Features of Amphiura filiformis embryonic development and skeletogenesis
In this study, we compare development and gene expression between the brittle star Amphiura filiformis and indirectly developing echinoids with elaborated skeleton [2, 18, 19]. To understand larval skeletogenesis in ophiuroids, we first analyzed the tempo and the mode of A. filiformis development, providing the most complete picture of ophiuroid development to date (Fig. 1). A. filiformis differs from euechinoid sea urchin development in the lack of micromeres at the vegetal pole (Fig. 1a, 6hpf), and in the early appearance of morphological evidence for animal–vegetal and oral–aboral axes (Fig. 1a, 16hpf and 23hpf). The fertilized egg undergoes 8 rounds of cleavages in the first 10 h of development producing a blastula of approximately 250 cells with a clearly visible blastocoel and still encased in the fertilization membrane (Fig. 1a, 10hpf ). Immediately after hatching (Fig. 1a, 16 hpf), the embryos elongate along the animal–vegetal axis and the cells in the vegetal half are distinctly thicker than the ones in the animal half. At the beginning of gastrulation (Fig. 1a, 27 hpf), A. filiformis embryos flatten along the oral–aboral axis. Furthermore, morphogenetic movements occur at a faster pace, although following a similar sequence of events (Fig. 1). Despite these differences, in both organisms skeletogenesis is preceded by the ingression of mesenchymal cells prior to gastrulation (Fig. 1a, 23 hpf), and the two bilaterally arranged spicules are formed just underneath the ectoderm within two clusters of mesenchymal cells located at the boundary with the invaginating endoderm as identified by calcein staining (Fig. 1a, 30 hpf) (for comparison with sea urchin see [33]).
Skeleton-specific genes, therefore, should be expressed in the cells tightly associated with skeletal elements in the two lateral patches of the A. filiformis gastrula where the spicules appear. We selected genes whose zygotic expression is present exclusively in skeletogenic cells throughout development in sea urchin and cloned their orthologs in Amphiura. These genes are encoding for transcription factors, such as alx1, jun [17, 34], and for differentiation proteins identified by proteomic studies in the bio-mineralized matrix of both larval and adult skeleton, such as p19, p58a and p58b [35–37]. Using whole mount in situ hybridization (WMISH), we observe their expression in the cells where A. filiformis skeleton primordia first appear (Fig. 1c). The expression of these genes remains associated only with the growing skeleton also later in development, as shown by the staining in the two mesenchymal clusters of cells at the base of the archenteron throughout gastrulation, where the skeleton becomes evident, and in a chain of mesenchymal cells distributed in a pattern that mirrors the elaborated skeletal structure of the pluteus (Additional file 1: Figure S1). Similar to sea urchin, these genes are exclusively expressed in cells surrounding the position of skeleton formation. Moreover, their co-expression over developmental time allows us to use their combination here as a marker for skeletogenesis. To establish the onset of expression of these skeletogenic lineage specific genes, we analyzed high-resolution time-courses using QPCR (Fig. 1b). Afi-alx1 and Afi-p19 start to be expressed at early blastula (12 hpf), while the maternally abundant Afi-jun decreases in the first 9 h of development until its zygotic expression is activated at 12 hpf (Fig. 1b). WMISH of Afi-alx1 at early blastula shows expression in 8 (±1) cells (n = 3) grouped together on one side of the embryo (Fig. 1c, Additional file 1: Figure S2) and is later expressed in 18 (±3) cells (n = 17) in the vegetal plate of the late blastula (Fig. 1c). Importantly, none of these three genes show any localized expression at earlier stages of development (Additional file 1: Figure S2), consistent with the QPCR data. At early blastula stage, next to similar cell counts, double fluorescent in situ hybridization (FISH) shows complete co-expression of Afi-alx1 and Afi-p19 (Additional file 1: Figure S2C). At mesenchyme blastula stage staining for these genes, as well as Afi-p58a and Afi-p58b, is detected only in the mesenchymal cells ingressing first, and later at gastrula stage in cells surrounding the position of calcium carbonate deposition (compare Fig. 1a, c). Taken together these high-resolution expression data suggest that, as in euechinoids, the primary mesenchyme cells might be the precursors of the skeletal cells and are here referred to as skeletogenic mesodermal cells (SM). These data also identify a specific regulatory program already present at blastula stage in a subset of the vegetal cells of the A. filiformis embryo. This is characterized by the presence of the transcription factors Afi-alx1 and Afi-jun and their co-expression with the differentiation genes Afi-p19, Afi-p58a and Afi-p58b for which sea urchin orthologs are participating in the formation of the bio-mineral matrix of the skeleton. The exact function of these genes in A. filiformis development and their role in the formation of the skeleton need to be tested in knock-down experiments in future investigations.
