Abstract
The limb is widely used as a model developmental system and changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limb malformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouse E11.5 limbs that fluorescently mark the ZPA or AER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AER-expressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.
Highlights
The tetrapod limb has long been used as a model of embryonic development in three dimensions
Embryos were harvested at E11.5, and zone of polarizing activity (ZPA) or apical ectodermal ridge (AER) cells were isolated from dissected limbs by fluorescenceactivated cell sorting (FACS)
The third AER enhancer, AER4460, showed weak posterior AER staining and is located in the first intron of the protein S gene (Pros1), 15 kb upstream of ADP-ribosylation factor-like 13b (Arl13b), which is involved in the localization of Shh receptor pathway components in the cilium (Larkins et al, 2011). We believe that these datasets will be valuable to researchers studying evolutionary changes in limb morphology and human congenital limb malformations
Summary
The tetrapod limb has long been used as a model of embryonic development in three dimensions. The limb bud begins as a small protrusion of cells from the flank of the embryo. As it develops, the limb bud must establish polarity along three axes: anterior-posterior (AP), proximal-distal (PD) and dorsal-ventral (DV). The AP axis is controlled by a small region in the posterior limb bud called the zone of polarizing activity (ZPA). These cells express sonic hedgehog (Shh), which acts as a morphogen to determine the AP axis. The PD axis is maintained by the apical ectodermal ridge (AER), which develops from the ectoderm at the distal edge of the limb bud. There is significant crosstalk between the ZPA and AER, with each
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