89 - Lmx1b-mediated Tcf4 Regulation in the Dorsalization of Limb Musculature

Poster Board Number: 89
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Abstract Body : LIM homeobox transcription factor 1-β (Lmx1b) dorsalizes the limb during development. In the normal limb, the dorsal muscle mass is less than the ventral. In the absence of Lmx1b, the dorsal limb muscle mirrors the ventral limb, increasing the overall dorsal muscle mass. Since asymmetrical development of dorsal and ventral musculature is imperative for proper balanced limb function, a better understanding of Lmx1b-mediated muscle patterning is needed. To elucidate the pathways targeted by Lmx1b during limb dorsalization, an Lmx1b ChIP-Seq analysis was performed on E12.5 mouse limbs that identified conserved non-coding regions associated with muscle development genes. Tcf4, also known as ITF2, is an E protein involved in the development of several organs including muscles. Tcf4 isomers have been shown to dimerize with MyoD1, a muscle-specific bHLH transcription factor, to activate or inhibit muscle differentiation. Four conserved potential cis-regulatory modules (pCRMs) were found upstream of the Tcf4 promoter. Thus, we hypothesized that Lmx1b regulates Tcf4 expression via these pCRMs to pattern dorsal muscles.

In silico genomic analysis was used to determine the regulatory potential of these pCRMs as well as their relationship to Tcf4. To characterize pCRM activity, pCRM-reporter constructs were transfected into developing chicken embryo forelimbs via targeted electroporation. The region of activity was compared to LMX1B and TCF4 expression using in-situ hybridization. We also performed qPCR on limbs of Lmx1b-knockout mice to further examine how Lmx1b may be related to muscle development through Tcf4 regulation.

Hi-C and chromatin marks suggest that all four pCRMs interact with the Tcf4 promoter region; however, pCRMs 3 and 4 appear to be more accessible with greater regulatory potential. In line with these predictions, pCRMs 1 and 2 exhibited weak activity without a dorsoventral bias, whereas pCRMs 3 and 4 had more robust activity that was accentuated in the dorsal limb mesoderm, overlapping LMX1B and TCF4 expression. Tcf4 expression was also significantly reduced in Lmx1b knockout mouse limbs compared to wildtype. Altogether, these data support a role for Lmx1b-mediated Tcf4 regulation to limit dorsal limb musculature during development.