/dev.124503.View ArticlePubMedGoogle Scholar
  6. Strathmann RR. Time and extent of ciliary response to particles in a non-filtering feeding mechanism. Biol Bull. 2007;212:93–103.View ArticleGoogle Scholar
  7. Satterlie RA, Andrew CR. Electrical activity at metamorphosis in larvae of the sea urchin Lytechinus pictus (Echinoidea: Echinodermata). J Exp Zool. 1985;235:197–204.View ArticleGoogle Scholar
  8. Mackie G, Spencer A, Strathmann R. Electrical activity associated with ciliary reversal in an echinoderm larva. Nature. 1969;223:1384–5.View ArticleGoogle Scholar
  9. Burke RD, Moller DJ, Krupke OA, Taylor VJ. Sea urchin neural development and the metazoan paradigm of neurogenesis. Genesis. 2014;52:208–21.View ArticleGoogle Scholar
  10. Wei Z, Angerer RC, Angerer LM. Direct development of neurons within foregut endoderm of sea urchin embryos. Proc Natl Acad Sci. 2011;108:9143–7. https://doi.org/10.1073/pnas.1018513108.View ArticlePubMedGoogle Scholar
  11. Consortium S.U.G.S. The genome of the sea urchin. Science (80-). 2007;314:941–52.View ArticleGoogle Scholar
  12. Burke RD, Angerer LM, Elphick MR, Humphrey GW, Yaguchi S, Kiyama T, et al. A genomic view of the sea urchin nervous system. Dev Biol. 2006;300:434–60.View ArticleGoogle Scholar
  13. Wei Z, Angerer LM, Angerer RC. Neurogenic gene regulatory pathways in the sea urchin embryo. Development. 2016;143:298–305. https://doi.org/10.1242/dev.125989.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Mcclay DR, Miranda E, Feinberg SL. Neurogenesis in the sea urchin embryo is initiated uniquely in three domains. Development. 2018;145:dev167742.View ArticleGoogle Scholar
  15. Yaguchi J, Angerer LM, Inaba K, Yaguchi S. Zinc finger homeobox is required for the differentiation of serotonergic neurons in the sea urchin embryo. Dev Biol. 2012;363:74–83. https://doi.org/10.1016/j.ydbio.2011.12.024.View ArticlePubMedGoogle Scholar
  16. Cary GA, Cameron RA, Hinman VF. EchinoBase: tools for echinoderm genome analyses. In: Kollmar M, editor. Eukaryot genomic databases methods and protocols. New York: Springer; 2018. p. 349–69. https://doi.org/10.1007/978-1-4939-7737-6_12.View ArticleGoogle Scholar
  17. Swiatek PJ, Gridley T. Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes Dev. 1993;7:2071–84.View ArticleGoogle Scholar
  18. Bradley LC, Snape A, Bhatt S, Wilkinson DG. The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest. Mech Dev. 1992;40:73–84.View ArticleGoogle Scholar
  19. Weber D, Wiese C, Gessler M. Hey bHLH transcription factors. Curr Top Dev Biol. 2014;1:1. https://doi.org/10.1016/B978-0-12-405943-6.00008-7.View ArticleGoogle Scholar
  20. Monastirioti M, Giagtzoglou N, Koumbanakis KA, Zacharioudaki E, Deligiannaki M, Wech I, et al. Drosophila Hey is a target of Notch in asymmetric divisions during embryonic and larval neurogenesis. Development. 2010;137:191–201. https://doi.org/10.1242/dev.043604.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Mukhopadhyay A, Jarrett J, Chlon T, Kessler JA. HeyL regulates the number of TrkC neurons in dorsal root ganglia. Dev Biol. 2009;334:142–51. https://doi.org/10.1016/j.ydbio.2009.07.018.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Jalali A, Bassuk AG, Kan L, Israsena N, Mukhopadhyay A, Mcguire T, et al. HeyL promotes neuronal differentiation of neural progenitor cells. J Neurosci Res. 2011;89:299–309.View ArticleGoogle Scholar
