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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Dev Biol. 2012 Dec 2;374(1):245–254. doi: 10.1016/j.ydbio.2012.11.013

Gene Regulatory Control in the Sea Urchin Aboral Ectoderm: Spatial Initiation, Signaling Inputs, and Cell Fate Lockdown

Smadar Ben-Tabou de-Leon 1,*, Yi-Hsien Su 2,*, Kuan-Ting Lin 2, Enhu Li 3, Eric H Davidson 3,*
PMCID: PMC3548969  NIHMSID: NIHMS426667  PMID: 23211652

Abstract

The regulation of oral-aboral ectoderm specification in the sea urchin embryo has been extensively studied in recent years. The oral-aboral polarity is initially imposed downstream of a redox gradient induced by asymmetric maternal distribution of mitochondria. Two TGF-β signaling pathways, Nodal and BMP, are then respectively utilized in the generation of oral and aboral regulatory states. However, a causal understanding of the regulation of aboral ectoderm specification has been lacking. In this work control of aboral ectoderm regulatory state specification was revealed by combining detailed regulatory gene expression studies, perturbation and cis-regulatory analyses. Our analysis illuminates a dynamic system where different factors dominate at different developmental times. We found that the initial activation of aboral genes depends directly on the redox sensitive transcription factor, hypoxia inducible factor 1α (HIF-1α). Two BMP ligands, BMP2/4 and BMP5/8, then significantly enhance aboral regulatory gene transcription. Ultimately, encoded feedback wiring lock-down the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.

Keywords: gene regulatory networks, cis-regulatory analysis, ectoderm specification, sea urchin, developmental control

Introduction

Cell fate specification and differentiation are controlled by complex regulatory networks encoded in the genome (Davidson, 2006; Davidson, 2010). The architecture of gene regulatory networks (GRNs) determines their information processing properties, and defines the temporal order of specification events (Ben-Tabou de-Leon and Davidson, 2007). Thus the structure-function relations of GRNs as they progress through developmental time provide systems level understanding of the progressive molecular control of developmental processes. The construction of experimentally based models of gene regulatory networks requires advanced experimental tools and methodologies.

The sea urchin embryo presents key experimental and theoretical advantages for the study of developmental GRNs: a relatively simple developmental program that has been used to address fundamental questions in development (Horstadius, 1939), easy gene transfer technology coupled with quantitative perturbation analysis of gene expression and a novel high throughput method for testing the expression level of more than a hundred cis-regulatory reporters in one experiment (Nam et al., 2010; Nam and Davidson, 2012). Predictive mathematical models were developed based on the knowledge gained from sea urchin regulatory studies, simulating the dynamics of gene regulatory circuits (Bolouri and Davidson, 2003; Ben-Tabou de-Leon and Davidson, 2009; Ben-Tabou de-Leon, 2010; Ben-Tabou de Leon and Davidson, 2010) and more recently, computing how the logic functions utilized by GRNs give rise to unique spatial regulatory states (Peter et al., 2012). These experimental and theoretical methodologies enabled the construction of one of the most elaborate models of developmental GRNs, a model of the control system governing specification of endomesoderm in the sea urchin embryo (Oliveri et al., 2008; Peter and Davidson, 2009; Peter and Davidson, 2011). It has the capacity to explain the molecular control of various developmental phenomena, such as spatial and temporal patterning of gene expression (Smith and Davidson, 2008; Peter and Davidson, 2011), the endoderm–mesoderm cell fate decision (Ben-Tabou de Leon and Davidson, 2010; Peter and Davidson, 2011), and the activation of sets of differentiation and structural genes in a specific cell lineage (Oliveri et al., 2008; Smith and Davidson, 2009). The recent computational model mentioned above, demonstrates that the endomesoderm GRN in fact includes sufficient information to compute predicatively almost all known regulatory gene expression in space and time, in this portion of the embryo up to gastrulation (Peter et al.). Experimentally based models of the GRNs governing specification of the various fate and regulatory state domains of the sea urchin embryo ectoderm are now being constructed and carry the promise to illuminate the regulatory control of ectodermal patterning (Su et al., 2009; Li et al., 2012).

