Single embryo-resolution quantitative analysis of reporters permits multiplex spatial cis-regulatory analysis.

Cis-regulatory modules (CRMs) control spatiotemporal gene expression patterns in embryos. While measurement of quantitative CRM activities has become efficient, measurement of spatial CRM activities still relies on slow, one-by-one methods. To overcome this bottleneck, we have developed a high-throughput method that can simultaneously measure quantitative and spatial CRM activities. The new method builds profiles of quantitative CRM activities measured at single-embryo resolution in many mosaic embryos and uses these profiles as a 'fingerprint' of spatial patterns. We show in sea urchin embryos that the new method, Multiplex and Mosaic Observation of Spatial Information encoded in Cis-regulatory modules (MMOSAIC), can efficiently predict spatial activities of new CRMs and can detect spatial responses of CRMs to gene perturbations. We anticipate that MMOSAIC will facilitate systems-wide functional analyses of CRMs in embryos.

GATA-6 gene enhancer contains nested regulatory modules for primary myocardium and the embedded nascent atrioventricular conduction system.

The cGATA-6 gene is flanked by an enhancer that selectively marks the atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information very early in the cardiogenic program and remains active in progressively more restricted regions of primary myocardium leading up to the emergence of a histologically distinct AVCS. We undertook to parse this enhancer to resolve how the respective AVCS-specific transcription program is regulated at the molecular level. We determined that this AVCS enhancer includes a 102 bp module that is sufficient to restrict expression to primary nonchamber myocardium. This offers a novel tool to analyze the early molecular delineation of primary and chamber myocardium, which subsequently give rise to components of the central and peripheral conduction system, respectively. Furthermore, we show that this 102 bp module in turn contains a nested 47 bp core module that has the potential to direct expression specifically to the AVCS domain of primary myocardium, albeit with low efficiency. Accordingly, we show that a GATA site and a GC-rich site in the 102 bp region bolster the activity of the nested 47 bp AVCS core region even within the context of the parental 1,478 bp enhancer. These are the first functional elements to be reported for a cardiac conduction system-specific control region.