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Book Chapter Notochord GRN-Fig. 4-FINAL-
The molecular switches that turn on gene expression in the notochord 
The gene regulatory network underlying notochord formation 
Discovery of evolutionarily conserved notochord genes
      Cis-regulatory modules (enhancers, or CRMs) are regions of DNA that contain the information for the proper spatial and temporal onset of gene expression. When CRMs do not work properly, gene expression is compromised or lost. These elusive sequences are difficult to find and test in humans and other vertebrates, but they can be easily identified and characterized in Ciona. We hunt these molecular switches along the genome and "dissect" them to learn what makes them work. We found that what makes CRMs work is a combination of binding sites for one or more transcription factors.
We have identified, fully characterized and published 32 notochord CRMs, the largest collection of regulatory modules in any animal with a notochord. We have cataloged these CRMs into different categories, based upon the transcription factors that activate them, and we have uncovered intriguing similarities between some of these Ciona CRMs and those that activate notochord gene expression in vertebrates. 
To read more about these findings, check out our publications: Di Gregorio and Levine, 1999; Dunn and Di Gregorio, 2009; Passamaneck et al., 2009;  Katikala et al., 2013; Jose’-Edwards et al., 2015.
      A gene regulatory network (GRN) is similar to a circuitry of genes that work together to power up gene expression in an organ or a tissue. A GRN consists of several transcription factors, regulatory proteins that bind the CRMs of their target genes (or each other's CRMs, or their own CRMs) and regulate gene expression. Transcription factors are DNA-binding proteins of different families that can either activate or repress expression of their target genes by binding their CRMs. Each transcription factor controls up to hundreds of genes. 
This diagram summarizes our contribution to the elucidation of the Ciona notochord GRN. The story of the Ciona notochord GRN began with the identification of Ci-Bra and Ci-Foxa2 by Corbo et al. (1997a,b) in the Levine lab, and with the fundamental work by the Satoh lab and the Satou lab, which have identified hundreds of genes and transcription factors expressed in the notochord (Imai et al., 2004, Satou et al., 2001, to name only a few). Our lab has identified the transcription factors in colors in the diagram, with the exception of bHLH1 (identified by Imai et al., 2004; characterized in our lab by Kugler et al., 2019). Through microarray screens and RNA-Seq we have identified notochord target genes for some of these factors (listed in black) and in some cases we have characterized their respective notochord CRMs as well (listed in blue). Regulatory interactions that could be either direct or indirect are interrupted by diagonal lines.
To read more about these findings, check out our publications: Kugler et al., 2008; Jose’-Edwards et al., 2011;
Jose’-Edwards et al., 2013;
Di Gregorio, 2020.
      We have identified over 150 genes expressed in the developing Ciona notochord. We have demonstrated that some 'relatives' (orthologs) of these genes are expressed in the notochord of a distantly related tunicate, Oikopleura dioica, as well as in the notochord of developing zebrafish and mouse embryos. In particular, using the knowledge gathered in Ciona, we have shown that three orthologs of a gene that encodes a crucial enzyme that acts as a collagen modifier, the prolyl 3-hydroxylase, are expressed in the mouse notochord. In humans, mutations in one of these three genes, called LEPRE1 (aka P3H1), have been associated with osteogenesis imperfecta (OI), or "brittle bone disease".
In 2007, we reported the first evidence of mosaic gene expression along the length of the notochord. This finding highlighted the existence of molecular heterogeneity in the simple, 40-cell long Ciona notochord. Later on, we showed the conservation of this type of mosaicism in notochord gene expression in another urochordate, the larvacean Oikopleura. These findings suggest the possibility that some genes might be expressed at different levels among neighboring cells also in the notochord of higher chordates, and that this mosaic expression might play a role in the development of embryonic structures associated with the notochord.
Together, these studies indicate that Ciona possesses an essential genetic toolkit of notochord genes that are evolutionary conserved across chordates, and therefore provides a simplified yet informative model for studies of genes involved in notochord development and malformations.
To read more about these findings, check out our publications: Oda-Ishii and Di Gregorio, 2007; Kugler et al., 2008, 2011; Capellini, Dunn et al., 2008.