Ascidian Zic Genes
6.2 Expression Pattern and Function of Zic-r.a
6.2.1 Expression and Function of Maternal Zic-r.a
6.2.1.1 Localization of Maternal mRNA
The number of cells that constitute the ascidian embryo is small, and cell lineages are invariant among individuals. Because of these features, cell lineages are pre- cisely determined, and gene expression patterns are traceable at the single cell level (Fig. 6.3).
1 2 3 4 5 6 7 8 9 1011121314 Position
0 0.5 1 1.5 2
Information content
human Zic1
1 2 3 4 5 6 7 8 9 10111213 Position
0 0.5 1 1.5 2
Information content
C. intestinalis Zic-r.a
1 2 3 4 5 6 7 8 Position
0 0.5 1 1.5 2
Information content
C. intestinalis Zic-r.b
Fig. 6.2 Sequence logos representing binding sequences of human ZIC1, C. intestinalis Zic-r.a, and C. intestinalis Zic-r.b, which were made using data from in vitro selection (Jolma et al. 2013;
Yagi et al. 2004a, b).
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A4.1 anterior vegetal
anterior animal
posterior vegetal
posterior animal
A5.1 A6.2 A7.3 notochord
A7.4 nerve cord A6.1 endoderm
A5.2
A6.4
A7.5 endoderm A7.6 mesenchyme A6.3
A7.7 notochord A7.8 nerve cord
B4.1
B5.1 B6.2 B7.3 mesenchyme
B7.4 muscle B6.1 endoderm
B5.2
B6.4
B7.5 muscle + trunk ventral cells B7.6 germ line cells
B6.3
B7.7 mesenchyme B7.8 muscle a4.2
a5.3
a6.6 epidermis a7.9
a7.10 a6.5
a5.4
a8.17 brain a8.18 palp a8.19 brain a8.20 palp
a7.13 a7.14 epidermis
a8.25 brain
a8.26 epidermis + epidermal neurons a6.8 epidermis
a6.7
b4.2
b5.3
b6.6 epidermis b7.9
b7.10 b6.5
b5.4 epidermis
b8.17 nerve cord
b8.18 epidermis + epidermal neurons b8.19 nerve cord
b8.20 epidermis + epidermal neurons 8-cell 16-cell 32-cell 64-cell early gastrula stage
Localized Zic-r.a mRNA Zic-r.b mRNA
Fig. 6.3 The cell lineage of Ciona embryos and the expression pattern of two Zic genes. Note that the ascidian embryo is bilaterally symmetrical, and each pair is designated with unique names 6 Ascidian Zic Genes
Zic-r.a mRNA is present in unfertilized eggs of Halocynthia and Ciona. After fertilization, it is prominently localized in the posterior vegetal region (Nishida and Sawada 2001; Satou et al. 2002). This localized mRNA is inherited by a pair of posterior-most cells in subsequent cell divisions (Figs. 6.3 and 6.4). Many mRNAs are similarly localized in the posterior pole (Yoshida et al. 1996; Satou 1999; Satou and Satoh 1997; Paix et al. 2009; Sasakura et al. 1998a, b; Yamada et al. 2005). Such posteriorly localized mRNAs are referred to as postplasmic or posterior-end-mark mRNAs. The posterior-most cells that retain maternal Zic-r.a mRNA give rise to the germ line cells. The sister cells (B5.1) of the posterior-most cells at the 16-cell stage
Ant.
Ani.
Veg.
Post.
Ant.
Post.
Ant.