Functional differences in the A. filiformis ortholog of the sea urchin skeletogenic initiator gene
In euechinoid sea urchin, the skeletogenic program is initiated by the zygotic expression of the paired-like homeodomain transcriptional repressor pmar1/micro1, which starts to be expressed only in micromeres as early as 4th cleavage [38, 39]. Pmar1 dominantly represses the globally expressed hesC and separates the skeletogenic lineage from the rest of the embryo forming the first element of the DNG (Fig. 2c). A potential brittle star pmar1 has been mentioned in the study of the Ophiocoma wendtii developmental transcriptome [30]. Using a reciprocal blast approach in an A. filiformis developmental transcriptome, which includes four developmental time-points from cleavage to gastrula stage, we identified a sequence with closest similarity to Spu-pmar1c, here referred to as Afi-pplx. We validated its true evolutionary relationship with the pmar1 genes through maximum likelihood and bayesian phylogenetic trees in the context of several classes of paired-like homeodomain (HD) sequences identified in different echinoderms and non-vertebrate deuterostomes. Using an alignment of the HD alone and independent of methodology, Afi-Pplx always grouped with good support as sistergroup of euchinoid Pmar1/Micro1 transcription factors (posterior probability of 0.93 and bootstrap of 63; Fig. 2a, Additional file 1: Figure S3A, C). On the other hand, the inclusion of other conserved domains in our analysis of the Pmar1/Micro1 proteins (i.e., engrailed repressor domains, eh1), resulted either in a highly supported polychotomy of Afi-Pplx, Phb1, and Pmar1/Micro1 genes using Bayesian inference (posterior probability 0.99; Additional file 1: Figure S3B) or in a low supported independent grouping of Afi-Pplx with brittle star and sea star (Patiria miniata, Pmi) Phb1 genes, whereas Pmar1/Micro1 genes group with Spu-Phb1 (bootstrap <60; Additional file 1: Figure S3D). Interestingly, all trees supported a monophyletic grouping of Afi-Pplx with Phb1 and Pmar1/Micro1 genes with high confidence (posterior probability 0.99 and bootstrap >83; Fig. 2a, Additional file 1: Figure S3). For this reason, we decided to name the Afi gene pmar1-phb1-like-homeobox (pplx). Interestingly, we were unable to find any close pmar1 hit in the available cidaroid (Eucidaris tribuloides), sea star (Patiria miniata) and hemichordate (Saccoglossus kowalevskii) genomes, although a phb1-related sequence was present. Our phylogeny clearly reveals (1) the phb1 and the pmar1 + pplx1 genes form a strongly supported distinct class of paired-like homeodomain; (2) the long branch to the euechinoid pmar1/micro1 genes and the absence of a clear cidaroid pmar1 gene suggest a recent evolution of these genes only in euechinoids; and (3) in euechinoids the pmar1 genes have been extensively duplicated.