  23. Price MA, Rogers AE, Treisman R. Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET). EMBO J. 1995;14:2589–601.View ArticleGoogle Scholar
  24. Sgambato V, Pages C, Rogard M, Besson M, Caboche J. Extracellular signal-regulated kinase (ERK) controls immediate early gene induction on corticostriatal stimulation. J Neurosci. 1998;18:8814–25.View ArticleGoogle Scholar
  25. Besnard A, Galan-Rodriguez B, Vanhoutte P, Caboche J. Elk-1 a transcription factor with multiple facets in the brain. Front Neurosci. 2011;5:1–11.View ArticleGoogle Scholar
  26. Rizzo F, Coffman JA, Arnone MI. An Elk transcription factor is required for Runx-dependent survival signaling in the sea urchin embryo. Dev Biol. 2016;416:173–86. https://doi.org/10.1016/j.ydbio.2016.05.026.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Bauer R, McGuffin ME, Mattox W, Tainsky MA. Cloning and characterization of the Drosophila homologue of the AP-2 transcription factor. Oncogene. 1998;17:1911–22.View ArticleGoogle Scholar
  28. Monge I, Mitchell PJ. DAP-2, the Drosophila homolog of transcription factor AP-2. Mech Dev. 1998;76:191–5.View ArticleGoogle Scholar
  29. Monge I, Krishnamurthy R, Sims D, Hirth F, Spengler M, Kammermeier L, et al. Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development. Development. 2001;1252:1239–52.Google Scholar
  30. Hoffman T, Javier A, Campeau S, Knight R, Schilling T. Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution. J Exp Zool. 2007;308B:679–91.View ArticleGoogle Scholar
  31. Knight RD, Nair S, Nelson S, Afshar A, Javidan Y, Geisler R, et al. Lockjaw encodes a zebrafish Tfap2a required for early neural crest development. Development. 2003;130:5755–68. https://doi.org/10.1242/dev.00575.View ArticlePubMedGoogle Scholar
  32. Luo T, Lee Y-H, Saint-Jeannet J-P, Sargent TD. Induction of neural crest in Xenopus by transcription factor AP2. Proc Natl Acad Sci. 2003;100:532–7. https://doi.org/10.1073/pnas.0237226100.View ArticlePubMedGoogle Scholar
  33. Hsu T, Schulz RA. Sequence and functional properties of Ets genes in the model organism Drosophila. Oncogene. 2000;19:6409–16.View ArticleGoogle Scholar
  34. Rizzo F, Fernandez-Serra M, Squarzoni P, Archimandritis A, Arnone MI. Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus). Dev Biol. 2006;300:35–48.View ArticleGoogle Scholar
  35. Roark M, Sturtevant MA, Emery J, Vaessin H, Grell E, Bier E. Scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development. Genes Dev. 1995;9:2384–98.View ArticleGoogle Scholar
  36. Nakakura EK, Watkins DN, Schuebel KE, Sriuranpong V, Borges MW, Nelkin BD, et al. Mammalian scratch: a neural-specific Snail family transcriptional repressor. Proc Natl Acad Sci. 2001;98:4010–5. https://doi.org/10.1073/pnas.051014098.View ArticlePubMedGoogle Scholar
  37. Karalay O, Doberauer K, Vadodaria KC, Knobloch M, Berti L, Miquelajauregui A, et al. Prospero-related homeobox 1 gene (Prox1) is regulated by canonical Wnt signaling and has a stage-specific role in adult hippocampal neurogenesis. Proc Natl Acad Sci. 2011;108:5807–12. https://doi.org/10.1073/pnas.1013456108.View ArticlePubMedGoogle Scholar