Post.
vegetal viewvegetal viewlateral view
fertilized egg16-cell embryo32-cell embryo
Localized Zic-r.a mRNA
Siblings of cells with localized Zic-r.a mRNA B5.1
B6.4
muscle mesenchyme notochord endoderm
muscle mesenchyme
Genes regulated by Zic-r.a in B5.1 Tbx6.b, Wnttun5, Admp
Genes regulated by Zic-r.a in B6.4 Tbx6.b, Admp, Hes.b, Snail, Otx, Lefty, Wnt3, Wnt5 Fig. 6.4 Illustrations
showing localization of Zic-r.a mRNA in early embryos. In sister cells (gray) of cells in which Zic-r.a is localized, several genes are activated by Zic-r.a
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contribute to the endoderm, mesenchyme, notochord, and muscle. The sister cells (B6.4) at the 32-cell stage contribute to mesenchyme and muscle. The sister cells (B7.5) at the 64-cell stage contribute to muscle and precursor cells of the adult heart and muscle, which are called trunk ventral cells.
6.2.1.2 Developmental Function of Maternal Zic-r.a
Maternal Zic-r.a was first identified as a muscle determinant (Nishida and Sawada 2001). Existence of a muscle determinant in ascidian eggs was first shown by a clas- sical embryological experiment in 1905 (Conklin 1905). Its molecular identity was then revealed to be Zic-r.a mRNA almost 100 years later (Nishida and Sawada 2001). Prior to this finding, several mRNAs localized in the posterior pole were identified and are called posterior end mark mRNAs (Yoshida et al. 1996; Satou 1999; Satou and Satoh 1997). Among them, only Zic-r.a plays a role in specification of muscle fate. Zic-r.a was later revealed to also be required for specification of the posterior mesenchyme lineage.
The blastomeres, in which Zic-r.a mRNA is localized, give rise to the germ line cells, and transcription is generally suppressed by a protein encoded by another localized mRNA, pem-1. Hence, Zic-r.a protein cannot activate its target genes in the posterior-most cells but activates them in sister cells of the posterior-most cells (Fig. 6.4) (Shirae-Kurabayashi et al. 2011; Kumano et al. 2011).
At the 16-cell stage, the posterior-most cells are named B5.2, and their sister cells are B5.1. Although Zic-r.a mRNA is localized in B5.2, Zic-r.a protein is pres- ent in both of these sister cells and activates its targets in B5.1 (Oda-Ishii et al.
2016). These targets include Tbx6.b, which encodes a T-box transcription factor, Wnttun5, which encodes a Wnt signaling molecule, and Admp, which encodes a signaling molecule of the BMP (bone morphogenetic protein) family. Zic-r.a works with Tcf7 and β-catenin to activate these target genes. Tcf7 is a transcription factor with an HMG-box and acts as an activator by forming a complex with β-catenin. At the 16-cell stage, β-catenin is translocated into nuclei of cells in the vegetal hemi- sphere. Thus, a collaboration of Tcf7/β-catenin and Zic-r.a activates Tbx6.b, Wnttun5, and Admp in the posterior vegetal blastomeres (Oda-Ishii et al. 2016).
Tcf7/β-catenin activates its targets, including Foxd and Fgf9/16/20, in the ante- rior and posterior vegetal cells. How are Tbx6.b and Wnttun5 activated specifically in the posterior vegetal cells? In other words, why are Tbx6.b and Wnttun5 not activated in the anterior vegetal cells? The most likely hypothesis is that Tcf7/β- - catenin binding sites are qualitatively different between these two groups; namely, Tcf7/β-catenin binding sites in the Tbx6.b and Wnttun5 enhancers could not activate gene expression alone and would require the help of Zic-r.a. However, this is not the case; when Tcf7 binding sites in the Tbx6.b enhancer are replaced with those in the Fgf9/16/20 enhancer, the chimeric Tbx6.b enhancer drives gene expression specifi- cally in the posterior vegetal cells (Oda-Ishii et al. 2016). Instead, there is a repres- sor element in each of the enhancers of Tbx6.b and Wnttun5. Without these repressor elements, Tbx6.b and Wnttun5 are expressed in the anterior and posterior vegetal
6 Ascidian Zic Genes
cells. What then is the function of Zic-r.a? Currently, the most likely hypothesis is that the repressor that binds to the repressor elements is present in the anterior and posterior vegetal cells, and Zic-r.a suppresses the function of the repressor.
