Temporal and spatial requirements for Nodal‐induced anterior mesendoderm and mesoderm in anterior neurulation

Abstract

Zebrafish with defective Nodal signaling have a phenotype analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory found that anterior open neural tube defects in Nodal signaling mutants were caused by defects in mesendodermal/mesodermal tissue. Defects in these mutants are already apparent at neural plate stage, before the neuroepithelium starts to fold into a tube. Consistent with this, we found that the requirement for Nodal signaling maps to mid-late blastula stages. This timing correlates with the timing of prechordal plate mesendoderm and anterior mesoderm induction, suggesting these tissues act to promote neurulation. To further identify tissues important for neurulation, we took advantage of the variable phenotypes in Nodal signaling-deficient sqt mutant and Lefty1-overexpressing embryos. Statistical analysis indicated a strong, positive correlation between a closed neural tube and presence of several mesendoderm/mesoderm-derived tissues (hatching glands, cephalic paraxial mesoderm, notochord, and head muscles). However, the neural tube was closed in a subset of embryos that lacked any one of these tissues. This suggests that several types of Nodal-induced mesendodermal/mesodermal precursors are competent to promote neurulation.

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

“…SB505124 blocks function of Nodal and Activin receptors, the Activin receptor-Like Kinases (ALKs) 4, 5, and 7, by binding to their ATP binding site (DaCosta Byfield et al, 2004; Hagos et al, 2007) at different stages of development. Our previous studies found that embryos treated with SB505124 starting at developmental stages approximately 40 minutes apart resulted in different neural tube phenotypes (Gonsar et al, 2016). Treatment with SB505124 beginning at sphere stage (mid-blastula, 4.0 hpf) resulted in an open neural tube, as indicated by an elongated or divided pineal anlage (Figure 1).…”

“…For instance, embryos treated at the earlier stage had a greater loss of mesodermal tissue, as indicated by the shorter tail and fewer number of somites (Figure 1). Our previous studies found that embryos with a greater loss of mesendoderm/mesoderm had an increased chance of having an open anterior neural tube (Gonsar et al, 2016). In case changes in gene expression within mesendoderm/mesoderm or other tissues are important for anterior neurulation, our RNAseq experiments used whole embryos.…”

“…Several genes encoding FGF ligands are expressed in the anterior mesendoderm/mesoderm during the period when anterior neurulation is occurring, including fgf8a and fgf17 , which were differentially expressed in our screen. fgf8a is expressed in the cephalic paraxial mesoderm, a tissue involved in promoting anterior neural tube closure, during gastrulation (Gonsar et al, 2016; Thisse and Thisse, 2004). fgf17 is expressed in the presumptive dorsal mesoderm at sphere stage (Reifers et al, 1998; Shimizu et al, 2006; Thisse and Thisse, 2004; Warga et al, 2013).…”

“…Using a similar approach, the requirement for Nodal signaling in anterior neurulation was mapped to up to late blastula stages (4.3 hpf) (Gonsar et al, 2016). In this study, inhibition of FGFR activity starting before the onset of gastrulation (6.0 hpf) caused anterior NTD.…”

“…For instance, in zebrafish, the genes fgf3 , fgf8 , and fgf17b can be induced by ectopic activation of Nodal signaling and expression is lost in the anterior regions of embryos that lack Nodal signaling, suggesting FGF signaling is in part downstream of Nodal (Cao et al, 2004; Mathieu et al, 2004). Our previous studies suggested that both mesoderm and mesendoderm were involved in anterior neurulation (Gonsar et al, 2016). However, loss of FGF signaling affects only mesoderm, suggesting that a severe loss of mesoderm is sufficient to cause an open neural tube.…”