Cell-specific alterations inPitx1regulatory landscape activation caused by the loss of a single enhancer

Abstract

Most developmental genes rely on multiple transcriptional enhancers for their accurate expression during embryogenesis. Because enhancers may have partially redundant activities, the loss of one of them often leads to a partial loss of gene expression and concurrent moderate phenotypic outcome, if any. While such a phenomenon has been observed in many instances, the nature of the underlying mechanisms remains elusive. We used the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion in vivo of one of its enhancers (Pen), which normally accounts for 30 percent of Pitx1 expression in hindlimb buds. By combining single cell transcriptomics and a novel in embryo cell tracing approach, we observed that this global decrease in Pitx1 expression results from both an increase in the number of non- or low-expressing cells, and a decrease in the number of high-expressing cells. We found that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. The resulting increase in Pitx1 non/low-expressing cells eventually affects the proximal limb more severely than the distal limb, leading to a clubfoot phenotype likely produced through a localized heterochrony and concurrent loss of irregular connective tissue. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.

baredSC: Bayesian Approach to Retrieve Expression Distribution of Single-Cell

“…Indeed, in the FL Pitx1 +/+ sample in black, the fluorescence level has a relatively wide scale. Rouco et al [2021] show that the expression of Pitx1 in the HL wild-type (red curve) is trimodal (Fig. 6A and Fig.…”

“…6A and Fig. 3A of Rouco et al [2021]). The first mode is included in the range of the FL Pitx1 +/+ so it corresponds to cells which do not express Pitx1.…”

“…6C or Fig. 4A of Rouco et al [2021]), one cannot distinguish the three modes in the HL Pitx1 +/+ . However, classical density plots are very sensitive to sampling noise, and do not allow a precise determination of the expression distribution.…”

“…This gene is not expressed in the forelimb and a gain of expression in this domain induces an arm-to-leg phenotype [DeLaurier et al, 2006;Kragesteen et al, 2018]. Its expression is controlled by many enhancers, one of them is Pen which accounts for 30-50% of the expression [Kragesteen et al, 2018;Rouco et al, 2021]. Rouco et al [2021] used scRNA-seq and flow cytometry to investigate whether Pitx1 was homogeneously expressed across hindlimb cells.…”

“…Its expression is controlled by many enhancers, one of them is Pen which accounts for 30-50% of the expression [Kragesteen et al, 2018;Rouco et al, 2021]. Rouco et al [2021] used scRNA-seq and flow cytometry to investigate whether Pitx1 was homogeneously expressed across hindlimb cells. They also studied the changes induced by the deletion of the Pen enhancer.…”