In its external developmental morphology, the sea urchin embryo ectoderm consists of four prominent territories: the oral ectoderm, within which the mouth forms through fusion of the foregut and the ectoderm; the aboral ectoderm, which differentiates into squamous epithelium; the ciliary band, positioned at the border between oral and aboral ectodermal domains; and the apical neurogenic domain (Fig. 1A left panel). The neurons of the sea urchin larva are mostly localized within the apical domain, in the ciliary band, and around the mouth (Burke et al., 2006). Oral/aboral diversification along the secondary embryonic axis of the sea urchin embryo forms as the result of an asymmetric redox gradient that derives originally from uneven maternal mitochondrial distribution (Coffman and Davidson, 2001; Coffman et al., 2004; Coffman et al., 2009). At the high end of this gradient, the future oral side of the embryo, the nodal gene is activated where its cis-regulatory control system responds to a redox sensitive transcription factor (Coffman and Davidson, 2001; Coffman et al., 2004; Nam et al., 2007; Range et al., 2007; Coffman et al., 2009; Range and Lepage, 2011). Nodal signaling then controls the specification and differentiation of the oral ectoderm domain (Duboc et al., 2004). Targets of Nodal signaling within the oral ectoderm include genes encoding both the ligand BMP2/4 and its antagonist Chordin (Duboc et al.; Duboc et al., 2004). The BMP ligand diffuses to the aboral ectoderm, to which its signaling activity is confined, due to inhibition of BMP reception by Chordin within the oral ectoderm (Duboc et al., 2004; Chen et al., 2011).

Fig 1.

Fig 1

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Spatio-temporal patterning of the sea urchin aboral ectoderm. A. Diagram of sea urchin embryogenesis marking the different embryonic territories and ectodermal sub-domains. Left – lateral view, right – aboral view. B. Temporal expression profiles of the aboral ectoderm regulatory genes (based on (Materna et al., 2010)). C. Spatial expression patterns of aboral ectoderm transcription factors at different time points at early development. Dark blue marks high expression level in this region, light blue marks low expression in this region, and gray marks regions where the gene is not detectable in WMISH (based on (Chen et al., 2011)).

Recent work shows that the oral ectoderm contains an unexpectedly complex, bilaterally arranged set of spatial regulatory state subdomains, (Li et al., 2012) and the same is true of the aboral ectoderm despite its seemingly uniform morphology (Chen et al., 2011). Prior to gastrulation the aboral ectoderm specifically expresses a set of regulatory genes encoding the transcription factors Tbx2/3, Irxa, Dlx, Msx, Hmx and Hox7 (Su et al., 2009; Chen et al., 2011). Although these genes turn on within a narrow time window (Fig. 1B (Materna et al., 2010)), their spatial expression is differential, defining regulatory state sub-regions within the aboral ectoderm (Chen et al., 2011). The dynamic changes in spatial subdomain expression of these genes between early and late blastula stages are shown diagrammatically in Fig. 1C (Chen et al., 2011). Previous analyses revealed that the reception of BMP2/4 enhances expression of the aboral ectoderm regulatory genes (Duboc et al., 2004; Su et al., 2009; Saudemont et al., 2011). However, the effect of BMP2/4 knock-down was less severe than the effect of BMP receptor knock-down (Yaguchi et al., 2010), and this suggests that there are additional BMP ligands operating at this time. Furthermore the aboral tbx2/3 gene is expressed before the phosphorylation of the BMP mediator, SMAD1/5/8, is detectable (Chen et al., 2011), indicating the presence of an early activator of aboral ectoderm genes not related to BMP signaling. Thus, a redox-dependent aboral ectoderm transcription factor was predicted to initiate the expression of tbx2/3 (Su et al., 2009). While intricate positive regulatory interactions were suspected to exist among the aboral ectoderm transcription factors (Su et al., 2009; Saudemont et al., 2011), prior to the present work temporal resolution of observations indicating such interactions was lacking, particularly for early time points.

Here we study the dynamic regulatory control of the aboral ectoderm specification by combining gene expression studies, perturbation analysis, and cis-regulatory analyses. We show that the redox sensitive transcription factor HIF1α (Hypoxia Inducible Factor 1α) is directly activating the early expression of aboral ectoderm regulatory genes, possibly mediating the primordial redox gradient. Both BMP2/4 and BMP5/8 then contribute to the magnitude of expression of the aboral ectoderm regulatory genes, and their inputs were verified for key genes at the cis-regulatory level. As typical for specification GRNs, the system later graduates to the use of encoded cross-regulatory feedback interactions, which thereafter internally control its transcriptional functions.

Materials and methods

Gene expression and perturbation analysis

Morpholino-substituted antisense oligonucleotides (MOs) specific to the ectoderm regulatory genes were obtained from Gene Tools (Philomath, OR), the sequences of which are shown in Supplementary Information, Table 1. For perturbation analyses, eggs were injected with 100–400 µM MO. RNA from uninjected control and MO injected embryos was isolated by RNeasy Micro kit (Qiagen). RNA samples were reverse transcribed by iScript cDNA synthesis kit (BioRad) for quantitative PCR (QPCR). To monitor the quantitative effects of each perturbation, data were normalized to the amount of ubiquitinm RNA as described (Revilla-i-Domingo et al., 2004). QPCR primers used in this study are also listed in Table S1. Whole mount in situ hybridization was performed as described in (Oliveri et al., 2006).

