CR405884, a hypothetical uncharacterized coding sequence, exhibits a peculiar expression pattern and FGF responsiveness during limb development (541.4)

Abstract

CR405884, a hypothetical uncharacterized coding sequence, exhibits a peculiar expression pattern and FGF responsiveness during limb development.Gabrielle N Lujan, Christopher Armstrong, Charmaine U. Pira, Kerby C. Oberg: Division of Human Anatomy, Loma Linda University, Loma Linda, CA During limb development, the apical ectodermal ridge (AER) secretes fibroblast growth factors (FGFs) which maintain limb outgrowth, in part, by regulating Sonic Hedgehog (SHH) expression. SHH from the zone of polarizing activity (ZPA) in the distal posterior aspect of the limb bud is responsible for posterior limb growth and patterning. The molecules and pathways used by FGF to regulate SHH expression are not known. Application of ectopic FGF in the posterior limb mesoderm can induce SHH expression. Using this capacity, we performed microarray analysis on the tissue surrounding an FGF soaked bead, 24 hrs after application. Interestingly, CR405884, an uncharacterized hypothetical coding sequence, exhibited an 11‐fold increased expression in response to FGF and thus, was analyzed further.We isolated CR405884 from chick cDNA by PCR and generated mRNA probes for whole mount in situ hybridization. We then determined the expression profile of CR405884 during limb development in stage 17‐27 embryos. To verify the microarray data, we implanted FGF‐soaked beads into stage 23 embryo limb buds, harvested the tissue after 24 hours, and evaluated CR405884 expression.Expression of CR405884 was first evident in stage 21 limb buds as a focal, 100 um diameter region just anterior to the SHH expression domain. Over the next several stages examined, the expression expanded anteriorly and distally. Furthermore, CR405884 expression increased in response to FGF.The focal expression of CR405884 anterior to and coincident with the expression of SHH may indicate a role for CR405884 in localizing SHH’s expression domain. This concept is further supported by its responsiveness to FGF, a known regulator of SHH expression.