Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/ Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1 + cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis.

How the positional values along the proximo-distal axis (stylopod-zeugopod-autopod) of the limb are specified is intensely debated. Early work suggested that cells intrinsically change their proximo-distal positional values by measuring time. Recently, however, it is suggested that instructive extrinsic signals from the trunk and apical ectodermal ridge specify the stylopod and zeugopod/autopod, respectively. Here, we show that the zeugopod and autopod are specified by an intrinsic timing mechanism. By grafting green fluorescent protein-expressing cells from early to late chick wing buds, we demonstrate that distal mesenchyme cells intrinsically time Hoxa13 expression, cell cycle parameters and the duration of the overlying apical ectodermal ridge. In addition, we reveal that cell affinities intrinsically change in the distal mesenchyme, which we suggest results in a gradient of positional values along the proximo-distal axis. We propose a complete model in which a switch from extrinsic signalling to intrinsic timing patterns the vertebrate limb.

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.