Microinjection and QPCR Measurement of GFP mRNA in Eggs Expressing GFP Constructs

PCR products were purified with the Qiagen Qiaquick PCR purification Kit or Zymo DNA clean and concentrator. The fragments were then microinjected into fertilized S. purpuratus eggs as described (Rast, 2000). Linearized BAC constructs were desalted by drop dialysis into TE buffer on a 0.025 µm VSWP filter (Millipore). Approximately 500–800 molecules of the desired reporter construct were injected, along with a 3–6 fold molar excess of HindIII-digested carrier sea urchin DNA per egg, in a 4 pl volume of 0.12 M KCl. A similar injection solution was made for BAC reporters but with 100–200 copies of the BAC per 4 pl. Embryos were collected at different stages for assessment of spatial activity by fluorescence microscopy, or for quantitative analysis of transcript prevalence by QPCR. For QPCR measurements RNA and DNA of about 150 injected embryos was isolated using Qiagen Allprep kit. The RNA was then reverse transcribed to cDNA using iScript cDNA synthesis Kit of Bio-Rad. QPCR for both DNA and cDNA was performed as described (Revilla-i-Domingo et al., 2004). The level of cDNA was computed by comparison to an internal standard (ubiquitin) cDNA, and the level of injected DNA was computed in comparison to Nodal DNA.

Constructs

Standard PCR and fusion PCR techniques using the High Fidelity PCR Kit (Roche) and Long template High Fidelity PCR Kit (Roche) were used to build reporter constructs. Binding-site sequences were mutated by PCR, and the resulting constructs were checked by sequencing. The mutation PCR primers were designed with about 20 bp sequences surrounding both ends of the target site. The target site was changed into a mutant form of the candidate transcription factor binding sites. Whole mount in situ hybridization was performed as described in (Oliveri et al., 2006).

Results

Effects of BMP2/4 and BMP5/8 perturbations on sea urchin embryo development and on aboral ectoderm patterning

The gene encoding the ligand BMP2/4 is expressed in the oral ectoderm, starting at about 12 hours post fertilization (hpf) (Fig. 2A; (Duboc et al., 2004; Su et al., 2009)). A second TGFβ gene encoding BMP5/8 is expressed maternally at a very low level and then at much higher levels zygotically, beginning at about 8 hpf. Its zygotic expression is uniform in the ectoderm, spanning all the ectodermal territories (Figs. 2A, B). To study the developmental roles of the different BMP ligands we injected fertilized sea urchin eggs with Morpholino antisense oligonucleotides (MO) that block the translation or the splicing of given genes (For MO sequences see Supplemental Information, Table S1). Embryos injected with BMP2/4 MO display severely reduced aboral expansion; while skeletal rods are formed, even at 72hpf they are not extended at the aboral vertex to form the distinct structure of the pluteus larva (Fig. S1A, B; (Lapraz et al., 2009; Saudemont et al., 2011)). When the embryos are co-injected with BMP2/4 and BMP5/8 MO’s, in addition to the reduced aboral expansion and skeletal patterning defects, the partitioning of the gut is affected (Fig. S1C). The effect of BMP5/8 MO injection is a mild reduction of the aboral expansion (Fig. S1E). Skeletal patterning depends on ectodermal signaling (Armstrong et al., 1993), and it is likely that the BMP input is indirectly required for skeletogenic ectodermal signaling; thus the regulatory programs that it drives are ultimately important for correct morphological patterning of the embryo.

Fig 2.

Fig 2

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Expression profiles of the ligands BMP2/4 and BMP5/8 and the effect of their perturbation on aboral ectoderm gene expression. A. BMP2/4 and BMP5/8 temporal profiles (based on (Materna et al., 2010)). B. BMP5/8 Spatial expression (WMISH), lv- lateral view, vv-vegetal view. C. The effect of BMP2/4 and BMP5/8 MO on the spatial expression patterns of tbx2/3, irxa and dlx at 24h. BMP2/4 MO strongly reduces the expression of tbx2/3, irxa and dlx, (panels 2, 6, 10), but the expression of irxa at the ectoderm–endoderm border remains (panel 2). BMP5/8 MO has weaker effect on the genes expression (panels 3,7, 11). Co-injection of BMP2/4 and BMP5/8 MO almost eliminates the expression of tbx2/3 and dlx, (panels 4, 8, 12) while still not affecting the vegetal ectoderm expression of irxa (panel 4). All embryos are shown in lateral view where the aboral side is to the left.

