3 - The Presence of ets2, etv4, and etv5 in lhx2+ Cells of the Distal Limb Provide a Mechanism for Fgf-regulation of lhx2

Poster Board Number: 3
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Co-authors:

Ethan Vyhmeister, Student Physician - Department of Pathology and Human Anatomy - Loma Linda University; Charmaine Pira - Department of Pathology and Human Anatomy - Loma Linda University; Kerby Oberg - Department of Pathology and Human Anatomy - Loma Linda University

Abstract Body : During limb development, fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) coordinate proximodistal outgrowth, while, sonic hedgehog (SHH) secreted from the zone of polarizing activity (ZPA) directs anteroposterior patterning and expansion. SHH and FGF both regulate each other’s expression in a positive feedback loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate in the Fgf-to-Shh loop and plays a role in maintaining FGF-regulation of Shh expression during limb development. We have identified two Lhx2-associated subAER cis-regulatory modules (LASARM1 and LASARM2) active within the Lhx2 expression domain. Binding sites for ETS transcription factor proteins are present in LASARM1/2 and are necessary for activity, suggesting that ETS transcription factors expressed in the subAER domain regulate Lhx2 expression. Therefore, we hypothesized that the ETS transcription factors that regulate Lhx2 expression through binding to LASARM1/2 would be co-expressed in Lhx2+ cells.

To determine which ETS transcription factors are co-expressed in Lhx2+ cells, we analyzed published mouse forelimb single-cell RNA sequencing data (scRNA-seq) and evaluated co-expression in subpopulations of cells using t-distributed stochastic neighbour embedding (t-SNE). Localization of co-expressed ETS transcription factors was further demonstrated by whole mount in situ hybridization (WMISH) in chicken limb buds.

Results of the scRNA-seq analysis revealed that Ets2, Etv4 and Etv5 are expressed in subpopulations of Lhx2+ cells and WMISH showed that the expression of these ETS transcripts overlaps LHX2 expression distally in the chicken limb bud. Investigation of the binding of ETS proteins to LASARM1/2 is in progress.

Our findings show that ETS2, ETV4 and ETV5 proteins are present in cells expressing Lhx­2+ and are thus capable of binding to ETS sites within LASARM1/2 to regulate activity. Moreover, this data provides a mechanism by which FGF, through ETS transcription factors, activates LASARM1/2 to regulate LHX2 expression.