Intriguingly, for this function, direct binding of Zic-r.a may not necessarily be required (Oda-Ishii et al. 2016). First, there are no clear Zic-r.a binding sites in the enhancers of Tbx6.b and Wnttun5. Second, no clear binding was found in these enhancers by chromatin immunoprecipitation (ChIP) followed by microarray and deep-sequencing assays; only weak binding was found in the Tbx6.b enhancer.
Lastly, in the ascidian embryo, Zic-r.a protein can physically interact with Tcf7.
Hence, it is possible that Zic-r.a might bind to the enhancer indirectly through Tcf7 (Fig. 6.5). Similar examples, in which Zic protein acts as a cofactor of transcription factors, are known in vertebrates (Koyabu et al. 2001; Sanchez-Ferras et al. 2014).
At the 32-cell stage, genes activated under the control of Zic-r.a include Hes.b, Snail, Otx, Admp, Bmp3, Lefty, Nodal, Wnt3, and Wnt5. These genes are all expressed in B6.4 cells, which are sister cells of the posterior-most cells at this stage. Tbx6.b and Wnttun5 are also activated in this pair of cells. The expression of Tbx6.b in B6.4 at the 32-cell stage is likely to be regulated differently from the expression in B5.1 at the 16-cell stage, because a different enhancer, which contains putative Zic-r.a binding sites, is responsible for the expression in B6.4 (Kugler et al.
Off
β-catenin/Tcf7 complex Tcf7 binding site
Zic-r.a Zic-r.
a
Zic-r.
a
Repressor (unidentified) Repressor element including 5’ -acacgaatcagcagg-3’
Anterior vegetal cells (A5.1, A5.2)Posterior vegetal cells (B5.1)
target genes of Zic-r.a and β-catenin/Tcf7
including Tbx6.b and Wnttun5 β-catenin/Tcf7 target genes including Foxd and Fgf9/16/20
On
On On
Fig. 6.5 Zic-r.a works together with β-catenin and Tcf7 to activate its targets in posterior vegetal cells. Zic-r.a is thought to suppress the function of a repressor, which has not yet been identified, and for this function, Zic-r.a does not necessarily bind directly to DNA
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2010). Therefore, it is possible that Zic-r.a is bound directly to enhancers of the above genes at the 32-cell stage.
Tbx6.b encodes an essential transcription factor for specification of muscle cells, because it regulates muscle-specific genes including genes encoding myosin light chain, myosin regulatory light chain, muscle actin, troponin I, troponin C, troponin T, and tropomyosin (Yagi et al. 2005). In addition, it regulates a gene encoding a myogenic factor, Mrf, which has a basic helix-loop-helix motif and is the sole ortho- log for vertebrate MyoD, Myogenin, MRF4 and Myf5, and another Zic gene, Zic-r.b (Yagi et al. 2005). Mrf and Zic-r.b are also required for proper differentiation of muscle cells. Thus, Zic-r.a begins the muscle specification gene pathway.
Zic-r.a is also required for mesenchyme specification (Kobayashi et al. 2003).
Zic-r.a activates Otx in the descendants of B5.1 at the 32-cell stage (Yagi et al.
2004a). Because Otx expression does not begin in B5.1 at the 16-cell stage, Zic-r.a might activate Otx indirectly or might activate it cooperatively with transcription factors activated at the 16-cell stage. At later stages, Otx is required for expression of Twist-r.a, which is an essential transcription factor for mesenchyme specification (Imai et al. 2003, 2006).
The B5.1 lineage also contributes to endoderm. In Halocynthia embryos, Zic-r.a function is suppressed in the posterior endodermal lineage by Fgf and Bmp signal- ing (Kondoh et al. 2003). The molecular mechanism of this suppression has not been fully elucidated, and it is not clear whether this suppression is required in Ciona embryos.