1. Conclusions 1.Synapsin IIa is expressed in the brain of adult zebrafish, however we did not find expression in the eye, gut, muscle, or heart. 2.The data presented here represents the first analysis of temporal expression of the synapsin IIa gene in developing zebrafish embryos. 3.Sequence comparisons and syntenic analysis supports the identification of a novel zebrafish synapsin II homolog. Objectives 1.Determine if there is expression of the synapsin IIa gene in adult zebrafish tissues. 2.Determine if there is expression of synapsin IIa gene during different developmental timepoints in zebrafish embryos. The Identification and Expression of Synapsin IIa in Zebrafish Kelly Welsh and Wendy Boehmler, Department of Biological Sciences York College of Pennsylvania Introduction Synapsins (I, II, and III) are a family of neuronal phoshoproteins that play an important role in neurotransmitter release by releasing synaptic vesicles in the presynaptic terminal to move towards the plasma membrane for exocytosis. Synapsins have also been implicated in neurite outgrowth, the stabilization of synapses, and synaptic vesicle docking, fusion, and recycling. It has been reported that synapsin II expression levels are decreased in post-mortem brain samples of schizophrenic patients. It is unclear whether the decrease in expression is the cause or result of the disease or whether antipsychotic drug treatment effects the expression of synapsin II. Synapsins and synapsin-like genes have been characterized in a variety of invertebrates and vertebrates, however the synapsin genes have yet to be characterized in zebrafish. Because zebrafish are now being increasingly used as a genetic model and to model a variety of human diseases, we focused our studies on the cloning and characterization of zebrafish synapsin IIa. Methods Searches of expressed sequence tag databases (http://www.ncbi.nlm.nih.gov/) led to the identification of a potential zebrafish Synapsin IIa homolog (Gene Accession number NM001002597). RT-PCR was conducted using RNA collected from adult zebrafish and embryos. Future Experiments Determine the spatial expression of synapsin IIa gene during different developmental timepoints in zebrafish embryos Perform a whole-mount in situ hybridization on embryonic zebrafish using the cloned synapsin IIa gene to determine its spatial and temporal expression pattern. Determine whether antipsychotic drug treatment alters the expression levels of synapsin IIa in zebrafish. Literature Cited Cesca, F., Baldell, P., Valtorta, F., & Benefenat, F. (2010). The synapsin key actors of synapse function and plasticity. Progress in Neurobiology, 91, 313-348. Chong, V.Z., Skoblenick, K., Morin, F., Xu, Y., Mishra, R.K., 2006. Dopamine-D1 and -D2 receptors differentially regulate synapsin II expression in the rat brain. Neuroscience, 138, 587–599. Gene Name Zebrafish