To study the contribution of the BMP ligands to the establishment of the aboral ectoderm regulatory state we investigated the effect of BMP2/4 and BMP5/8 MO injection on the spatial expression of tbx2/3, irxa and dlx at 19hpf and 24hpf by whole mount in-situ hybridization (WMISH) (Figs. 2C and S1). Neither MO alone nor the two in combination has a strong visible effect on expression of irxa at the vegetal/aboral ectoderm boundary, though both obliterate irxa expression farther up in the aboral ectoderm indicating that irxa is driven by additional activators in vegetal portion of its domain of aboral ectoderm expression (Fig. 2C, compare panels 1 and 4). The expression of tbx2/3 and dlx is significantly reduced at these times by BMP2/4 MO injection, or by co-injection of BMP2/4 and BMP5/8 MO. The injection of BMP5/8 MO alone reduces the expression of dlx, but has only a weak effect on the expression of tbx2/3. These results agree with previously published results of BMP2/4 perturbations (Saudemont et al., 2011). The additional finding is that BMP5/8 is also necessary for normal expression of both irxa and dlx.

Expression and developmental role of the hypoxia-dependent transcription factor HIF1α

As described in Introduction, maternal anisotropy of the mitochondrial distribution generates a redox gradient along which oral-aboral polarization occurs (Coffman and Davidson, 2001; Coffman et al., 2004; Coffman et al., 2009). High mitochondrial concentration and the resulting high redox potential and oxidizing environment are correlated with oral fate (Coffman and Davidson, 2001; Coffman et al., 2004; Coffman et al., 2009). A redox sensitive factor responding to the reducing environment on the opposite aboral side had been predicted to initiate the early expression of tbx2/3 (Su et al., 2009), but the responsible factor had not been identified. Here we suggest that the well known oxygen sensitive transcription factor HIF1α (hypoxia-inducible factor 1α) executes this role in aboral ectoderm specification early in sea urchin embryogenesis. HIF is a heterodimeric transcription factor which in vertebrates consists of one of three different redox-sensitive HIFα subunits (HIF1α, HIF2α, and HIF3α) complexed with a common constitutive subunit, HIFβ (Wenger et al., 2005). HIF1αβ and HIF2αβ heterodimers function as transcriptional activators of oxygen-regulated target genes. Under normoxic conditions the HIFα subunits are hydroxylated by a family of redox-sensitive prolyl hydroxylases (PHD) and the hydroxylated forms are targeted for degradation (Ivan et al., 2001). Hypoxic conditions decrease the activity of PHD, resulting in rapid HIFα subunit accumulation (Jewell et al., 2001). Recent studies have shown that a reducing environment is required to stabilize HIF-1α under hypoxic conditions (Chen and Shi, 2008; Guo et al., 2008). Thus HIF1α is a likely candidate for a transcription factor that operates specifically in conditions of reducing environment and low redox potential. Sea urchin has a single HIF1α gene encoded in the genome which is structurally similar to the vertebrate HIF1α and HIF2α (Loenarz et al., 2011; Rytkonen et al., 2011). SpHIF1α is maternal and its transcripts are present in all the cells of the embryo, at least up to 12 hpf (Figs.3A, B). Its zygotic expression starts at about 18hpf (Fig.3A). The HIF1α gene is then expressed specifically in the non-skeletogenic mesoderm, small micromeres, and their postgastrular coelomic pouch descendants (Fig. 3B), so only the maternal phase of expression is relevant to this study. Injection of HIF1α MO reduces aboral expansion at 72hpf, (Fig.S2) and severely reduced the expression tbx2/3 at 19hpf, less so at 24hpf (Fig.3C, S3–S5). The effect of HIF1α MO on irxa expression was less pronounced at these times (Fig.3C, S3–S5).

Fig. 3.

Fig. 3

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HIF1α expression profiles and the effect of HIF1α perturbation on the spatial expression of tbx2/3 and irxa. A. HIF1α temporal expression measured by QPCR. B. HIF1α spatial expression at different time points (WMISH). All embryos are shown in lateral view where the aboral side is to the left. C. Effect of HIF1α MO on the spatial expression pattern of irxa and tbx2/3 at19h and 24h. The expression of irxa is somewhat reduced at these times while the expression of tbx2/3 is strongly reduced at 19hpf and less so at 24hpf. All embryos are shown in lateral view where the aboral side is to the left.

Early drivers of the aboral ectoderm regulatory state, as revealed by quantitative perturbation analysis

To study gene interactions through time, particularly those responsible for initiation of gene expression when the levels are at first low, a sensitive quantitative analysis is required. To that end we injected fertilized sea urchin eggs with specific MO and measured the expression levels of selected regulatory genes in the aboral ectoderm and in other embryonic territories at three time points, using quantitative PCR (QPCR) as described in many earlier GRN analyses (Oliveri et al., 2008; Peter and Davidson, 2011) (See table S1 for MO and QPCR primer sequences). A summary of these perturbation results is presented in Fig. 4. The matrices in Fig. 4A–C indicate perturbations that reduced the average ratio between the expression level of a gene and the expression level at control MO to less than 0.4, at 16hpf (Fig. 4A, S3), 19hpf (Fig. 4B, S4) or 24hpf (Fig. 4C, S5). At these early times all interactions observed were positive. Average ratios between the expression level of the aboral ectoderm transcription factors in specific MO and control MO for all perturbations are presented in Figs. S3–5. The effect of all perturbations on all tested genes is presented in Fig. S6.

