HUDSON, Terry-Ann*; MCADORY, Brenda; TOWNSEL, James G.; Medgar Evers College; Tennessee State University; Meharry Medical College: Localization of Semaphorin 2a in the Central Nervous System (CNS) of Limulus polyphemus by in situ Hybridization

Semaphorins are classified as membrane-bound and as secreted proteins. Some have been shown to be vital for guidance of axons during the development of the nervous system while other classes are involved in immune system development. Semaphorins are grouped into eight classes based on similarities of domains and species of origin. An approximately 500 bp SEMA domain is characteristic of all semaphorins. The main object of this investigation is to synthesize an RNA probe for the localization and detection of semaphorin 2a in Limulus polyphemus (horseshoe crab) utilizing in situ hybridization. Previously, a full length DNA sequence of semaphorin 2a was cloned into the pCR BLUNT II TOPO vector. We used this sequence to design primers specific for a 247 bp region in the SEMA domain. T3 and T7 promoter regions were appended to the primers. PCR products were produced using a forward (T3F2) and reverse (T7R1) primer. The predicted size of the PCR product (247 bp) was confirmed on a 1% agarose gel. The PCR product was then used as a template for the in vitro transcription of both sense and antisense RNA probes. The probes were non-isotopically labeled with digoxigenin (DIG)-11-UTP during transcription. Prior to being used in in situ studies, the probes along with pTRI- actin controls, were analyzed by gel electrophoresis and labworks software. Following analysis, the probes were hybridized to Limulus Central Nervous System (CNS) tissue sections that previously had been fixed in paraformaldehyde and embedded in paraffin. It is believed that localization of semaphorin 2a by in situ hybridization will provide insights into the biological significance of semaphorin in the CNS of the horseshoe crab. These studies were supported by NIH grants P20MD00261, RR03032, and MH57067.