Identification of a proteoglycan‐associated regulatory region active during central nervous system development

Lmx1b is a homeodomain transcription factor necessary for limb, kidney, eye and central nervous system (CNS) development. Lmx1b KO mice show reduced cerebellum and tectum formation, loss of dopaminergic/serotonergic neurons, ocular malformations, and distal ventral‐ventral limb symmetry. Direct downstream targets of Lmx1b in the brain and limb remain unknown. Recent microarray studies have implicated the proteoglycans Keratocan, Lumican, and Decorin as possible downstream targets of Lmx1b. Interestingly, these proteoglycans cluster to a locus in the genome (KLD). Using in silico analysis, we identified a conserved non‐coding region with a known Lmx1b binding site (CNR Peak 3). We hypothesize that Lmx1b directly regulates KLD via CNR Peak 3. We isolated CNR Peak 3 and generated a green fluorescent protein (GFP) reporter construct. This construct was electroporated into the chick embryo at Hamburger‐Hamilton (HH) 4 and HH10. GFP was visualized under fluorescent microscopy. GFP expression was detected in the chick HH10 neural tube and HH19 surrounding the developing midbrain and cerebellum. At HH19, Lmx1b and KLD mRNA expression overlap GFP expression. The data suggests CNR Peak 3 plays a role in the regulation of KLD during CNS development. We suspect that Lmx1b mediates KLD regulation through the Lmx1b binding site in CNR Peak 3. Further studies are underway investigating Peak 3 CNR in other Lmx1b patterned tissues. Grant Funding Source : National Institutes of Child Health and Human Development