12 - Despite Apparent Overlapping Roles, the Expression and Regulation of lhx9 Differs from Its Paralog lhx2 in the Developing Chick Wing
Monday, March 25, 2024
10:15am – 12:15pm US EDT
Location: Sheraton Hall
Poster Board Number: 12
There are separate poster presentation times for odd and even posters.
Odd poster #s – first hour
Even poster #s – second hour
Co-authors:
Jean Kim - Loma Linda University; Ethan Vyhmeister - Loma Linda University; Jessica Britton - Loma Linda University; Charmaine Pira - Loma Linda University; Kerby Oberg - Loma Linda University
SURF Intern Loma Linda University Escondido, California, United States
Abstract Body :
The distal tip of the developing limb is covered by a rim of ectoderm called the apical ectodermal ridge (AER). Fibroblast growth factors (Fgfs) secreted from the AER control limb outgrowth and patterning. Lhx9, a LIM homeodomain transcription factor, is expressed in the distal mesoderm, along with its paralog Lhx2. In mice, Lhx9 and Lhx2 play seemingly redundant roles downstream of Fgf, with both expressed in the anterior and posterior sub-AER mesoderm of the early limb bud. In chickens, however, the expression of LHX9 is sharply restricted to the anterior domain suggesting differences in regulation despite similarities in the genetic landscape. We suspect that the differential expression is due to differences in the cis-regulatory modules (CRMs) regulating LHX9’s temporospatial expression. Work in our lab suggests that Fgf uses ETS transcription factors to regulate LHX2 expression in chicken limb buds. Our lab has also identified a chicken CRM (labeled -11) associated with LHX9 that has activity mirroring the differential expression of LHX9 in the limb. In silico analysis of CRM(-11) identified a conserved, 10bp ETS binding site. Thus, we hypothesized that Fgf mediates CRM(-11)’s activity via ETS transcription factors.
To map the differential expression of LHX9 and LHX2 throughout limb development we used in situ hybridization. We also performed site-directed mutagenesis of the ETS binding site on a CRM(-11)-tk-GFP reporter construct. The normal and mutated CRM(-11) constructs were transfected into distal chick limb mesoderm by electroporation at a stage with robust LHX9 expression.
LHX9 expression was restricted to the anterior sub-AER mesoderm throughout limb development (HH17-HH33), unlike LHX2. Activity (fluorescence) was determined 24 hours post- transfection. Interestingly, mutation of the ETS binding site within CRM(-11) did not affect activity suggesting that Fgf does not regulate LHX9 through ETS transcription factors.
These data indicate that the expression pattern and regulation of LHX9 is different from LHX2 in chickens, despite apparent redundant roles in mice.