Notch signalling regulates the contribution of progenitor cells from the chick Hensen's node to the floor plate and notochord

Abstract

Hensen's node of the chick embryo contains multipotent self-renewing progenitor cells that can contribute to either the floor plate or the notochord. Floor plate cells are a population of epithelial cells that lie at the ventral midline of the developing neural tube, whereas the notochord is a rod of axial mesoderm that lies directly beneath the floor plate. These two tissues serve as a source of a potent signalling morphogen, sonic hedgehog (Shh), which patterns the dorsoventral axis of the neural tube. We show, through both gain- and loss-of-function approaches, that Notch signalling promotes the contribution of chick axial progenitor cells to the floor plate and inhibits contribution to the notochord. Thus, we propose that Notch regulates the allocation of appropriate numbers of progenitor cells from Hensen's node of the chick embryo to the notochord and the floor plate.

Fig. 1. Notch inhibition prevents progenitor cells from populating the chick floor plate

(A) Schematic of the assay: GFP-expressing donor cultured HH1-4 in DMSO or DAPT. The medial sector of donor Hensen’s node is grafted to a homochronic site in a non-GFP HH4 non-treated host and cultured overnight. (B) Boxplot showing that Notch inhibition significantly reduces the contribution of progenitors to the floor plate (FP) along the embryonic axis. (C,I) Host embryos after culture. (D-H) Transverse sections of C showing GFP cells contributing to the FP for most of the axis and notochord over the entire axis. (D′-H′) Same sections as D-H showing Foxa2. (D″-H″) Overlay of D-H with D′-H′. (J-N) Sections of I showing GFP cells contributing to the notochord over the entire axis; FP contribution is absent throughout the majority of the axis with FP contribution limited to the most caudal end of the embryo. (J′-N′) Same sections as J-N showing Foxa2. (J″-N″) Overlay of J-N with J′-N′.

Fig. 2. Notch inhibition throughout the host prevents progenitor cells from populating the floor plate

(A) Schematic of the assay: same as Fig. 1 except that the host was cultured overnight in DMSO or DAPT. (B) Boxplot showing that Notch inhibition significantly reduces the contribution of progenitor cells to FP. (C,H) Host embryos after culture. The DAPT-treated host (H) displays severe somite defects. (D-G) Transverse sections of C showing GFP cells contributing to the FP and notochord. (D′-G′) Same as D-G showing Foxa2. (D″-G″) Overlay of D-G with D′-G′. (I-L) Sections of H showing GFP cells contributing to the notochord over the entire axis; FP contribution is absent throughout the majority of the axis with FP contribution limited to the most caudal end of the embryo. (I′-L′) Same as I-L showing Foxa2. (I″-L″) Overlay of I-L with I′-L′.

Fig. 3. Notch inhibition does not prevent progenitor cells from becoming notochord

(A) Schematic of the assay: same as Fig. 1 except the graft made at the boundary between the area opaca and area pelucida at the level of Hensen’s node in a non-GFP-expressing HH3⁺ host and cultured overnight. Serial sections were analyzed using Foxa2 or 3B9 antibodies. (B-G) Transverse sections of the control. (B′-G′) Same as in B-G showing Foxa2 or 3B9 expression. (H-M) Sections of DAPT-treated sample. (H′-K′) Same as in H-K showing lack of Foxa2 and presence of 3B9. (L′,M′) At the caudal end of these explants some GFP cells are Foxa2 positive.

Fig. 4. Notch activation in Hensen’s node progenitors promotes the contribution of these cells to the floor plate and inhibits them from populating the notochord

(A) Schematic of the assay. (B-E′) Serial transverse sections of embryo electroporated with pCIG-GFP showing contribution of electroporated cells to FP and the notochord. (F-I′) Sections of embryo electroporated with pCIG-dnRBPjκ showing a minor contribution to the FP and predominant contribution to notochord. (J-M′) Sections of embryo electroporated with pCIG-NICD showing exclusive contribution to the FP.

Fig. 5. Following Notch inhibition in a whole chick embryo, a larger notochord develops

(A-B′) Foxa2 in embryos cultured overnight in DAPT or DMSO and their corresponding sections. Foxa2 in the floor plate (A,A′) is not affected in a DAPT-treated embryo (B,B′). DAPT-treated embryos have a larger notochord.