Microarray Analysis of Prothrombin Knockdown in Zebrafish

Prothrombin is the zymogen of thrombin that plays a pivotal role in coagulation. Knockdown of prothrombin in the zebrafish was previously shown to cause defects embryonic brain development and hemorrhage. To find genes affected by potential thrombin signaling in embryogenesis, microarray was performed using total RNA prepared from prothrombin antisense morpholino-injected embryos exhibiting brain abnormalities versus control morpholino-injected embryos. A total of 52 upregulated and downregulated genes were identified in duplicate microarray. Real time RT-PCR for 10 selected genes identified by the microarray confirmed the expression changes in these genes. One particular gene phlda3, was at least eleven fold upregulated and in situ hybridization revealed expansion of phlda3 expression in the central nervous system, brachial arches and head endoderm in knockdown embryos. Phlada3 gene encodes a small protein containing pleckstrin homology domain that is involved in regulating IP3 release. The only transcription factor identified on the array, the sry-related HMG box transcription factor, sox21a was shown to be down regulated approximately twofold. In situ hybridization showed a reduction at the midbrain-hindbrain boundary expression compared to control embryos. We propose that these two genes regulated by thrombin are critical for brain development in zebrafish embryos.