  38. Choksi SP, Southall TD, Bossing T, Edoff K, de Wit E, Fischer BEE, et al. Prospero acts as a binary switch between self-renewal and differentiation in drosophila neural stem cells. Dev Cell. 2006;11:775–89.View ArticleGoogle Scholar
  39. Poustka AJ, Kühn A, Groth D, Weise V, Yaguchi S, Burke RD, et al. A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks. Genome Biol. 2007;8:R85.View ArticleGoogle Scholar
  40. Materna SC, Ransick A, Li E, Davidson EH. Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos. Dev Biol. 2013;375:92–104. https://doi.org/10.1016/j.ydbio.2012.11.033.View ArticlePubMedGoogle Scholar
  41. Ohtoshi A, Nishijima I, Justice MJ, Behringer RR. Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos. Mech Dev. 2001;110:241–4.View ArticleGoogle Scholar
  42. Kawahara A, Bin CC, Dawid IB. The homeobox gene mbx is involved in eye and tectum development. Dev Biol. 2002;248:107–17.View ArticleGoogle Scholar
  43. Avivi C, Goldstein RS. Differential expression of Islet-1 in neural crest-derived ganglia: Islet-1 + dorsal root ganglion cells are post-mitotic and Islet-1 + sympathetic ganglion cells are still cycling. Dev Brain Res. 1999;115:89–92.View ArticleGoogle Scholar
  44. Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell. 1996;84:309–20.View ArticleGoogle Scholar
  45. Sun Y, Dykes IM, Liang X, Eng SR, Evans SM, Turner EE. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nat Neurosci. 2008;11:1283–93.View ArticleGoogle Scholar
  46. Wagner E, Stolfi A, Gi Choi Y, Levine M. Islet is a key determinant of ascidian palp morphogenesis. Development. 2014;141:3084–92. https://doi.org/10.1242/dev.110684.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Hong CS, Park BY, Saint-Jeannet JP. The function of Dmrt genes in vertebrate development: it is not just about sex. Dev Biol. 2007;310:1–9.View ArticleGoogle Scholar
  48. Huang X, Hong C-S, O’Donnell M, Saint-Jeannet J-P. The doublesex-related gene, XDmrt4, is required for neurogenesis in the olfactory system. Proc Natl Acad Sci U S A [Internet]. 2005;102:11349–54. http://www.ncbi.nlm.nih.gov/pubmed/16061812.
  49. Wagner E, Levine M. FGF signaling establishes the anterior border of the Ciona neural tube. Development. 2012;139:2351–9.View ArticleGoogle Scholar
  50. Jung C-G, Kim H-K, Kawaguchi M, Khanna KK, Hida H, Asai K, et al. Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation. Development. 2005;132:5137–45. https://doi.org/10.1242/dev.02098.View ArticlePubMedGoogle Scholar
  51. Saudemont A, Haillot E, Mekpoh F, Bessodes N, Quirin M, Lapraz F, et al. Ancestral regulatory circuits governing ectoderm patterning downstream of nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. PLoS Genet. 2010;6:1–31.View ArticleGoogle Scholar
  52. Sun KLW, Correia JP, Kennedy TE. Netrins: versatile extracellular cues with diverse functions. Development. 2011;138:2153–69. https://doi.org/10.1242/dev.044529.View ArticleGoogle Scholar
  53. Yazdani U, Terman JR. The semaphorins. Genome Biol. 2006;7(3):211.View ArticleGoogle Scholar
  54. Priller C, Bauer T, Mitteregger G, Krebs B, Kretzschmar HA, Herms J. Synapse formation and function is modulated by the amyloid precursor protein. J Neurosci. 2006;26:7212–21. https://doi.org/10.1523/JNEUROSCI.1450-06.2006.View ArticlePubMedGoogle Scholar
