Lmx1b Transcriptional Regulation in Limb Development

Abstract Body : INTRODUCCTION AND OBJECTIVE
Dorso-ventral (DV) patterning depends on Lmx1b, a transcription factor expressed in the dorsal limb mesoderm. Lmx1b is considered the dorsal determinant as it is both necessary and sufficient to specify dorsal fates. However, little is known about Lmx1b regulation and function.
Recently, we reported that Lmx1b expression in the limb depends on two enhancers termed LARM1 and LARM2. Their removal recapitulates the Lmx1b-null phenotype exclusively in the limbs, consistent in double ventral limbs with no detectable Lmx1b expression. Remarkably, deletion of each enhancer individually results in the double ventral phenotype restricted to the posterior (ΔLARM1) or to the anterior half (ΔLARM2) of the limb unveiling anterior-posterior (AP) spatial modularity.
Here we aim for a comprehensive and systematic investigation of the Lmx1b transcriptional apparatus, to understand the mechanisms underlying Lmx1b’s control of dorsalization.
MATERIAL AND METHODS
We follow a multidisciplinary strategy that integrates genomic analyses, mouse genetics, and in vivo functional examination of regulatory elements.
RESULTS
To dissect the regulatory logic of the Lmx1b locus, we first explored its genomic landscape in detail. The Lmx1b gene is surrounded by several non-coding transcripts, two of them, the C130021I20Rik and the AK020435, classified as lncRNAs. C130021I20Rik faithfully recapitulates the Lmx1b expression pattern while AK020435 shows distal restriction and a ventral domain. Interestingly, both are regulated by the LARMs, including the AK020435 ventral domain. This demonstrates their integration in the Lmx1b regulatory landscape and reveal a previously unnoticed ventral activity of the LARMs.
We were also interested in identifying the LARM trans-acting regulatory factors. By combining literature-based evidence and ChIP-seq datasets we found βcatenin, Gli3 and Hox13 paralogs as plausible candidates for further studies. Considering the AP specificity of LARMs, we started by exploring the role of Gli3 in dorsalization. Indeed, the link between Lmx1b and AP patterning has been noticed both in humans and mice. The major regulator of limb AP patterning is Shh that, emanating from the zone of polarizing activity in the posterior limb bud, forms a gradient to polarize the limb by suppressing Gli3 processing to its repressor form Gli3R. Thus, it is possible that the Shh pathway differentially regulates each enhancer, positively LARM2 and negatively LARM1. This is supported by the phenotype of Gli3-/-;ΔLARM double mutants. In the absence of LARM1, the removal of Gli3 results in an extension of the double-ventral phenotype all along the AP axis, indicating lack of LARM2 activity. Likewise, Gli3-/-;ΔLARM2 mutants show rescue of Lmx1b expression indicating anterior LARM1 activity. This reflects the influence of AP identity factors in differential enhancer usage.
CONCLUSION and SIGNIFICANCE
We demonstrate that Lmx1b expression in the limb is subject to a complex and modular transcriptional regulation connected with AP patterning. Further work focus in the analysis of the differential enhancer usage and its functional relevance during limb development.
FUNDING SOURCES
Spanish Ministry of Science and Innovation (MCIN) Grant PID-2020-114525-GB-I-00 to MR. PhD fellowship of the University of Cantabria to SZ and PhD fellowship of MICN (FSE/PRE2018-083421) to ACI.