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-mediated 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 the pan-neuronal marker 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 produced dosage-dependent phenotypes. At a higher concentration it was lethal, suggesting that this gene has a 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 early embryo. At a well-tuned lower concentration, the embryos survived and both SynB signal and serotonin signal were greatly reduced. Therefore Brn1/2/4 functions downstream of SoxC and is required for differentiation of all neurons. 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 processes that regulate allocation of ectoderm, endoderm and mesoderm fates along the animal-vegetal embryonic axis. By labeling SoxC-expressing cells with anti-phospho-histone H3 antibody we found that some SoxC expressing cells were still dividing suggesting that SoxC-positive cells are in a neuronal precursor state. The rare observation of SoxC message in one of two adjacent dividing cells further suggested that the two daughter cells could take alternative fates with one cell remaining in the precursor state and the sister cell differentiating. We proposed a model in which Six3 is involved in neuro-ectoderm domain specification and SoxC is involved in commitment of neural precursors, while Brn1/2/4 and Z167 function downstream in commitment of neurons to serotonergic and non-serotonergic neuronal fates. In a collaboration with Larry Tabak's lab to study developmental functions of protein glycosylation in sea urchin embryos, we identified 13 SpGalNAc-T genes. Using morpholino knockdown methods we showed that UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase SpGalNAc-T13 expression was detected by whole mount in situ hybiridzation in skeletogenic mesenchyme cells (SMCs) of early gastrulae. When SpGalNAc-T13 was knocked down by morpholino oligo injection, embryos failed to secrete skeletal spicules normally produced by SMCs and neurons failed to differentiate. Further study revealed that SoxC expression was reduced in SpGalNAc-T13 injected embryos, suggesting a block upstream in the gene regulatory pathway for neurons. The fact that development of neurons in ectoderm is affected, but SpGalNac-T13 mRNA was not detected there suggests that the mRNA is expressed broadly at low levels, or alternatively that it is expressed maternally and SpGalNac-T13 protein is supplied maternally.
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