New regulatory circuit controlling spatial and temporal gene expression in the sea urchin embryo oral ectoderm GRN

Abstract

The sea urchin oral ectoderm gene regulatory network (GRN) model has increased in complexity as additional genes are added to it, revealing its multiple spatial regulatory state domains. The formation of the oral ectoderm begins with an oral-aboral redox gradient, which is interpreted by the cis-regulatory system of the nodal gene to cause its expression on the oral side of the embryo. Nodal signaling drives cohorts of regulatory genes within the oral ectoderm and its derived subdomains. Activation of these genes occurs sequentially, spanning the entire blastula stage. During this process the stomodeal subdomain emerges inside of the oral ectoderm, and bilateral subdomains defining the lateral portions of the future ciliary band emerge adjacent to the central oral ectoderm. Here we examine two regulatory genes encoding repressors, sip1 and ets4, which selectively prevent transcription of oral ectoderm genes until their expression is cleared from the oral ectoderm as an indirect consequence of Nodal signaling. We show that the timing of transcriptional de-repression of sip1 and ets4 targets which occurs upon their clearance explains the dynamics of oral ectoderm gene expression. In addition two other repressors, the direct Nodal target not, and the feed forward Nodal target goosecoid, repress expression of regulatory genes in the central animal oral ectoderm thereby confining their expression to the lateral domains of the animal ectoderm. These results have permitted construction of an enhanced animal ectoderm GRN model highlighting the repressive interactions providing precise temporal and spatial control of regulatory gene expression.

Keywords: Gene regulatory network; Oral ectoderm; Sea urchin; ets4; sip1.

Figure 1

Dynamic spatial gene expression patterns and territories of the oral ectoderm. A) Expression of pax4l, ets4, sip1 and emx. pax4l transcripts are localized in the oral ectoderm during the blastula stage, similar to nodal. Initially ets4, sip1, and emx transcripts cover both oral and aboral ectoderm. Oral expression of these genes fades at mid-blastula and becomes complementary to the central oral ectoderm (marked by lefty expression in the double WMISH) after18 h. B) Diagrams illustrating ectodermal gene expression domains shown in lateral view. Developmental stages are the early blastula stage (12h), late blastula stage (18h), and mesenchyme blastula stage (24h). For simplicity, endomesodermal domains are not indicated. Ectodermal domains are color-coded and labeled on the left; domain-specific genes are shown in Table S1. Apical—apical plate; Ec—ectoderm. lv—lateral view; vv—vegetal view; av—apical view. All embryos in lateral or vegetal views were shown with the oral ectoderm facing left. C) Expression matrix for ectodermal genes during the blastula stage. Three time points were included representing the early blastula stage (12h), late blastula stage (18h), and mesenchyme blastula stage (24h). A graphic presentation of the expression patterns is shown in Table S1.

Figure 2

Temporal expression profiles of selected ectodermal genes establishing oral-aboral polarity at the blastula stage. A) Time courses for nodal, not, vegf3, gsc, and foxg. These genes are activated sequentially between 8 hr and 18 hr. B) Time courses for ets4 and sip1. Zygotic activation of ets4 and sip1 is concurrent with nodal transcription; ets4 is also transcribed maternally. After 11–12 h, transcript levels of sip1 and ets4 undergo a sharp decline.

Figure 3

The gene regulatory network (GRN) model of the animal ectoderm up to mesenchyme blastula stage. This GRN model includes features relevant to the step-wise establishment of regulatory states. The circuitry shows direct and indirect Nodal signaling effects involved in ectodermal gene expression, and the double negative gate logic mediated by ets4 and sip1 clearance, which provides both spatial and temporal restriction of oral ectodermal and stomodeal gene expression. The targets of these double negative gates include gsc, foxg and bra.