Fig 4.

Fig 4

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Summary of the quantitative perturbation analysis conducted. Each perturbation analysis matrix represents the interactions between regulatory genes at a given time point. The change in the output-gene expression after injection of a MO targeting the input gene is denoted “+” if the average ratio between the expression level in the input gene MO and in random MO is decreased below 0.4 (that is, average of [output gene level at input MO/output gene level at random MO]<0.4, Figs. S3–S5). In that case, the input gene is an activator of the output gene. A – 16hpf, B – 19hpf, C – 24hpf.

A glance at the matrices of interaction effects at 16, 19, and 24 hpf in Fig.4 shows that there is relatively rapid change in the inputs to given downstream genes. Tbx2/3 and HIF1α are early drivers of the aboral ectoderm regulatory genes. Prominent effects of interference with translation of these factors are seen at the two earliest time points (Figs. 4A, B, S3, 4, 6). At 16hpf and 19hpf HIF1α MO strongly reduces the expression level of tbx2/3, dlx, and msx, and has a significant effect on the level of hmx (Fig. 4A,B, S3,4). But by 24hpf the effect of HIF1α MO on the expression level of the aboral ectoderm transcription factors is much weaker (Fig. S5). Since, as we note above, only HIF1α translated from the ubiquitous maternal mRNA (Fig.3A, B) can be present in ectodermal cells, the disappearance of the maternal message at 18h predicts this result. Tbx2/3 MO significantly reduces the levels of dlx, hox7 and msx at 16hpf and these genes and irxa at 19hpf (Figs. 4A, B, S3,4,6). At 24hpf it affects only hox7 (Figs. 4C, S5, 6). These results indicate that the tbx2/3 gene is an important driver of the aboral ectoderm genes, particularly in the early phase of their expression. The decreasing response to Tbx2/3 MO at 24h (Fig. 4C) could be due to the clearance of tbx2/3 expression from the animal domain of the aboral ectoderm at this time, as this reduces overlap of its spatial expression with other aboral ectoderm genes (Fig. 1C). Since these target genes continue to be expressed, additional regulatory inputs are evidently coming into play.

Roles of the BMP inputs

The effects of BMP2/4 and BMP5/8 MO, individually and together, were similarly assessed by QPCR (Figs. 4, S3–6). At 16hpf co-injection of BMP2/4 and BMP5/8 MO decreases the expression of the aboral ectoderm regulatory genes, but the variation in the measurement are quite significant at this time except for the genes tbx2/3 and hox7 (Fig, 4A, S3). At this time, the injection of BMP2/4 MO alone does not produce any significant effect on the aboral genes (Fig. S3) while the injection of BMP5/8 MO does reduce the expression of hox7, msx and dlx (Fig. 4A, S3C, E). This relatively weak effect of the BMP perturbation at 16hpf agrees with the weak phosphorylated SMAD1/5/8 signal at this time (Chen et al., 2011). The stronger effect of BMP5/8 MO compared to BMP2/4 MO at this early time point correlates with the earlier expression of BMP5/8 compared to BMP2/4 (Fig. 2A). However, at 19hpf co-injection of BMP2/4 MO and BMP5/8 MO together significantly decreases the expression level of the aboral ectoderm genes except from hmx (Fig. 4B, S4, S6). At this time injection of BMP2/4 MO or BMP5/8 MO alone also affects expression levels but less so than when the two reagents are combined. Even stronger effects are seen at 24hpf (Fig.4). These results agree with the distinct phosphorylated SMAD1/5/8 signal at 18hpf and the stronger signal at 24hpf (Chen et al., 2011). In summary, the BMP pathway enhances the expression of all the aboral ectoderm regulatory genes studied, starting at about 19hpf and supplying a sharp boost in the expression levels at 24hpf. Apparently, the BMP pathway does not reach its full activation strength before the ligand BMP2/4 is at work, at about 18–19hpf.

Positive regulatory interactions

Injections of irxa and dlx MO's show positive regulatory interactions among the aboral ectoderm regulatory genes at 19hpf and 24hpf (Figs. 4B–C). The results indicate positive feedback loops among the aboral ectoderm regulatory genes. For example we see that dlx has a positive input to irxa while irxa also has a positive input to dlx. Nested feedbacks among some of these genes were indicated in our initial draft GRN (Su et al., 2009) and proposed to be necessary for the maintenance of expression of the aboral ectoderm regulatory genes.

