Identification of transcripts potentially involved in neural tube closure using RNA sequencing

Abstract

Anencephaly is a fatal human neural tube defect (NTD) in which the anterior neural tube remains open. Zebrafish embryos with reduced Nodal signaling display an open anterior neural tube phenotype that is analogous to anencephaly. Previous work from our laboratory suggests that Nodal signaling acts through induction of the head mesendoderm and mesoderm. Head mesendoderm/mesoderm then, through an unknown mechanism, promotes formation of the polarized neuroepithelium that is capable of undergoing the movements required for closure. We compared the transcriptome of embryos treated with a Nodal signaling inhibitor at sphere stage, which causes NTDs, to embryos treated at 30% epiboly, which does not cause NTDs. This screen identified over 3,000 transcripts with potential roles in anterior neurulation. Expression of several genes encoding components of tight and adherens junctions was significantly reduced, supporting the model that Nodal signaling regulates formation of the neuroepithelium. mRNAs involved in Wnt, FGF, and BMP signaling were also differentially expressed, suggesting these pathways might regulate anterior neurulation. In support of this, we found that pharmacological inhibition of FGF-receptor function causes an open anterior NTD as well as loss of mesodermal derivatives. This suggests that Nodal and FGF signaling both promote anterior neurulation through induction of head mesoderm.

Keywords: RNA sequencing; neural tube defects; neurulation; nodal; zebrafish.

Figure 1:. ONT and CNT cause open and closed neural tube phenotypes, respectively.

Treatment with SB505124 at sphere stage always results in an open neural tube while treatment at 30% epiboly stage always results in a closed neural tube. Top row: Lateral views, animal pole to top. Middle row: Lateral views, anterior towards top. Bottom row: Dorsal view, anterior towards top.

Figure 2:. Differential expression of transcripts encoding components of the BMP and Nodal signaling pathways.

A) Danio rerio Nodal/Activin and BMP signaling pathways. Blue stars indicate the corresponding gene had higher expression in CNT embryos. Yellow stars indicate the corresponding gene had lower expression in CNT embryos. B) Table of differentially expressed genes in these pathways and the corresponding expression ratios from the RNA-sequencing data analysis.

Figure 3:. Differential expression of transcripts encoding portions of the Wnt signaling pathway.

A) Danio rerio Wnt signaling pathway. Colored stars used as in Figure 2. B) Table of differentially expressed genes in the Wnt pathway and corresponding expression ratios from the RNA-sequencing data analysis.

Figure 4:. Differential expression of transcripts encoding portions of the FGF signaling pathway.

A) Danio rerio FGF signaling pathway. Colored stars used as in Figure 2. B) Table of differentially expressed genes represented in the FGF pathway and corresponding expression ratios from the RNA-sequencing data analysis.

Figure 5.. FGF signaling at or before the onset of gastrulation is required for anterior neural tube closure.

Embryos were treated with SU5402 starting at time points indicated and then analyzed for anterior neural tube phenotype by pineal morphology. First column: lateral views of whole embryos with anterior to the left and dorsal to the top. Scale bar: 250 μm. Second-fourth columns: dorsal view with anterior to the top. Scale bar: 50 μm. Refer to Table 5 for quantitative data.

Figure 6:. Expression of the oep gene is decreased in embryos with deficient FGF signaling.

Embryos were treated with SU5402 starting at dome stage and then fixed at 75% epiboly and assayed for expression of the Nodal receptor complex gene oep and the Nodal ligand gene ndr2. oep expression has a dose dependent decrease in the midline tissue (white arrows) and prechordal plate/anterior neuroectoderm (black arrowheads). In contrast, expression levels of ndr2 remain high, although the shape of the midline tissue is disrupted at the highest concentration. Scale bar: 100 μM.

Figure 7.. Correlation between notochord presence and neural tube closure in FGF signaling deficient embryos.

Embryos were assayed with the notochord marker col2a1 to determine the amount of notochord (nc) present and the pineal marker otx5 to report the neural tube phenotype. Representative images of embryos treated at 30% epiboly with DMSO or the FGFR inhibitor SU5402. Pineal phenotype is indicated but not shown. Dorsal views of deyolked embryo trunks with anterior to the left. Scale bar: 150 μm. Refer to Table 6 for quantitative data.

Figure 8:. Comparison of embryos treated with a variety of FGFR inhibitors reveals a common phenotype of loss of posterior structures.

WT embryos were treated with the indicated FGFR inhibitor or vehicle alone (DMSO) starting at dome stage and continuing until fixation at ~30 hpf. ndr1;ndr2 mutants were produced by natural spawnings. Embryos treated with PD161570 and PD173074 treatment have phenotypes that similar to those caused by SU5402, including loss of hindbrain rhombomerers (r’s, black arrows) that increases in severity with increasing phenotypic class & inhibitor concentration. In contrast, ndr1;ndr2 mutants have no evidence of loss of hindbrain tissues even though they have a similar shortening of the anterior-posterior axis as the FGFR inhibitor treated embryos and more disrupted morphology (compare region of the midbrain/hindbrain boundary (MHB)). Pineal precursors are indicated by white arrowheads. First column, lateral views. Second-fourth columns, dorsal views. Scale bars: First column 0.5 mm, remaining columns 0.1 mm. Refer to Table 8 and Supplemental Table 2 for quantitative data.

Figure 9:. Differential expression of transcripts encoding portions of Adherens Junctions.

A) Schematic of an adherens junction. Colored stars used as in Figure 2. B) Table of differentially expressed adherens junction genes and corresponding expression ratios from the RNA-sequencing data analysis.

Figure 10:. Differential expression of transcripts encoding portions of the Tight Junction pathway.

A) Schematic of a tight junction. Colored stars used as in Figure 2. B) Table of differentially expressed tight junction genes and corresponding expression ratios from the RNA-sequencing data analysis.