Importantly, the temporal and spatial expression of Afi-pplx is highly similar to the sea urchin Spu-pmar1 (Fig. 2b, c). Afi-pplx is transiently expressed only in the zygote, starting its expression at late cleavage in a group of 10 (±3) cells (n = 7). It has a maximum level of expression at early blastula, when it is expressed in 20 (±5) cells (n = 4), then in 35 (±6) cells (n = 7) at the vegetal plate, to drop down to undetectable levels by mesenchyme blastula (Fig. 2b, c). It is important to notice that the number of cells expressing Afi-pplx at blastula stage is twice as much as the number of SM cells marked by Afi-alx1. A detailed analysis of protein domains using sequence comparison revealed that Afi-Pplx lacks two eh1 motifs, which are necessary for the Spu-Pmar1 repressive function [40]. It has been shown that this short protein motif can easily be acquired and lost throughout evolution [41]. In Afi-Pplx, moreover, amino acid (aa) 50 of the homeodomain, known as the recognition aa, is an H instead of a Q (Additional file 1: Figure S4A). On this basis, we hypothesize that Afi-Pplx is not functionally similar to Spu-Pmar1 and unlikely acts as transcriptional repressor, despite a similar domain of expression. To test this, we injected sea urchin fertilized eggs with equimolar amount of a synthetic mRNA encoding for Afi-Pplx and Spu-Pmar1 as already described [40]. We also injected a GFP only as negative control for injection artifacts (Spu-5′pmar1-gfp; Fig. 2e, Additional file 1: Figure S4) and controlled for correct translation of the synthetic Afi-pplx mRNA using a fusion with GFP (Afi-pplx-gfp; Additional file 1: Figure S4A), which indeed is translated and localized in all nuclei of the sea urchin embryos (Additional file 1: Figure S4 B). Whereas ectopic expression of Spu-pmar1 leads to the re-specification of every cell of the embryo to a skeletogenic fate [40], ectopic expression of Afi-pplx does not show any re-specification of cells towards the skeletogenic fate, as shown also at molecular level with the lack of expansion of Spu-delta expression (Fig. 2e). To better characterize at molecular level the effects of Afi-pplx injection on the sea urchin skeletogenic program and exclude any potential compensatory effects, we quantified the level of expression of ten sea urchin skeletogenic genes, using QPCR, in Afi-pplx- and Spu-pmar1-injected embryos and compared them with GFP-injected controls. The sea urchin genes analyzed include all the immediate downstream genes of the double negative gate (Spu-delta, Spu-tbr, Spu-ets1/2, and Spu-alx), as well as late specification genes (Spu-foxb), signaling receptor (Spu-vegfr), and skeleton matrix genes (Spu-sm50, Spu-p19, Spu-p58a, Spu-p58b). In agreement to what has been already published by [2], the Spu-pmar1-injected embryos show upregulation of all skeletogenic genes above threshold levels (ΔΔCt > 1.6); on the contrary, Afi-pplx-injected embryos show little or no effect on all genes analyzed (Additional file 1: Figure S4F). Our results indicate that Afi-pplx is not capable of repressing the Spu-hesC gene and, thus, operates differently from Spu-pmar1. Interestingly, ectopic expression of Afi-pplx-mRNA shows specific and reproducible phenotypic effects on the development of the S. purpuratus skeleton at a later stage (Additional file 1: Figure S4D, E). This could be the result of Afi-Pplx having an activator function, opposite to Pmar1 repression, or a consequence of different interactions of these two transcription factors with other regulatory partners and/or cis-regulatory sequences. In summary, these data suggest that although Afi-pplx is expressed in a very similar spatio-temporal pattern of Spu-pmar, it might provide a different regulatory function in the brittle star skeletogenic program.