Perturbation analysis kinetics predict direct inputs of the BMP pathway

The perturbation analysis reveals a dynamic system where different factors dominate at different time points (Fig. 4). In Fig. 5A–F we portray the expression levels of the aboral ectoderm transcription factors at 16hpf, 19hpf and 24hpf, at control MO, Tbx2/3 MO and BMP2/4+BMP5/8 MO. At 16hpf tbx2/3 expression is severely reduced due to the perturbation of the BMP pathway (Fig. 5A). At this time tbx2/3 perturbation significantly affects most of the aboral ectoderm genes (Fig. 5B–F). This raises the following question: does the BMP reception directly activate all the aboral ectoderm transcription factors through the phosphorylated SMAD1/5/8, or does the BMP pathway affects the aboral ectoderm genes indirectly, through the activation of key regulators, such as Tbx2/3? To gain insights into the expected dynamic response to BMP perturbation at different network architectures, we applied a mathematical model for network kinetics that was developed earlier (Davidson, 1986; Bolouri and Davidson, 2003; Ben-Tabou de-Leon and Davidson, 2009; Ben-Tabou de-Leon, 2010).

Fig 5.

Fig 5

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Kinetics of perturbation analysis and mathematical modeling. A–F, Average expression levels of the aboral ectoderm transcription factors at control (random) MO, Tbx2/3 MO and BMP2/4+BMP5/8 MO at 16hpf, 19hpf and 24hpf. Expression levels were measured by QPCR. Here we included only batches where both Tbx2/3 MO and BMP2/4+BMP5/8 MO were injected, 16h n=3 (tbx2/3, dlx and, msx) and n=4 (irxa, hox7 and hmx), 19h n=4, 24h n=3. Error bars show standard error. G–I, Mathematical model of the effect of BMP and Tbx2/3 perturbation on a downstream gene (Sup. Information 1, model based on (Bolouri and Davidson, 2003; Ben-Tabou de-Leon and Davidson, 2009; Ben-Tabou de-Leon, 2010)). G. Simulated protein concentration per cell of the inputs, Tbx2/3 and pSMAD1/5/8. H. Simulated mRNA level per cell for a downstream gene, TF, that is indirectly activated by the BMP pathway through the activation of the transcription factor Tbx2/3. TF mRNA level in the intact circuit is plotted in blue, at BMP MO is plotted in green and at Tbx2/3 MO is plotted in red. I. Simulated mRNA level for cell for a downstream gene, TF that is directly activated by the BMP pathway in a feedforward structure with Tbx2/3. Similar color code as in H.

We simulated the response of a downstream gene to the perturbations of the BMP pathway and the transcription factor Tbx2/3 at direct and indirect network connectivity, (Fig. 5G–I. The model equations are presented in the supplemental information.). The model was constructed based on the following experimental observations: tbx2/3 is responding to HIF1α and BMP perturbations at 16hpf (Fig. S3, 6), and therefore in the model we assume that tbx2/3 is activated by two inputs, pSMAD1/5/8 (BMP) and A1 (e.g., HIF1α). The expression of tbx2/3 precedes the phosphorylation of SMAD1/5/8 (Chen et al., 2011) and we included that in the model (Fig. 5G). Additionally, the effect of BMP perturbation on the expression level of the aboral ectoderm genes is strongest compared to other perturbations at 19hpf and 24hpf (Fig. 5A–F, Fig S4, 5), and particularly on tbx2/3 expression at 16hpf (Fig. 4A, S3A). Therefore we assumed that the activation strength of pSMAD1/5/8 is larger than of the other inputs. The simulations of the effect of BMP and Tbx2/3 perturbation on a downstream gene are presented in Fig. 5H (indirect connectivity) and in Fig. 5I (direct feedforward connectivity). The simulation of direct activation of the downstream gene by BMP in a feedforward structure with Tbx2/3 agrees well with the dynamic perturbation data (Fig. 5A–F), strengthening the prediction of direct activation of these genes by pSMAD1/5/8.

Cis-regulatory control of tbx2/3 and dlx, two key activators of the aboral ectoderm GRN

To verify the predictions of the perturbation analysis (Figs 4, 5) we conducted cis-regulatory analyses of the genes tbx2/3 and dlx, two of the important activators in the aboral ectoderm GRN (Figs. 4A–C, (Su et al., 2009)). High through-put screening analysis identified functional conserved regulatory elements in the vicinity of tbx2/3 and dlx (Nam et al., 2010). We constructed GFP reporter constructs driven by these elements and compared their expression profiles with those of GFP recombinant BACs containing the tbx2/3 and dlx genes plus large genomic regions surrounding these genes (BAC and short construct maps, Fig. 6A, tbx2/3; 6D, dlx). The comparison shows similarity of spatial expression as well as of levels of expression between the recombinant BACs and the short reporter constructs. Thus the short constructs contain sufficient regulatory information to drive the expression of tbx2/3 and dlx, (Figs. 6B,C,E,F S7A,B,E,F).

