Clonal analysis in mice underlines the importance of rhombomeric boundaries in cell movement restriction during hindbrain segmentation

Abstract

Background: Boundaries 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 findings: Sophisticated 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.

Conclusions: Our 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.

Figure 1. Clones respect the rhombomeric boundaries upon early labelling.

(A) Scheme depicting the embryonic stage upon TM administration (5.5 dpc), the embryonic stage of Cre-induction (6.0 dpc), and the stage when the observation was carried out and the hindbrain dissected (9.5 dpc). Note the dramatic change in embryo morphology between the time of TM administration and the time of embryo observation. Blue areas in 5.5–6.5 dpc depict the embryonic tissue. r1-r7, rhombomere 1–7. (B) Immunostaining to reveal GFP-positive clones in whole embryos (in toto) and in flat-mounted hindbrains.

Figure 2. Estimated and theoretical probabilities for a clone to cross the boundary as a function of its position along the AP axis.

(A) The scatter plot shows the size of the observed clones versus its centre position along the AP axis of the rhombomere. Radii have been measured as relative to the AP length of the rhombomere (see Materials and Methods). Colour of each clone has been chosen to reflect if it respects the boundary (red circles) or it crosses the boundary (green circles). (B) Estimated and theoretical probabilities for a clone to cross the boundary as a function of its position along the AP axis. Red circles show the horizontal position of the centre of observed clones along the AP axis of the rhombomere (it has been rescaled to [0,1]). Clones crossing the boundary have been placed in vertical position 1, clones respecting the boundary in vertical position 0. The red line is the probability of crossing the boundary as given by a generalized additive model fitted to data. Grey step lines show a discrete version of the estimated probability as given by the fraction of clones contained in each interval that cross the boundary. The black line is the probability of crossing the boundary as given by the theoretical model that assumes no boundary effect on the behaviour of clones.

Figure 3. Measure of the clone size and position.

Clone radius was measured as the half-AP length of the clone (c/2) relative to the rhombomere AP length (b), (radius = (c/2)/b. Position of the clone was established by measuring from the centre of the clone to its immediately anterior rhombomeric boundary (a). Position of the clone was then referred as a percentage of the length of the rhombomere at the level of the clone centre (b) (a/b x 100).