In A. filiformis HesC is unlikely to be a repressor of delta, ets1/2, and tbr
In euechinoids, the second element of the DNG consists of the globally expressed gene hesC, which is excluded from the skeletogenic lineage by the repressive action of Pmar1. HesC directly represses a cohort of genes encoding for TFs (ets1/2, alx1, tbr, and soxC) and signaling molecules (delta) [2, 38, 42]. This cohort of genes will drive forward the skeletogenic program up to the activation of differentiation gene batteries (Fig. 2d) [2]. On the contrary, in cidaroids a great variability in the expression and function of hesC has been recently reported [18, 19]. Although we showed that Afi-pplx is probably not working as a repressor and thus is not part of a DNG logic, we cannot exclude that Afi-hesC might still spatially restrict the expression of the same downstream genes via its repressive action. Therefore, we cloned and analyzed the spatio-temporal expression of Afi-hesC and its immediate downstream genes Afi-ets1/2, Afi-alx1, Afi-tbr, and Afi-delta. At the aa level, Afi-HesC shows conservation of all its distinctive domains, including the VRPW repressor domain, making it likely to retain a transcriptional repressor function. Afi-hesC is not detectable throughout cleavage stages (Additional file 1: Figure S5A), and begins to be expressed at blastula stage in a ring of cells towards the vegetal pole (Fig. 3a), thereby acting as a local rather than a global regulator. Importantly, A. filiformis orthologs of the DNG downstream genes, tbr, ets1/2, and delta, are partially co-expressed with Afi-hesC (Fig. 3b) at blastula stage. This co-expression becomes even more extensive at mesenchyme blastula stage, when Afi-hesC occupies the vegetal plate together with ets1/2, delta, and tbr, making a repressive action of Afi-HesC on these genes highly unlikely (Fig. 3a). Furthermore, double FISH shows complete co-expression of Afi-hesC with Afi-foxA at this stage (Fig. 3b), while at mesenchyme blastula stage Afi-hesC will occupy the center of the vegetal plate delimited by a ring of Afi-foxA expressing cells (Fig. 3, Additional file 1: Figure S6). In sea urchin, the Spu-foxA gene is initially expressed in the entire endomesoderm territory apart from the SM lineage, while later in development it gets restricted specifically to the endoderm lineage only [9].
Double FISH shows that Afi-pplx, Afi-ets1/2, Afi-tbr, and Afi-delta are all expressed in a larger domain than Afi-alx1 (Fig. 3, Additional file 1: Figure S7), a result additionally supported by the counts of positively labeled cells in several embryos. At blastula stage, Afi-ets1/2 is expressed in 32 (±2) cells (n = 6), Afi-tbr in 40 (±3) cells (n = 5), and Afi-delta in 32 (±3) cells (n = 5), which indicates that they are likely expressed in the whole mesoderm and possibly partially in the endoderm territories and are not restricted only to the skeletogenic precursor cells marked by Afi-alx1 18 (±3) cells (n = 17) as in sea urchin. This is consistent also with the earlier expression of Afi-delta and Afi-pplx in a wider domain of 20 (±5) cells (n = 5) compared to 8 (±1) cells (n = 2) of Afi-alx1 at early blastula (Additional file 1: Figure S5). Afi-HesC, however, could still repress Afi-alx1 into a small domain in the center of the vegetal plate of the blastula. Assuming that Afi-HesC is the main direct repressor of Afi-alx1 in the vegetal plate, as shown in euechinoid and in a species of cidaroid sea urchins (S. purpuratus [43] and E. tribuloides [18]), the expression pattern of those genes should be mutually exclusive. The double FISH identifies some cells that do not express Afi-alx1 within the domain delimited by Afi-hesC, suggesting either a non-direct relationship between Afi-alx1 and Afi-hesC or the presence of another repressor of Afi-alx1 in these cells (Additional file 1: Figure S7).
Altogether our analysis suggests the absence of a pplx/hesC DNG in brittle star as a mechanism of initial specification of the subdomain of mesoderm expressing skeleton-specific genes and is supported by the following: (1) Partial co-expression at blastula stage and complete co-expression at mesenchyme blastula stage of Afi-hesC with Afi-tbr, Afi-ets1/2, and Afi-delta support that the cis-regulatory apparatuses of these genes are insensitive to the repression by Afi-HesC and are thus different from euechinoid sea urchin. (2) Afi-pplx is expressed in 35 (±6) cells (n = 7) similar to Afi-tbr, Afi-ets1/2, and Afi-delta suggesting co-expression with Afi-hesC and, hence, the absence of a repressive action of Afi-pplx1 on Afi-hesC at this stage, consistent with the absence of known protein repressive domain. (3) The incomplete mutual exclusive expression of Afi-hesC with Afi-alx1 makes a role of Afi-hesC as sole repressor of Afi-alx1 unlikely.
