Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown

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 lockdown the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.

Fig 1

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)).

Fig 2

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.

Fig. 3

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.

Fig 4

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.

Fig 5

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.

Fig 6

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.

Fig 7

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.