  55. Lewin G, Carter B. Neurotrophic Factors [Internet]. 2014. http://link.springer.com/10.1007/978-3-642-59920-0_1.
  56. Zhou P, Qian L, D’Aurelio M, Cho S, Wang G, Manfredi G, et al. Prohibitin reduces mitochondrial free radical production and protects brain cells from different injury modalities. J Neurosci. 2012;32:583–93.View ArticleGoogle Scholar
  57. Schneider M, Schambony A, Wedlich D. Prohibitin1 acts as a neural crest specifier in Xenopus development by repressing the transcription factor E2F1. Development. 2010;137:4073–81. https://doi.org/10.1242/dev.053405.View ArticlePubMedGoogle Scholar
  58. Li S, Mattar P, Dixit R, Lawn SO, Wilkinson G, Kinch C, et al. RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis. J Neurosci. 2014;34:2169–90. https://doi.org/10.1523/JNEUROSCI.4077-13.2014.View ArticlePubMedGoogle Scholar
  59. Dasgupta B, Gutman DH. Neurofibromin regulates neural stem cell proliferation, survival, and astroglial differentiation in vitro and in vivo. J Neurosci. 2005;25:5584–94. https://doi.org/10.1523/JNEUROSCI.4693-04.2005.View ArticlePubMedGoogle Scholar
  60. Li X, Newbern JM, Wu Y, Morgan-Smith M, Zhong J, Charron J, et al. MEK is a key regulator of gliogenesis in the developing brain. Neuron. 2012;75:1035–50. https://doi.org/10.1016/j.neuron.2012.08.031.View ArticlePubMedPubMed CentralGoogle Scholar
  61. Geiman TM, Durum SK, Muegge K. Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh). Genomics. 1998;54:477–83.View ArticleGoogle Scholar
  62. Von Eyss B, Maaskola J, Memczak S, Möllmann K, Schuetz A, Loddenkemper C, et al. The SNF2-like helicase HELLS mediates E2F3-dependent transcription and cellular transformation. EMBO J. 2012;31:972–85.View ArticleGoogle Scholar
  63. Weihe E, Tao-Cheng J-H, Schafer MK-H, Erickson JD, Eident LE. Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles. Proc Natl Acad Sci. 1996;93:3547–52.View ArticleGoogle Scholar
  64. Erickson JD, Varoqui H. Molecular analysis of vesicular amine transporter function and targeting to secretory organelles. FASEB J Off Publ Fed Am Soc Exp Biol. 2000;14:2450–8.Google Scholar
  65. Lynagh T, Mikhaleva Y, Colding JM, Glover JC, Pless SA. Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes. Proc Natl Acad Sci. 2018. https://doi.org/10.1073/pnas.1806614115.View ArticlePubMedGoogle Scholar
  66. Angerer LM, Yaguchi S, Angerer RC, Burke RD. The evolution of nervous system patterning: insights from sea urchin development. Development. 2011;138:3613–23. https://doi.org/10.1242/dev.058172.View ArticlePubMedPubMed CentralGoogle Scholar
  67. Hinman VF, Burke RD. Embryonic neurogenesis in echinoderms. Wiley Interdiscip Rev Dev Biol. 2018;7:1–15.View ArticleGoogle Scholar
  68. Barsi JC, Li E, Davidson EH. Geometric control of ciliated band regulatory states in the sea urchin embryo. Development. 2015;142:953–61. https://doi.org/10.1242/dev.117986.View ArticlePubMedPubMed CentralGoogle Scholar
  69. Chia FS, Burke RD. Echinoderm metamorphosis: fate of larval structures. New York: Elsevier-North Holland; 1978.Google Scholar
  70. Bolouri H, Davidson EH. Transcriptional regulatory cascades in development: Initial rates, not steady state, determine network kinetics. Proc Natl Acad Sci. 2003;100:9371–6. https://doi.org/10.1073/pnas.1533293100.View ArticlePubMedGoogle Scholar
  71. Sherwood DR, McClay DR. Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation. Development. 1997;124:3363–74.PubMedGoogle Scholar
  72. Sweet HC, Gehring M, Ettensohn CA. LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties. Development. 1955;2002:1945–55.Google Scholar
  73. Yankura KA, Koechlein CS, Cryan AF, Cheatle A, Hinman VF. Gene regulatory network for neurogenesis in a sea star embryo connects broad neural specification and localized patterning. Proc Natl Acad Sci U S A. 2013;110:8591–6.View ArticleGoogle Scholar
  74. Mellott DO, Thisdelle J, Burke RD. Notch signaling patterns neurogenic ectoderm and regulates the asymmetric division of neural progenitors in sea urchin embryos. Development. 2017. https://doi.org/10.1242/dev.151720.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2019

Advertisement