The Transcription Factor Brachyury Links NMP Maintenance with Segmentation
The Brachyury gene plays a key role in both maintaining NMPs and promoting proper segmentation, as mutations in Brachyury result in truncated embryos with fewer than normal somites, and the somites that do form have irregular borders (Martin, 2016). A mutation in the mouse Brachyury gene (also called T, an abbreviation of the word “tailless”) was first identified in 1927 (Dobrovolskaia-Zavadskaia, 1927). Animals that are heterozygous have the obvious phenotype of having a short tail relative to siblings, and the mutation was aptly named Brachyury, which is Latin for “short tail.” The molecular identity of the mutated gene was discovered in 1990 (Herrmann et al., 1990), which became the founding member of the T-box transcription factor family, for which there are now 17 members identified in the mouse and human, and 26 in zebrafish (Ahn et al., 2012; Papaioannou, 2014). The Brachyury gene is often referred to now by the name Tbxt. NMPs are defined by the co-expression of Brachyury and the Sox2 transcription factor, and complete loss of Brachyury function in the mouse or zebrafish results in severely truncated embryos, with only 8-12 of the anteriormost somites forming (Chesley, 1935; Halpern et al., 1993; Martin and Kimelman, 2008; Schultemerker et al., 1994). The truncated axis phenotype occurs because Brachyury directly regulates the expression of genes that are essential for the maintenance of the NMP population. Brachyury, along with the caudal homeobox transcription factor Cdx2, directly activates the expression of canonical Wnt signaling ligands (Amin et al., 2016; Evans et al., 2012; Martin and Kimelman, 2008; Morley et al., 2009), and Wnt signaling is involved in the specification and expansion of NMPs, as well as their maintenance. Wnt signaling in turn activates Brachyury and Cdx2 expression, creating an autoregulatory loop in the NMPs (Amin et al., 2016; Martin and Kimelman, 2008; Yamaguchi et al., 1999).
Another Brachyury direct target that supports NMP maintenance is the cyp26al enzyme, which is responsible for degrading the retinoic acid (RA) signaling molecule (Martin and Kimelman, 2010). RA is produced in the most recently formed somites by the enzyme Aldhla2. After production, RA then diffuses into neighboring cells and binds to retinoic acid receptors, which act as transcription factors to regulate downstream gene expression (Duester, 2008). Retinoic acid normally functions in the mouse to induce an initial population of NMPs (although this is species- specific, as this is not observed in zebrafish), and subsequently to promote neural differentiation from the NMPs (Berenguer et al., 2018; Gouti et al., 2017). Due to this later role, it is important to keep the majority of NMPs in a low retinoic acid environment, so they can be sustained in an undifferentiated state. Mutations in the cyp26al enzyme in mouse and zebrafish result in axial truncations with fewer somites than normal due to the failure to maintain the NMPs, with truncated embryos exhibiting expanded posterior neural tissue (Abu-Abed et al., 2001; Emoto et al., 2005; Martin and Kimelman, 2010). In the chick, the increased proximity of the retinoic acid source to the NMPs as the presomitic mesoderm shortens during late axial extension is hypothesized to be important for terminating axis extension through NMP depletion via neural induction (Olivera-Martinez et al., 2012). These studies reveal that an essential role of Brachyury is to create an environment of low retinoic acid through cyp26al activation, and together with its activation of Wnt ligand expression, Brachyury establishes a molecular niche critical for NMP maintenance (Martin and Kimelman, 2010).
The poorly formed anterior somites present in Brachyury mutants indicate it also plays a role in the segmentation process. Indeed, Brachyury directly activates several key components of the segmentation process and has been implicated as a factor causing human congenital scoliosis when mutated (Ghebranious et al., 2008). Notch signaling is required for segmentation by coupling the oscillating gene expression in neighboring cells (Horikawa et al., 2006; Jiang et al., 2000; Mara et al., 2007; Okubo et al., 2012; Ozbudak and Lewis, 2008; Riedel-Kruse et al., 2007). The zebrafish brachyury orthologue tbxta (ntla) directly activates the expression of the Notch ligands deltaC and deltaD (Garnett et al., 2009; Jahangiri et al., 2012; Morley et al., 2009). Mutations in either of these genes result in severe somitogenesis defects (Holley et al., 2000; Julich et al., 2005; vanEeden et al., 1996). Direct regulation of Delta ligand expression by Brachyury is conserved in the mouse, where Brachyury activates Dill expression. Dili has t-box binding sites in its regulatory region that are essential for expression, and like the zebrafish delta mutants, mouse Dill mutants have severe somitogenesis defects (Concepcion and Papaioannou, 2014; Hofmann et al., 2004; Hrabe de Angelis et al., 1997). Notch/Delta signaling coordinates the oscillations of the Hes/her transcription factors in neighboring cells, which are core components of the segmentation process (Horikawa et al., 2006; Jiang et al., 2000; Mara et al., 2007; Okubo et al., 2012; Ozbudak and Lewis, 2008; Riedel-Kruse et al., 2007). The Hes/her genes are also directly regulated by Brachyury. Zebrafish Tbxta binds to the -9kb to +3kb genomic regions of both her1 and her7 (Morley et al., 2009), which are together required for proper somitogenesis (Henry et al., 2002; Holley et al., 2002; Oates and Ho, 2002). There is a functional t-box binding site in the herl promoter that is required for her! expression (Brend and Holley, 2009). The her7 gene has one of the largest clusters of t-box biding sites of any gene in the zebrafish genome, but these sites have not been tested functionally (Garnett et al., 2009). Similarly in the mouse, the Hes7 oscillatory gene is a direct transcriptional target of Brachyury, and segmentation in Hes7 mouse mutants does not occur properly (Bessho et al.. 2003; Bessho et al., 2001; Faial et al., 2015). The direct regulation of Notch ligands and hairy enhancer of split transcription factors by Brachyury in both mouse and zebrafish shows that Brachyury has a conserved role in the segmentation process independent of its conserved role in NMP maintenance and mesoderm specification.