Laser capture microdissection of gonads from juvenile zebrafish.
BACKGROUND
Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.


METHODS
The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.


RESULTS
The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.


CONCLUSIONS
The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.
Jørgensen
Anne
A
Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. annejoer@ruc.dk
Nielsen
John E
JE
Morthorst
Jane E
JE
Bjerregaard
Poul
P
Leffers
Henrik
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006066:Gonads / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D006801:Humans; D017403:In Situ Hybridization; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
D000469:Alkaline Phosphatase