Chromosome Human Chromosome SynII233 rhoa233 craf233 plxnbl233 tkt233 her9233 slc41a3233 Figure 4: Comparison of zebrafish and human Synapsin II. Human and zebrafish SynapsinII amino acid sequences were aligned using CLUSTALW. Dashes in sequences allow optimal alignment for amino acid insertions/deletions. Identical amino acids are shown and similar amino acids are highlighted by plus signs. (Identities = 314/480 (66%) ) Domains A and C are highlighted, Domain A and B contain phosphorylation sites and Domain C stabilizes the interaction. Results Table 1: Markers and Genes Syntenic with SynII Gene in Zebrafish and Humans. Used tissues from zebrafish brain, eye, gut, heart & muscle and 24, 42, and 72 hours post fertilization (hpf) embryos RNA IsolationcDNA PCR Denatured at 95°C Annealed at 46°C Elongated at 72°C 30 cycles Zebrafish Synapsin IIa gene cloned addiction-dirkh.blogspot.com Figure 1: RT-PCR analysis of Synapsin IIa from zebrafish embryos. The SynIIa gene was amplified from zebrafish brain, 24 hpf, and 72 hpf. The primers used encompassed the predicted ATG start codon and stop codon to produce an amplicon of 1420 base pairs (bp). Ladder PCR Purification of Synapsin IIa Ligation Transformation (Cesca et al. 2010) ZF 2a 1 MNYLRRRLSDSTFISNLPNGYMSDLQKPDPPQPPPPGPAPVPAPASGPAPVSPRQ--ERK 58 MN+LRRRLSDS+FI+NLPNGYM+DLQ+P+P QPPPP P A ++ AP + ER+ Human2b2 MNFLRRRLSDSSFIANLPNGYMTDLQRPEPQQPPPPPPPGPGAASASAAPPTASPGPERR 61 ZF 2a 59 PIQPQP---------GLGTGFFSSITNVVKQTAASAGLVEQTSVPASSKR-IKILLVIDE 108 P +G+ FFSS++ VKQTAASAGLV+ + ++ R K+LLV+DE Human2b62 PPPASAPAPQPAPTPSVGSSFFSSLSQAVKQTAASAGLVDAPAPAPAAARKAKVLLVVDE 121 ZF 2a 109 PQHDWAKLFRGKKLNGDYDIKVEQAEFGDISIVAHANGSCNVAVQVLQNENKVLRSFKPD 168 P DWAK FRGKK+ GDYDIKVEQAEF ++++VAHA+G+ V +QVL+N KV+RSF+PD Human2b122 PHADWAKCFRGKKVLGDYDIKVEQAEFSELNLVAHADGTYAVDMQVLRNGTKVVRSFRPD 181 ZF 2a 169 FVLIRQHAFSMTENEDFRNLIIGLQYAGIPSINSLESVYNLCDKPWAFACLINTYKKLGQ 228 FVLIRQHAF M ENEDFR+LIIG+QYAG+PSINSLES+YN CDKPW FA L+ YK LG Human2b182 FVLIRQHAFGMAENEDFRHLIIGMQYAGLPSINSLESIYNFCDKPWVFAQLVAIYKTLGG 241 ZF 2a 229 EKFPLIEQTFYPNYKEMVTMPAFPVVVKIGHAHSGVGKVKVENHTKFQDIASVVAITQTY 288 EKFPLIEQT+YPN+KEM+T+P FPVVVKIGHAHSG+GKVKVENH FQDIASVVA+TQTY Human2b242 EKFPLIEQTYYPNHKEMLTLPTFPVVVKIGHAHSGMGKVKVENHYDFQDIASVVALTQTY 301 ZF 2a 289 STCEPFIDPKYDIRIQKIGNDYKAYMRTSVSGNWKSNTGTAMVEQVAMTDRYKLWVDTCC 348 +T EPFID KYDIR+QKIGN+YKAYMRTS+SGNWK+NTG+AM+EQ+AM+DRYKLWVDTC Human2b302 ATAEPFIDSKYDIRVQKIGNNYKAYMRTSISGNWKTNTGSAMLEQIAMSDRYKLWVDTCS 361 ZF 2a 349 EIFGGLEICAVKAINGKDGKDYITEVMGSSMPLMGEHQAQDQQLIADMVIAKMNHVMAQ- 407 E+FGGL+ICAVKA++GKDGKDYI EVM SMPL+GEHQ +D+QLI ++VI+KMN ++++ Human2b362 EMFGGLDICAVKAVHGKDGKDYIFEVMDCSMPLIGEHQVEDRQLITELVISKMNQLLSRT 421 ZF 2a 408 ---NPKRP-TAIQPKQITVENNTLVEVKNAPPQKAPPQGCLQYILDCNGVAVGPKLVQAN 463 +P+RP T QP+ T+++ + PPQ+ PPQGCLQYILDCNG+AVGPK VQA+ Human2b422 PALSPQRPLTTQQPQSGTLKDP---DSSKTPPQRPPPQGCLQYILDCNGIAVGPKQVQAS 478