The epiblast is the mammalian embryonic tissue that contains the pluripotent stem cells that generate the whole embryo. We have established a method for inducing functional genetic mosaics in the mouse. Using this system, here we show that induction of a mosaic imbalance of Myc expression in the epiblast provokes the expansion of cells with higher Myc levels through the apoptotic elimination of cells with lower levels, without disrupting development. In contrast, homogeneous shifts in Myc levels did not affect epiblast cell viability, indicating that the observed competition results from comparison of relative Myc levels between epiblast cells. During normal development we found that Myc levels are intrinsically heterogeneous among epiblast cells, and that endogenous cell competition refines the epiblast cell population through the elimination of cells with low relative Myc levels. These results show that natural cell competition in the early mammalian embryo contributes to the selection of the epiblast cell pool.

HP1 (Heterochromatin protein 1) is a conserved, non-histone chromosomal protein that is best known for its preferential binding to pericentric heterochromatin and its role in position effect variegation in Drosophila. Using immunolocalization, we show that HP1 is a constant feature of the telomeres of interphase polytene and mitotic chromosomes. This localization does not require the presence of telomeric retrotransposons, since HP1 is also detected at the ends of terminally deleted chromosomes that lack these elements. Importantly, larvae expressing reduced or mutant versions of HP1 exhibit aberrant chromosome associations and multiple telomeric fusions in neuroblast cells, imaginal disks, and male meiotic cells. Taken together, these results provide evidence that HP1 plays a functional role in mediating normal telomere behavior in Drosophila.

Developing vertebrate limbs initiate proximo-distal patterning by interpreting opposing gradients of diffusible signaling molecules. We report two thresholds of proximo-distal signals in the limb bud: a higher threshold that establishes the upper-arm to forearm transition; and a lower one that positions a later transition from forearm to hand. For this last transition to happen, however, the signal environment seems to be insufficient, and we show that a timing mechanism dependent on histone acetylation status is also necessary. Therefore, as a consequence of the time dependence, the lower signaling threshold remains cryptic until the timing mechanism reveals it. We propose that this timing mechanism prevents the distal transition from happening too early, so that the prospective forearm has enough time to expand and form a properly sized segment. Importantly, the gene expression changes provoked by the first transition further regulate proximo-distal signal distribution, thereby coordinating the positioning of the two thresholds, which ensures robustness. This model is compatible with the most recent genetic analyses and underscores the importance of growth during the time-dependent patterning phase, providing a new mechanistic framework for understanding congenital limb defects.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

Supplemental Digital Content is available in the text.