Notch signaling downstream of foxD5 promotes neural ectodermal transcription factors that inhibit neural differentiation

Abstract

We investigated the role of the Notch signaling pathway in regulating several transcription factors that stabilize a neural fate and expand the neural plate. Increased Notch signaling in a neural lineage via a constitutively activated form (NICD) up-regulated geminin and zic2 in a cell-autonomous manner, and expanded the neural plate domains of sox11, sox2, and sox3. Loss- and gain-of-function assays show that foxD5 acts upstream of notch1 gene expression. Decreasing Notch signaling with an anti-morphic form of a Notch ligand (X-Delta-1(STU)) showed that the foxD5-mediated expansion of the sox gene neural plate domains requires Notch signaling. However, geminin and zic2 appear to be dually regulated by foxD5 and Notch1 signaling. These studies demonstrate that: (1) Notch signaling acts downstream of foxD5 to promote the expression of a subset of neural ectodermal transcription factors; and (2) Notch signaling and the foxD5 transcriptional pathway together maintain the neural plate in an undifferentiated state. Developmental Dynamics 238:1358-1365, 2009. (c) 2009 Wiley-Liss, Inc.

Figure 1

Previous studies indicate that foxD5 acts upstream of the other neural ectodermal transcription factors that expand the neural plate. Some of these decrease neural differentiation genes (blue boxes) and some promote the onset of neural differentiation (green boxes). foxD5 directly activates the transcription of gem, sox11 and zic2, and these three genes coordinately regulate the remaining NE transcription factors. (Based on Yan et al., 2009)

Figure 2

foxD5 acts upstream of notch1. A. Percentage of the embryos with altered notch1 expression after injection of foxD5-MOs in a neural plate precursor, or injection of foxD5 mRNA in either a ventral epidermis precursor (V11) or a neural plate precursor (D11). Numbers in parentheses indicate sample sizes. B. Embryos at neural plate stages processed for in situ hybridization detection of notch1 mRNA. Local depletion of FoxD5 by foxD5-MOs causes a loss of notch1 expression (arrow). Locally increasing FoxD5 by mRNA injection in the ventral epidermis (red cells) does not induce ectopic notch1 expression (middle panel), but doing so in the neural plate (right panel) expands the notch1 expression domain (compare white bars on injected [right] versus uninjected [left] sides). The inset shows that the foxD5-expressing cells (red nuclei) do not express notch1 at levels greater than surrounding cells in the neural plate (np), nor induce it in the adjacent dorsal epidermis (ep).

Figure 3

Effects of increased Notch1 signaling on the NE genes. A. Percentage of embryos in which NICD, expressed in either the neural plate (D11) or ventral epidermis (V11) lineages, altered the expression of twelve NE genes. The table to the right reports the numbers of embryos examined for each experiment. B. Examples of the in situ hybridization assays for those NE genes altered by NICD expressed in the indicated lineages. geminin and zic2 are up-regulated in a cell autonomous manner (arrows); insets show that NICD-expressing cells (red nuclei) express elevated levels of the respective genes compared to adjacent cells. NICD causes the expansion of the neural plate domains of the three sox genes (compare white bars on injected [right] versus uninjected [left] sides). NICD induces the ectopic expression of geminin in the ventral epidermis (arrow), but does not induce any of the other NE genes in this tissue (red cells; see also 3A).

Figure 4

Decreased Notch signaling reduces the expression of the sox genes. A. Percentage of the embryos with altered NE gene expression after injection of X-Delta-1^STU mRNA in the D11 lineage. The numbers in parentheses indicate sample sizes. B. Examples of the in situ hybridization assays showing cell autonomous up-regulation of geminin and zic2 (arrows) in the neural plate, cell autonomous repression of sox11, sox2 and hes1 (arrows) and reduction in width of the sox2- and sox3-expression domains in the neural plate (white bars).

Figure 5

Decreased Notch signaling reverses foxD5-mediated effects on sox genes. A. Either foxD5 mRNA alone or foxD5 plus X-Delta-1^STU mRNAs were injected into the D11 lineage. The numbers in parentheses indicate sample sizes. X-Delta-1^STU interfered with foxD5-mediated expansion of the sox gene neural plate domains. The up-regulation of geminin and zic2 were not affected. B. Examples of the in situ hybridization assays showing reversal of foxD5 expansion of the sox3 domain and lack of reversal of the up-regulation of zic2 in the neural plate. zic2 expression is normally absent from the midline of the st14 neural plate (Brewster et al., 1998). When foxD5 is expressed in the midline by injecting mRNA into blastomere D11, zic2 is ectopically expressed (arrow). This ectopic expression is also observed when X-Delta-1^STU is co-expressed (arrow). Insets show the effect is cell autonomous; nearly all of the mRNA-expressing cells (red nuclei) show ectopic zic2 expression in both samples.

Figure 6

Notch signaling impacts the expression of NE transcription factors in two ways. 1. foxD5 directly up-regulates the expression of geminin (gem), sox11 and zic2 in both the neural plate and the ventral epidermis. Notch signaling, acting downstream of foxD5, co-regulates gem and zic2 expression levels, but only in the neural plate; it does not up-regulate sox11 expression levels. 2. Downstream of foxD5, Notch signaling is required for the expansion of the neural plate domains of the sox genes.