A Cluster of Noninvoluting Endocytic Cells at the Margin of the Zebrafish Blastoderm Marks the Site of Embryonic Shield Formation

Abstract

In zebrafish embryos, the nascent embryonic shield first appears as a thickening in the germ ring of the mid-epiboly blastoderm. This site defines the dorsal side of the developing embryo. In this paper, we report that the site of embryonic axis formation is marked earlier at the late-blastula stage by the appearance of a cluster of cells with unique endocytic activities. This cluster of cells is composed of enveloping layer epithelial cells and one to two layers of underlying deep cells. Unlike other marginal blastomeres, cells in this cluster do not participate in involution as the blastoderm undergoes epiboly. These noninvoluting endocytic marginal (NEM) cells can be selectively labeled by applying membrane impermeant fluorescent probes to pre-epiboly and mid-epiboly embryos. During embryonic shield formation, deep cells in the NEM cell cluster rearrange and are displaced forward to the leading edge of the blastoderm. As deep NEM cells move into this location, they become a group of cells known as "forerunner cells." Between 60%- and 80%-epiboly, the forerunner cells coalesce into a coherent cell cluster that forms a wedge-shaped cap at the leading edge of the blastoderm. During embryonic axis formation, deep cells migrate and converge toward the embryonic midline, which is defined by the center of the forerunner cell cluster. At approximately 90% epiboly, the forerunner cell cluster becomes overlapped by the constricting germ ring. At tailbud stage, forerunner cells form the dorsal roof of Kupffer's vesicle, which is located ventral to the nascent chordoneural hinge. On the basis of previous grafting studies and known dorsal gene expression patterns, we discuss possible roles that the NEM/forerunner cell cluster may play in teleost axis formation.

Canopy1, a positive feedback regulator of FGF signaling, controls progenitor cell clustering during Kupffer's vesicle organogenesis

“…DFCs are progenitor cells of KV, which is a key organ required for LR patterning (9)(10)(11). At midgastrulation, a cluster of ∼20 DFCs appears adjacent to the embryonic shield (12,13). The DFC cluster then moves to the vegetal pole and forms a more compact and oval-shaped cluster by late gastrulation (7,11,14).…”

“…The DFC cluster then moves to the vegetal pole and forms a more compact and oval-shaped cluster by late gastrulation (7,11,14). At the end of gastrulation, DFCs differentiate into ciliated epithelial cells of the KV, which generates the nodal flow required for LR patterning (7,12,13). Recent studies have shown that FGF signaling is required for morphogenesis and ciliogenesis of the KV as well as for LR patterning (2,8,15).…”

Xenopus, an ideal model system to study vertebrate left‐right asymmetry

“…A second noteworthy feature is the great variability of dimensions of the respective structures. While the PNC in mouse measures 50 m from left to right, this amounts to about 100 m in rabbit, and approximately 200 m across the Xenopus GRP, and KVs in fish range between 50 -150 m (Cooper and D'Amico, 1996;Melby et al, 1996;Okada et al, 2005;Blum et al, 2007Blum et al, , 2009Schweickert et al, 2007). The large size of the GRP not only allows for easier imaging, but also simpler preparation and handling compared to the PNC in mouse and rabbit.…”

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

“…KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages 8,9 . During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo 10,11 .…”

“…Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf 8 . Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells 12 .…”