Fig 6.

Fig 6

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Cis-regulatory analysis of tbx2/3 and Dlx. A. Sptbx2/3:GFP recombinant BAC map showing tbx2/3 exons (blue boxes), regulatory regions (orange boxes) and a map of tbx2/3 regulatory region U indicating the location of functional HIF1α and SMAD1/5/8 binding sites. B, C, Embryos injected with (B) SpTbx2/3 GFP recombinant BAC or (C) SpTbx2/3 U:B:P:GFP reporter construct show correct expression at the ectoderm. D. Spdlx:GFP recombinant BAC map showing dlx exons (blue boxes), regulatory regions (orange boxes) and a map of dlx regulatory regions U and B indicating the location of functional HIF1α, Tbx2/3 and SMAD1/5/8 binding sites. E, F, Embryos injected with (E) SpDlx GFP recombinant BAC or (F) SpDlx U:B:P:GFP reporter construct show correct expression at the ectoderm. G. The effect of the mutations of SMAD sites and BMP2/4 MO on tbx2/3 U:B:P:GFP construct at two time points. H. The effect of HIF1α site mutation and HIF1α MO on tbx2/3 U:B:P:GFP construct at two time points. I. The effect of the mutations of SMAD sites and BMP2/4+BMP5/8 MO on dlx U:B:P:GFP construct at two time points. J. The effect of HIF1α and Tbx2/3 site mutation and HIF1α MO and Tbx2/3 MO on Dlx U:B:P:GFP construct at two time points. Error bars show standard error, p values calculated by one tailed z-test.

Deletion of the 5' region annotated "U" from the tbx2/3 regulatory element reduces the expression level of the reporter by about 50% at 19hpf and 24hpf indicating that this region contains important regulatory information (Fig. S7C,D). The intact tbx2/3 U:B:P:GFP reporter construct responds to HIF1α MO at 19hpf and 24hpf (Fig. 6H, light blue bars) and to BMP 2/4 MO at 24hpf (Fig. 6G, light green bars). Mutations of the four consensus SMAD sites and of the single HIF1α consensus site in "U", reduces the reporter expression at the same time points at which the response to the relevant MO is observed, (SMAD, Fig. 6G, dark green bars, HIF1α, Fig. 6H, red bars. See supplemental data for the complete sequences of regulatory elements; Table S2 for binding site sequences, and Table S3 for mutation statistics). These results confirm that the BMP pathway and HIF1α directly activate tbx2/3 through their consensus target sites, in agreement with the perturbation predictions of Fig. 4.

Deletion of the 5' regions annotated "U" and "B" from the dlx regulatory element reduces the expression level of this reporter by about 40% at 24hpf indicating that these regions contain important regulatory information (Fig. S7H). The intact dlx U:B:P:GFP reporter construct responds to BMP2/4+BMP5/8 MO and to Tbx2/3 MO at 24hpf, (BMP, Fig. 6I, light green bars; Tbx2/3, Fig. 6J, purple bars) and to HIF1α MO at 19hpf (Fig. 6J, light blue bars). Mutations of the two SMAD consensus sites in "U", the two consensus Tbx2/3 sites in "U" and consensus HIF1α sites in "U" and "B" reduce the expression of the dlx reporter construct at the same time points where the response to the relevant MO is observed, (SMAD, Fig. 6I, dark green bars; HIF1α, Fig. 6J, red bars; Tbx2/3 Fig. 6J, blue bars). Thus BMP, Tbx2/3 and HIF1α provide direct inputs into dlx, in agreement with the perturbation results (Fig. 4). These direct BMP inputs were not predicted earlier (Su et al., 2009) though the tbx2/3 input into dlx was, and though its identity was not known, so was a redox sensitive activating input into tbx2/3.

The timing of the effect of mutations and perturbations on the level of reporter constructs, Fig. 6, seem to be delayed compared to the effects of the perturbations on the endogenous genes, Fig. 4. This is quite common when comparing reporter constructs and endogenous genes due to the experimental differences of the two assays and the mosaic incorporation of the reporters in the sea urchin embryos. Therefore we assume that the exact timing of the actual regulatory connection in the sea urchin embryo correspond to the timing observed by the perturbation of the endogenous genes.

Discussion

Specification of the aboral ectoderm is a progressive and spatially complex process. As shown diagrammatically in Fig.1, there are distinct regulatory state domains arrayed along the animal-vegetal dimension. In a way this is paradoxical, as the initial studies on the aboral ectoderm of sea urchin embryos depicted this territory as a uniform squamous epithelium in which downstream genes such as spec1, spec2a and cy3a actin are uniformly expressed (Cox et al., 1986; Hardin et al., 1988; Kirchhamer and Davidson, 1996). However, we are here examining regulatory functions, not embryonic cell differentiation, and it is now clear from other domains of this embryo that a progression of spatial regulatory states precedes the spatial resolution of the cell fates to which the territory eventually gives rise (Peter and Davidson, 2011). We may be here examining the initial stages of a complex partitioning of what we have viewed as a single territory into multiple different regulatory domains; the functional biological significance of these regulatory domains might not be played out until later stages of larval development.

