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la slota net Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus PMID: Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus Leslie A.
Slota Department of Biology, Duke University, 124 Science Dr.
Miranda Department of Biology, Duke University, 124 Science Dr.
McClay Department of Biology, Duke University, 124 Science Dr.
The Creative Commons Public Domain Dedication waiver applies to the data made available in this article, unless otherwise stated.
Figure S1: Sense probes of transcription factors expressed in the apical organ.
In situ hybridizations show expression patterns for the sense and antisense probes for egr, hey, and elk.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
Figure S2: Sense probes of transcription factors expressed in or near the ciliary band.
In situ hybridizations show expression patterns for the sense and antisense probes for ap2, ese, scratch, and prox.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
Figure S3: Sense probes of transcription factors expressed in the foregut.
In situ hybridizations show expression patterns for the sense and antisense probes for mbx, islet, dmrt, and atbf1.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
Figure S4: Sense probes of axon guidance molecules.
In situ hybridizations show expression patterns for the sense and antisense probes for netrin and semaa.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
Figure S5: Sense probes of genes involved in neural survival or proliferation in other species.
In situ hybridizations show expression patterns for the sense and antisense probes for app, trk, prohibitin, raso, and hells.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
Figure S6: Sense probes of neurotransmitter-related genes.
In situ hybridizations show expression patterns for the sense and antisense probes for vacht, drd1, and asicl4.
For each gene, in situ hybridization with sense probes was done side by side with antisense probes on embryos from the same time point and was left in color solution for the same amount of time.
GUID: CEA98296-8A06-4052-972B-5224739BC1FB All data underlying the current analyses are publicly available or are included in the Additional files.
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 click the following article 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 blackjack casino rules tier, 2 identify how subtypes of neurons are specified, 3 perform comparative studies with the sea urchin, protostome, and vertebrate organisms.
Electronic supplementary material The online version of this article 10.
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.
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 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.
Over the last 20 years, much effort has been put into creating a developmental gene regulatory network GRN for sea urchin development.
However, only a limited number of these neural molecules have been spatiotemporally and functionally characterized.
Expanding the sea urchin neurogenic GRN can be beneficial for many reasons.
The first is the evolutionary insights that can be gained.
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.
Furthermore, some transcription factors are check this out 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.
Perturbations of these transcription factors led to the construction of preliminary gene regulatory networks for neurogenesis.
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 la slota net 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.
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 see more 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.
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.
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.
Lv- egr is detected very faintly at 10—12 h postfertilization hpf in the anterior two-thirds of the embryo brackets in Fig.
This expression is then diminished Fig.
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.
Since expression of Lv- egr in the apical organ does not begin until 24 hpf early pluteus stagewhich 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.
Transcription factors expressed in the apical organ.
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, click here 24 hpf, Lv- hey is expressed in 2—4 cells in the apical organ t— x.
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.
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.
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.
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.
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.
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.
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 Please click for source 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.
In the sea urchin S.
Lv- elk is expressed at 10—18 hpf in the skeletogenic mesoderm Fig.
From 20 to 22 hpf, Lv- elk is expressed faintly in the non-skeletogenic mesoderm but not in the skeletogenic mesoderm Fig.
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.
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.
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.
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.
Perturbations of Lv- ap2 will be necessary to determine what role Lv- ap2 might be having in this region.
Expression of transcription factors in or near the ciliary band.
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 here neurons shown in inset images in v— x.
Cells expressing Lv- scratch increase in number through 32 hpf.
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.
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.
However, at 22 hpf, Lv- ese is also expressed in a salt and pepper pattern of cells in the lateral ciliary band Fig.
By 26 hpf, Lv- ese expression remains in the lateral ciliary band and extends to the postoral neurons Fig.
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.
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.
As time proceeds, more Lv- scratch expressing cells are found throughout the ciliary band Fig.
The restricted and relatively late expression pattern suggests that Lv- scratch has a function more similar to that in vertebrates.
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.
Through 30 hpf, Lv- prox remains in mesodermal cells Fig.
However, by 32 hpf, Lv- prox is scattered throughout the ciliary band Fig.
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.
Lv- mbx1 is expressed in a stripe of expression in the developing gut starting at 16 hpf Fig.
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.
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.
Transcription factors expressed in the foregut.
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.
Starting at 18 hpf, Lv- islet expression is not just in the foregut but in the stripe of ectoderm near the foregut q.
Islet is also required for the development of C.
Prior to 16 hpf, Lv- islet expression is not detected by in situ in Lytechinus Fig.
