Program/Abstract # 102 Evolution of Hox PG2 gene content in teleosts Adam Davis, Jean-Luc Scemama, Edmund J. Stellwag Department of Biology, East Carolina University, Greenville, NC, USA Based on the results from large-scale genomic sequencing, it was presumed that the Hox paralog group 2 (PG2) gene composition of the Japanese medaka (Oryzias latipes) consisted of three functional genes, hoxa2a, a2b and b2a (Kurosawa et al., 2006). We have cloned and sequenced the cDNAs corresponding to the three medaka Hox PG2 genes and showed that they were identical to the respective medaka genomic sequences. Sequence alignment of the hoxa2b cDNA insert with the annotated genomic sequence of medaka hoxa2b revealed that the exon splice junction reported in Genbank was misassigned. In silico translation of the correctly annotated genomic sequence revealed the existence of premature termination codons in the region corresponding to exon 2, which provided evidence that the medaka hoxa2b was a transcribed pseudogene. Results from whole-mount in situ hybridization revealed that Yhoxa2b was robustly expressed in the embryonic trunk, neural tube and pectoral fin buds, which are atypical domains for Hox PG2 expression, while exceedingly weak expression was observed in the canonical hindbrain and pharyngeal arch domains. Our identification of translational stop codons in the region encoding hoxa2b and the unusual embryonic expression pattern of the mRNA derived from the transcription of this region provided independent evidence for a Yhoxa2b gene in the medaka and suggested that the divergent expression of Yhoxa2b may be due to elimination or relaxation of selection among the Hox PG2 cis- regulatory elements required for expression within canonical domains. We are currently conducting functional genetic studies to understand the role of Yhoxa2b in embryonic development of medaka. doi:10.1016/j.ydbio.2008.05.113 Program/Abstract # 103 Evolutionary developmental biology of teleostean pharyngeal arch specification Pierre Le Pabic, Edmund J. Stellwag, Jean-Luc Scemama Department of Biology, East Carolina University, Greenville, NC, USA Our research involves functional genetic analyses of tilapia Hox PG2 genes. Independent microinjection of hoxa2a, hoxa2b or hoxb2a- directed antisense morpholino's (MO's) into tilapia zygotes resulted in phenotypes affecting all the pharyngeal arches; a striking difference from zebrafish in which 2nd to 1st arch homeosis was observed only in dual knock-downs of hoxa2b and b2a. Tilapia hoxa2a knock-down resulted in PA2 to PA1 homeosis and an anteriorizing homeotic shift of PA6 and 7 to PA5 and 6, respectively, which showed that, unlike zebrafish, tilapia hoxa2a acted alone as a selector gene of PA2 identity and had posterior pharyngeal arch-specifying activity. Knock-down of tilapia hoxa2b or hoxb2a resulted in additional phenotypes char- acterized by a misshapen Meckel's cartilage (PA1), the absence of a ceratohyal in PA2 and homeotic transformation of PA5 and 6 to PA4 and 5 identities, respectively. In situ hybridization of hoxa2a, a2b or b2a MO-induced morphant embryos with probes directed toward these three genes showed complex relationships. For example, hoxa2a morphants showed diminished expression of hoxa2a throughout the arches, undetectable expression of hoxa2b and b2a in the 2nd arch, but increased expression of hoxa2b and b2a in the posterior arches. By comparison, hoxa2b morphants showed dramatically increased hoxa2b expression over its expression domain, but unchanged hoxa2a or b2a expression. Our results are indicative of auto- and cross-regulatory activities among the tilapia genes within Hox PG2 and point to the extensive evolutionary divergence in Hox PG2 gene function between tilapia and zebrafish. doi:10.1016/j.ydbio.2008.05.114 Program/Abstract # 104 Investigating a role for trpm7 function in physiologic cation homeostasis Michael R. Elizondo a,b , David M. Parichy a a Department of Biology, University of Washington, Seattle, WA 98195, USA b Graduate Program in Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA TRPM7 is a divalent cation channel with a kinase domain that has roles in magnesium homeostasis, calcium transport, cell death, and regulation of the cytoskeleton. We have previously shown that zebra- fish mutant for trpm7 has severe growth defects and gross alterations in bone development, including bone malformations, vertebral co- lumn compression, changes in the timing of ossification for endo- chondral and intramembraneous bones, and develops mineralized deposits in the larval kidney. Strong expression of trpm7 in the kidney and the corpuscles of Stannius suggests a role in regulating phy- siologic cation homeostasis. In support of this we are evaluating changes in electrolyte levels in trpm7 mutants, and have identified changes in the expression of factors regulating ion homeostasis. To determine whether growth, bone or kidney phenotypes in trpm7 mutants may be mediated by the altered levels of these factors we are developing inducible transgenic lines of these factors to assess their effect in a wild-type background. Together these analyses will provide an improved understanding of the relationship of trpm7 function to physiological homeostasis and how changes in trpm7 function affect growth, bone development and kidney function. doi:10.1016/j.ydbio.2008.05.115 Program/Abstract # 105 Molecular mechanisms underlying skeletal variation in zebrafish Michelle H. Connolly, Brian K. Hall Department of Biology, Dalhousie University, Halifax, NS, Canada Zebrafish (Danio rerio) embryos were heat-shocked to determine whether embryonic stress contributes to the expression of phenotypic variation. Bone-specific staining among full siblings indicates a stage- specific increase in the number of caudal vertebrae. The mechanism underlying this meristic variation was explored by real time RT PCR. Fibroblast growth factor-8 (Fgf8), which is essential for somite boundary formation, was reduced among fish heat-shocked at 12 somite stage, the same stage that developed a greater number of caudal vertebrae. Lower levels of Fgf8 may be indicative of a slowed molecular clock, a potential underlying cause of increased caudal vertebrae number among fish heat-shocked at this stage. This research will contribute to our understanding of evolutionary change and give greater insight to the underlying causes of skeletal anomalies that affect many cultured fish. This work was supported by an FGS grant to M.H.C. and an NSERC grant to B.K.H. doi:10.1016/j.ydbio.2008.05.116 499 ABSTRACTS / Developmental Biology 319 (2008) 493–501