In Fig.7, we show the network of regulatory interactions indicated among the genes studied here at the aboral ectoderm, at three different times, 16, 19, and 24hpf. This model shows the significant findings of this work: These are the initial role of the redox sensitive maternal factor HIF1α in activating the earliest regulatory genes on this side of the embryo, particularly the “pioneer” gene, tbx2/3; the continued strong driver architecture in which Tbx2/3 and BMP erect feed forward inputs into other regulatory genes; and the web of cross-regulatory interactions that as we earlier proposed (Su et al., 2009) lock these circuits into place, after the HIF1α input has disappeared.

Fig 7.

Fig 7

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GRN diagrams at 16h, 19h and 24h showing the dynamic control of the aboral ectoderm spatio-temporal patterning. Thick lines and blue diamonds mark the regulatory connections that were verified by cis-regulatory analysis to be direct.

The new discovery of the role of HIF1α suggests that both ends of the redox asymmetry set up in the egg by unequal distribution of maternal mitochondria are utilized at the transcriptional regulatory level. As reviewed above, on the oral side, high redox state is used to activate the nodal cis-regulatory system (Nam et al., 2007; Range et al., 2007). Here we see that on the aboral side the redox sensitive factor, HIF1α, directly activates aboral transcription factors (Fig 6), possibly due to HIF1α stabilization induced by the reducing environment on this side (Coffman and Davidson, 2001; Coffman et al., 2004; Coffman et al., 2009). This could solve the problem of how the aboral ectoderm regulatory state is initiated in the right place in the embryo, an important implication here bolstered by direct cis-regulatory analysis. Further studies of the spatial pattern of the protein HIF1α at these early developmental stages are expected to shed light on this patterning mechanism. The BMP2/4/Chordin device ensures that though originating in the oral ectoderm, this activating input is experienced by cells only on the aboral side. But at the earliest times this input is not yet strongly functional. Most of the BMP input then comes from the ubiquitously but transiently expressed BMP5/8. Throughout, the role of BMP signaling in the aboral ectoderm is fundamentally that of a transcriptional booster and maintenance. This conclusion too is now based on cis-regulatory results which entirely confirm predictions from BMP perturbation results and from the subcircuit architecture shown in Fig.7. Thus, the view which assigns to the oral ectoderm the role of the primary organizer of both oral and aboral ectoderm specification, via expression of the diffusible BMP2/4 ligand, is probably incomplete. Our study implies that the cytoarchitecture of the egg might be utilized on both oral and aboral sides of the embryo to control the differential initial regulatory states.

As this work was in progress additional regulatory genes were discovered to be expressed in the aboral ectoderm at these and later stages. Thus the aboral ectoderm GRNs can be expected to become regionally diversified and progressively more complex as embryonic development proceeds. Further analysis will hopefully explain the differential spatial expression of the aboral regulatory genes along the animal-vegetal dimension (Fig. 1C, (Chen et al., 2011)). HIF1α and the BMP pathway activate genes that are expressed in all the spatial domains of the aboral ectoderm and therefore cannot explain this differential expression pattern. Neither can the positive interactions between the aboral ectoderm transcription factors explain the exclusion of hmx to the animal domain and msx and later tbx2/3 to the central and vegetal domains of the aboral ectoderm (Fig. 1C, (Chen et al., 2011)). Local repression is likely to be responsible for the spatial restriction of these genes to sub-domains within the aboral ectoderm (Fig. 7).

Conclusions

In the most general terms this work is another step toward a goal that only a few years ago would have seemed utterly unattainable: extension of GRN models to encompass the whole of an embryo. When this is achieved the inputs into the regulatory genes of the model will all be outputs from other genes of the model, and all signaling interactions will begin and end in the genomic regulatory apparatus. Explicit knowledge of the program for embryogenesis will thus arrive at a level of logical completeness, and this is now most likely to be attained for sea urchin embryos.

Supplementary Material

01

Highlights.

  • Aboral ectoderm specification in the sea urchin embryo is controlled by multiple mechanisms.

  • A redox-sensitive transcription factor initiates the aboral ectoderm regulatory state.

  • Two different BMP ligands provide inputs essential for continuing activity of aboral ectoderm regulatory genes.

  • The aboral ectoderm regulatory state is locked down by a positive feedback wiring.

Acknowledgments

Research was supported by NIH grant HD-037105 to EHD and by National Science Council grants 99-2627-B-001-003 and 101-2923-B-001-004-MY2 to YS.

Footnotes

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