Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Abstract

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The intrinsic and extrinsic effects of TET proteins during gastrulation

“…After whole-mount immunofluorescence, embryos were unfolded with forceps from the lateral side, placed onto a pre-treated glass slide with their ventral side (node/notochord) facing up, mounted with a drop of mounting medium (Thermo Scientific Shandon, 9990402), then sealed with a coverslip ( Xiao et al., 2018 ). Z-stack images were acquired with a Zeiss LSM 880 inverted confocal microscope (Carl Zeiss), using a Plan-Apochromat 10X/0.45 objective for node/notochord 5hmC imaging for embryo E4 (embryo from Figure 6 G), a Plan-Apochromat 20X/0.8 objective for embryos in Figure 6 B and embryo E1, E2, E3, and E5 in Figure 6 G. Z-stacks with a thickness that ranged approximately 15-22 μm made up of 16-25 images were collected.…”

Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

“…Whole‐mount immunofluorescence staining of intact mouse embryos was performed as previously described (Xiao et al , 2018). The embryos were then mounted in 1% low‐melting agarose (Lonza; Basel, Switzerland) on a glass bottom dish (Thermo Fisher Scientific), covered in VectaShield mounting medium (Linaris; Dossenheim, Germany) and kept cold and protected from light until imaging.…”

The Organizer and Its Signaling in Embryonic Development

“…A streak is located on the posterior side of the mouse embryo, wherein the AVE is located at the anterior side. The node located on the ventral surface (during embryonic day 7 (E7)) ( Figure 1 ) [ 30 , 31 ]. This species-specific organizer (s) secrete molecules are most common across the vertebrates, including BMP, Wnt, and Nodal inhibitors [ 6 ], and forms prechordal plate and notochord [ 12 , 32 ].…”