Lv- islet is expressed in some, but not all, embryos beginning at 16 hpf in the developing gut Fig.
By 18 hpf, Lv- islet is expressed in a transverse stripe in the anterior portion of the embryo that spans the la slota net ectoderm and foregut Fig.
This stripe of expression remains through 32 hpf Fig.
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.
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.
Lv- dmrt is detected by in situ hybridization beginning at 22 hpf in the foregut, where it remains through pluteus stage Fig.
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.
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.
At 22 hpf, Lv- atbf1 is also expressed in the ectoderm surrounding blastopore Fig.
By 24 hpf, Lv- atbf1 is expressed in the foregut, in a pattern very similar to Lv- dmrt and Lv- mbx1 Fig.
Additional sites of expression of Lv- atbf1 in the L.
Lv- netrin is expressed in a bilaterally symmetric pattern in blackjack with sidebets app vegetal plate from 12 to 16 hpf Fig.
At 18 hpf, vegetal expression of Lv- netrin remains in the vegetal plate and is expressed faintly in the apical organ Fig.
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.
The expression of Lv- netrin surrounding the ciliary band suggests that it is used in guidance of axons in the sea urchin embryo.
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.
By 16 hpf, Lv- semaa is expressed in the vegetal plate, apical organ domain, and in a band in the ventral ectoderm Fig.
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.
Apical and hindgut expression continues through pluteus stage Fig.
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.
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.
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.
At this time point, some embryos begin to express Lv- app in the postoral neurons arrowhead in inset image of Fig.
At 28 hpf, Lv- app remains faintly in the pouches and is expressed in the postoral neurons Fig.
From 30 to 32 hpf, Lv- app is expressed in the postoral neurons but is no longer detected in the coelomic pouches Fig.
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.
Expression of survival and proliferation genes in the nervous system.
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 i— l.
By 22 hpf, Lv- rasO expression is on a single side of the lateral ciliary band.
Expression then diminishes by 28 hpf.
Lv- trk is first detected read more the postoral neurons in some, but not a majority, of embryos by in situ hybridization at 22 hpf Fig.
By 24hpf, Lv- trk is expressed in the postoral neurons where it remains through 32 hpf Fig.
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.
In Lytechinus, expression of Lv- prohibitin is not detected by in situ hybridization from 10 to 16 hpf Fig.
Signal begins to accumulate for Lv- prohibitin in the hindgut starting at 18 hpf Fig.
Overtime, Lv- prohibitin continues to be expressed in the mid and hindgut Fig.
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 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 simple blackjack chart ectoderm Fig.
At 18 hpf, it is expressed in and around the developing blastopore as well as diffusely in the ectoderm Fig.
At 20 hpf, Lv- rasO is expressed in one or both lateral sides of the ciliary band Fig.
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.
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.
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.
It is not expressed from 10 to 18 hpf Fig.
Expression of neurotransmitter-related genes.
It remains expressed in those areas faintly until 32 hpf Fig.
In Lytechinus, Lv- asicl4 shows no expression from 10 to 22 hpf Fig.
Expression is detected by in situ beginning at 24 hpf in the apical organ where it remains through 32 hpf Fig.
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.
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.
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 visit web page and pepper pattern within the nervous system such as Lv- scratch, Lv- ese and Lv- prox, Fig.
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.
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.
Schematic of genes expressed in different regions of the ectoderm and endoderm at late gastrula and pluteus larva stages.
Expression within a region does not mean that the genes are necessarily co-expressed with one another.
Expression within a region does not mean that the genes are necessarily co-expressed.
A anterior, P posterior Rows indicate genes, and columns indicate areas of the embryo.
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.
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 la slota net 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.
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.
These include the transcription factors Lv- ese, Lv- prox, Lv- islet, See more atbf1, Lv- hey, article source 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.
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.
It is possible that genes expressed both in mesodermal tissues and in neurogenic territories are performing similar roles in both regions.
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.
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 please click for source 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 la slota net 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.
Adult animals and embryo culture Adult L.
Sugarloaf Key, FL, USA.
Gametes were harvested by injection of 0.
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.
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.
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.
RNA probes were synthesized in vitro and hybridized at 65 °C.
For each antisense probe, a sense probe was also tested and shown to show no expression pattern or background signal only Additional file : Figs.
LAS wrote manuscript and prepared 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.
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.
Availability of data and materials All data underlying the current analyses are publicly available or are included in the Additional files.
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 la slota net publication.
Contributor Information Leslie A.
McClay, Phone: 919-613-8188, Email:.
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