Figure 4

pax4l is an oral ectodermal gene controlled by the nodal pathway. A) WMISH observations on pax4l. This experiment shows that ectodermal, but not mesodermal, expression of pax4l is completely lost if the Nodal signaling pathway is inhibited with SB-431542. The embryos were shown with the oral ectoderm facing left. B) Quantitative perturbation results. pax4l transcript levels are reduced in response to the nodal MASO, and increased by lefty MASO. Changes in expression levels of ectodermal genes were quantified by QPCR relative to poly-ubiquitin. Results shown as arithmetic mean ± standard deviation (ddCt:ΔΔCt, i.e., QPCR cycle number normalized to control Ct and to polyubiqitin Ct; 1 ddCt = 1.9 fold difference).

Figure 5

Cis-regulatory analysis to uncover the regulatory inputs driving early sip1 expression. A) Diagram of the 16 kb region upstream of sip1 and reporter constructs shown earlier to have cis-regulatory activity. All constructs include a GFP reporter and sequence tag for expression analysis (Nam et al., 2010). B) Identification of active modules driving sip1 expression through a series of deletion constructs. C) Mutation of otx-binding sites, resulting in reduced expression level.

Figure 6

Clearance of sip1 and ets4 from oral ectoderm in response to nodal or not perturbation. A) Spatial effects of nodal and not MASOs. In nodal MASO treated embryos ets4 and sip1 transcription was detected in both oral and aboral ectoderm up to 24h. In not-MASO treated embryos, ets4 continues to be transcribed in the oral ectoderm, but oral clearance of sip1proceeded as in controls. All embryos were shown with the oral ectoderm facing left. B) Quantitative analysis of ets4 transcript levels. Increased Nodal signaling through knockdown of lefty led to a moderate reduction of ets4 abundance. This reduction was abolished by co-injection of not MASO.

Figure 7

Selective repression of oral ectoderm genes ets4 and sip1. Expression of both genes was inhibited using two different MASOs for each gene, in at least three batches of embryos. A) ets4 MASO; B) sip1 MASO. Results are shown in ddCt (cf Fig.4.) as arithmetic mean ± standard deviation.

Figure 8

Spatial effects of ets4 and sip1 MASOs. A) ets4 MASO. Transcripts of foxg and gsc were detected at early blastula stage (13 h) in embryos bearing ets4 MASO, but not in control embryos. Expression of gsc is still restricted to the oral ectoderm in ets4 morphants, but expression of foxg covers the whole ectoderm. B) sip1 MASO. Expression of foxg in sip1 morphants covers the oral and aboral ectoderm at the mid-blastula stage (15 hpf), similar to (A). lv—lateral view; av—apical view. All embryos were shown with the oral ectoderm facing left.

Figure 9

Transcriptional control of stomodeal bra expression. A) Endodermal and stomodeal expression of bra during blastula stage. While endodermal bra can be seen at early blastula, stomodeal bra expression (marked by “*”) starts later and can be observed at 20h early mesenchyme blastula stage. B) Diagram of assay protocol testing for direct nodal targets by temporarily blocking Nodal signaling. Nodal signaling pathway inhibitor SB-431542 (SB) was added to cultures of sea urchin embryos at 24h, and gene expression was analyzed at 28h. C, QPCR assessment of effects of temporary SB treatment at indicated concentrations. This treatment significantly reduced the expression levels of nodal and not, but had small effects on bra levels. D, WMISH observations of effects of treatment with SB. Stomodeal bra expression is wiped out by 4h SB treatment, but endodermal bra expression is not affected; foxa stomodeal expression is diminished and endodermal expression is not affected. E) Effect of ets4 plus sip1 MASO on early bra expression. WMISH shows that ets4 and sip1 are required for proper timing expression of stomodeal bra expression; ets4/sip1 MASOs resulted in abnormal ectodermal bra expression during the early blastula stage. lv—lateral view; av— apical view. * marks stomodeal bra or foxa. All embryos in lateral or vegetal views were shown with the oral ectoderm facing left.

Figure 10

Transcriptional regulation of the emx gene. A) Spatial emx expression changes in response to nodal, not, or gsc MASOs. B) soxb1 input to ectodermal emx expression. Expression levels of emx were measured by QPCR relative to poly-ubiquitin. Results are shown in ddCt (cf Fig.4) as arithmetic mean ± standard deviation. vv: vegetal view. All embryos were shown with the oral ectoderm facing left.