Clonal Analysis in Mice Underlines the Importance of Rhombomeric Boundaries in Cell Movement Restriction during Hindbrain Segmentation

Abstract

BackgroundBoundaries that prevent cell movement allow groups of cells to maintain their identity and follow independent developmental trajectories without the need for ongoing instructive signals from surrounding tissues. This is the case of vertebrate rhombomeric boundaries. Analysis in the developing chick hindbrain provided the first evidence that rhombomeres are units of cell lineage. The appearance of morphologically visible rhombomeres requires the segment restricted expression of a series of transcription factors, which position the boundaries and prefigure where morphological boundaries will be established. When the boundaries are established, when the cells are committed to a particular rhombomere and how they are organized within the hindbrain are important questions to our understanding of developmental regionalization.Methodology/Principal FindingsSophisticated experimental tools with high-resolution analysis have allowed us to explore cell lineage restriction within the hindbrain in mouse embryos. This novel strategy is based on knock-in alleles of ubiquitous expression and allows unrestricted clonal analysis of cell lineage from the two-cell stage to the adult mouse. Combining this analysis with statistical and mathematical tools we show that there is lineage compartmentalization along the anteroposterior axis from very early stages of mouse embryonic development.ConclusionsOur results show that the compartment border coincides with the morphological boundary in the mouse hindbrain. The restriction of the cells to cross rhombomeric boundaries seen in chick is also observed in mouse. We show that the rhombomeric boundaries themselves are involved in cell movement restriction, although an underlying pre-pattern during early embryonic development might influence the way that cell populations organize.

Yap/Taz-TEAD ACTIVITY LINKS MECHANICAL CUES TO CELL PROGENITOR BEHAVIOR DURING HINDBRAIN SEGMENTATION

“…This involves the segmentation of the tissue, which leads to the transitory formation of morphological buldges named rhombomeres (r1-r7). Each rhombomere constitute developmental units of gene expression and cell lineage compartments (Kiecker & Lumsden, 2005;Fraser et al, 1990;Jimenez-Guri et al, 2010).…”

A New Role of Hindbrain Boundaries as Pools of Neural Stem/Progenitor Cells Regulated by Sox2

“…1Aa,d; n = 10). At st. [16][17]Sox2 + cells are still present in rhombomeres, yet enhanced Sox2 expression can be detected in HBs ( Fig. 1Ab,e; n = 10).…”

“…Previous studies have shown that Sox2 is broadly expressed in the early chick hindbrain [52,71]. Here, we examined Sox2 expression at later stages (st. [15][16][17][18], when multiple other boundary markers are fully expressed [34,72]. Determination of the boundary regions was made based on the clear morphological bulges at these sites which contain an accumulation of cell bodies at the ventricular side compared to rhombomeres, as shown by DAPI nuclear staining, as well as by the specialized expression of different markers at these regions (Fig.…”

“…To quantify the changes in Sox2 expression from st. [15][16][17][18]Sox2 + cells were counted in eight comparable areas taken from boundaries of rhombomeres 3/4 and 4/5, or from rhombomeres 4,5 ( Fig. 1C; + cells within the hindbrain of the different stages was also measured using flow cytometry.…”

“…Individual rhombomeres are separated from their neighbors by well-defined boundaries. Contrary to rhombomeres, hindbrain boundaries (HBs) share the same molecular and cellular characteristics along the hindbrain, such as a unique fan-shaped morphology, enriched extracellular matrix (ECM), slow proliferation rate, and reduced interkinetic nuclear cell migration [5,7,[13][14][15][16][17][18][19]. Although HBs were identified decades ago, their role during hindbrain development remains largely unknown, especially in amniotes.…”