In previous work we had shown that the Six3 gene is required for development of all nerve cells in the embryo. Therefore, we carried out a morpholino oligonucleotide loss-of-function screen to identify genes dependent on Six3 for their expression during embryogenesis, one of which was SoxC. Morpholino knockdown of SoxC function similarly repressed differentiation of all neurons, placing it downstream from Six3 and upstream of commitment of neural precursors. The fact that SoxC was required for development of multiple neuronal types suggested that it is not the immediate driver of their terminal differentiation. Consistent with this idea, SoxC mRNA was not detectable in mature nerve cells expressing the terminal differentiation marker synaptotagmin B (SynB). Therefore, an RNA-Seq screen was carried out to identify SoxC-dependent genes. From the set of affected genes, we further tested those encoding two transcription factors, Z167 and Brn1/2/4. Double-labeled in situ hybridization revealed that the corresponding mRNAs were co-expressed in some cells with SoxC message and also in some cells expressing SynB, suggesting that these factors could link SoxC function to terminal differentiation. Z167 expression was confined to the anterior pole domain of the embryo and, in a loss-of-function experiment, we found that it is required for differentiation of serotonergic neurons that form only in this region. Morpholino knockdown of Brn1/2/4 was lethal, suggesting that this gene has a separate critical early function for the embryo. Consistent with this, analysis of Brn1/2/4 mRNA expression showed significant levels accumulate maternally and uniformly in the embryo. Misexpression of synthetic Brn1/2/4 mRNA produced a dramatic phenotype similar to that observed when all signaling is blocked in the embryo: endoderm and mesoderm fail to differentiate and the embryo consists of an epithelial ball with an excess of SynB-expressing neurons. This suggests that one early function of Brn1/2/4 could be in the pathways that regulate allocation of ectoderm, endoderm and mesoderm fates along the animal-vegetal embryonic axis (for example, see Range et al. www.ncbi.nlm.nih.gov/pubmed/23335859). We are testing a model in which Six3 and SoxC are involved in commitment of neural precursors, while Brn1/2/4 and Z167 function downstream in commitment of neurons to serotonergic and other neuronal fates.
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