Other differences between sea urchin and brittle star skeletogenic GRNs
Downstream of the initial tier of regulation activated by the DNG, the sea urchin SM network is stabilized by an interlocking loop (IL) engaging the genes, tgif, erg, and hex in a recursively wired positive feedback loop [2]. Interestingly, this IL is conserved in mesodermal cells of sea star, an echinoderm class that does not form any larval skeleton [11], suggesting an ancestral function not directly linked to larval skeletogenesis. In A. filiformis the genes Afi-tgif, Afi-erg and Afi-hex are expressed or enriched in a group of cells at the vegetal plate of the blastula, similar to sea urchin and sea star, but with the following differences: Afi-erg is expressed in a smaller domain nested within Afi-hex expressing cells; Afi-tgif is ubiquitously expressed at low levels and enriched only in the vegetal plate (Fig. 4a). This is consistent with a transient function of the IL in Afi-erg positive cells of the vegetal plate only at blastula stage. Whereas Afi-erg stays active in SM until gastrula stage (Fig. 4a, Additional file 1: Figure S8), Afi-hex is turned off from SM as soon as these cells enter into the blastocoel (Fig. 4a) and is unlikely to be a driver of skeletogenic genes at later stages. Importantly, at mesenchyme blastula stage these same three genes are now co-expressed in the vegetal plate, where the non-skeletogenic mesodermal cells (NSM) reside, and possibly reestablish the IL in these cells (Fig. 4a). Time-course comparisons between sea urchin and brittle star pinpoint differences of initial inputs responsible for the activation of these genes. In the only echinoderm species where the dynamic of gene expression is available, S. purpuratus, the three genes are activated in the following order: Spu-hex, Spu-erg, and Spu-tgif, in all cases needing the former for the activation of its subsequent. Conversely, in brittle star the order of activation is perfectly reverted (Fig. 4b) suggesting differences in initiation and potentially promoter logic of the IL in brittle star compared to sea urchin. It is important to notice that orthologs of the main drivers of the sea urchin and sea star IL genes, Tbr and Ets1/2, in brittle star are expressed not only in SM lineage, but also in a wider mesodermal area consistent with the expression of hex and tgif, but not erg. This implies that Afi-erg requires extra input(s) to be restricted to a subset of cells at blastula stage. These data are in agreement with an ancient pan-mesodermal role of the hex-erg-tgif IL, as seen in sea star [11], rather than performing a dedicated SM function as evolved in euechinoids.
In sea urchin skeletogenesis, two extra TFs, foxB and dri, directly regulate the expression of some differentiation genes [44–46]. Spu-foxB is only employed during larval skeletogenesis in S. purpuratus, whereas Spu-dri is also expressed in an adult skeletogenic domain [21]. In contrast, the brittle star orthologs of these are not involved in larval or adult skeletogenesis ([47] and unpublished data), as confirmed by WMISH and QPCR (Figs. 1b, 4).
Finally, recent transcriptomic screenings in S. purpuratus [16, 48] identified three additional transcription factors as specifically expressed in skeletogenic cells during development, although little is known about their role in the GRN for skeletogenesis. These are Spu-nk7, Spu-alx4, and Spu-mitf. From quantitative transcriptome data available in the EchinoBase (http://www.echinobase.org/Echinobase/), it is evident that none of them is expressed before SM ingress into the blastocoel (24 hpf), and spatial data confirm the expression in primary mesenchymal cells only for Spu-nk7 and Spu-mitf [49, 50]. In sea urchin, Spu-nk7 is expressed in skeletogenic cells from mesenchyme blastula throughout development, suggesting a role in late skeletogenesis. To understand the potential role of these genes in brittle star, we surveyed our transcriptome data; we identified only Afi-nk7 and Afi-alx4, and analyzed the expression of Afi-nk7. This gene showed expression already at blastula in the vegetal plate and in the SM cells of the mesenchyme blastula stage, but it is absent from skeletogenic cells at later stages of development and during skeleton deposition (Fig. 4a).
In summary, while the IL might still act in brittle star mesodermal cells, the late skeletogenic regulators, Afi-foxB and Afi-dri, are not responsible for driving the expression of any skeletogenic differentiation genes because they are never expressed at the analyzed stages (mitf) or never expressed in these cells (foxb and dri). Additionally, the expression of Afi-nk7 shows heterochronic expression between the two classes of echinoderms.
Dynamic regulatory states during A. filiformis mesoderm development
A recent study showed conservation of the blastula mesodermal regulatory state among different classes of echinoderms, excluding brittle stars [6], although the relative positioning of the different mesodermal cells within the vegetal plate showed a certain degree of variation.
To understand the timing of specification and the disposition of various mesodermal cells (i.e., skeletogenic and non-skeletogenic mesoderm) within the vegetal half of the embryo, we performed a series of in situ hybridizations on NSM regulatory genes, using Afi-alx1 as a landmark for SM and Afi-foxA for its outer boundary (Fig. 5, Additional file 1: Figure S6 and S7). We found no expression of NSM specification genes (gataE, gataC, and gcm) during blastula stage in A. filiformis (Fig. 5). At this stage, the SM is eccentric to the boundary delimited by Afi-foxA/Afi-hesC, establishing, thus, a third small mesodermal domain of unknown function (Additional file 1: Figure S7B and C). A few hours later, at mesenchymal blastula, once the SM cells ingress into the blastocoel, Afi-gataE and Afi-gataC are expressed in the entire vegetal plate (Fig. 5) along with Afi-hesC (Additional file 1: Figure S7) and other mesodermal genes (i.e.,
tbr, ets1/2, tgif, erg,
hex, and delta). In sea urchin, Alx1 represses the NSM driver gene gcm in the SM lineage to ensure spatial separation of these two types of mesoderm. The absence of expression of Afi-gcm in brittle star (confirmed both with WMISH Fig. 5, and QPCR, Fig. 1b) makes this network linkage unlikely to exist. The late expression of Afi-gataE and Afi-gataC also suggests that NSM specification and patterning might occur at later stages compared to sea urchin.
Cell specification is not a single-step process and several genes contribute to different aspects of this biological process emphasizing the importance of studying the dynamics of regulatory states. Therefore, we built a cellular resolution map (Fig. 6b) of the different mesodermal regulatory states up to mesenchyme blastula stage integrating all presented data. Our analysis revealed that (1) only Afi-pplx and Afi-delta have localized expression in a group of cells already visible by the end of cleavage stage (Fig. 2, Additional file 1: Figure S5). (2) Most of the mesodermal genes, including the SM genes, start their zygotic expression around early blastula stage (12 hpf), suggesting that the initiation of mesoderm specification might occur at this stage (Fig. 1b). (3) After hatching, a cohort of regulatory genes is expressed in all mesodermal cells and likely specifies a pan-mesodermal state. These are Afi-tbr, Afi-ets1/2,
Afi-tgif, Afi-hex, Afi-pplx, and Afi-delta. (4) At this stage, a unique combination of transcription factors characterizes at least three distinct mesodermal domains (Fig. 6b, light green). An SM domain marked by the expression of Afi-alx1, Afi-jun, Afi-nk7, and Afi-erg (Fig. 6b, dark green). A small domain expressing only the pan-mesodermal genes Afi-tbr, Afi-ets1/2,
Afi-tgif, Afi-hex, Afi-pplx, and Afi-delta (Fig. 6b, light green), and lastly, a one cell-wide ring of overlap between Afi-foxA and Afi-hesC and the pan-mesodermal genes (Fig. 6b, light green with blue dots). Additionally, expression of Afi-foxA and Afi-hesC spans towards the presumptive endoderm (Fig. 6b, blue). (5) By mesenchyme blastula, the SM and the NSM are now completely segregated although both express pan-mesodermal genes. The SM is now composed of mesenchymal cells, which have ingressed into the blastocoel and express Afi-alx1, Afi-nk7, and Afi-jun as distinctive markers. The NSM remains in the vegetal plate of the blastula epithelium and is distinguished by the expression of Afi-hesC, Afi-delta, Afi-hex, Afi-tgif, Afi-gataC, and Afi-gataE.