Ascidian Zic Genes
6.3 Expression Pattern and Function of Zic-r.b
6.3.1 Expression and Function at the 32-Cell Stage
6.3.1.1 Upstream Regulatory Mechanisms
Zic-r.b mRNA is not expressed maternally. It is first expressed at the 32-cell stage in marginal cells of the vegetal hemisphere (A6.2, A6.4, B6.2, and B6.4) (Figs. 6.3 and 6.7). Most mesodermal cells and the ventral and lateral rows of the nerve cord cells are derived from these Zic-r.b expressing cells.
The expression of Zic-r.b in the marginal cells at the 32-cell stage is regulated by Gata.a, Foxd, Fgf9/16/20, and Foxa.a (Hudson et al. 2016; Imai et al. 2016) (Fig. 6.8). Whereas Gata.a is a maternal protein, Foxd, Fgf9/16/20, and Foxa.a are zygotically expressed in all vegetal cells except cells with a germ cell fate at the 16-cell stage. These three genes are among the first genes activated from the zygotic genome. Although it is not known how Foxa.a is activated, the expression of Foxd and Fgf9/16/20 is known to be activated directly by β-catenin and Tcf7 (Oda-Ishii et al. 2016; Rothbächer et al. 2007). At the 16-cell stage, β-catenin is translocated to the nuclei of vegetal hemisphere cells (A5.1, A5.2, and B5.1), except the most pos- terior cells (B5.2) (Hudson et al. 2013), and a complex of β-catenin and Tcf7 directly activates Foxd and Fgf9/16/20 (Imai et al. 2002b; Oda-Ishii et al. 2016). At the same time, this β-catenin/Tcf7 complex suppresses Gata.a binding activity to its target sites through direct interaction between β-catenin/Tcf7 and Gata.a. In this manner, Gata.a activates its target genes specifically in the animal hemisphere, where β-catenin is not translocated to nuclei (Oda-Ishii et al. 2016). At the 32-cell stage, each of the three vegetal cell pairs that express Foxd divides into a marginal cell and an endodermal cell. β-catenin continues to be translocated into the nuclei of the endodermal cells, and therefore Gata.a function continues to be suppressed there.
Meanwhile, β-catenin is not translocated into the nuclei of the marginal cells, and therefore Gata.a begins to work as a transcriptional activator. Thus, a combination of Gata.a, Foxd, Foxa.a, and Ets1/2 that is activated by Fgf9/16/20 signaling pro- motes Zic-r.b expression specifically in the marginal cells.
Fig. 6.6 Zic-r.a is expressed in the anterior portion of the brain and in the dorsal and ventral rows of the nerve cord at the tailbud stage, although its function has not been studied
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Zic-r.b expression in B6.2 is likely regulated by an additional distinct mecha- nism, in which Tbx6.b, expressed under the control of Zic-r.a, is involved. The abovementioned mechanism dependent on Gata.a and the Tbx6.b-dependent
Ant.
Post.
vegetal view32-cell embryo
B6.4 B6.2
muscle mesenchyme A6.4 nerve cordnotochord A6.2 nerve cordnotochord
muscle mesenchyme notochord Fig. 6.7 Zic-r.b is
expressed in the margin of the vegetal hemisphere of the 32-cell embryo (gray).
These cells mainly contribute to mesodermal tissues
β-catenin/Tcf7 Gata.a
β-catenin/Tcf7 Gata.a Foxd Fgf9/16/20
Gata.a Gata.a Foxa.a Foxa.aFoxa.a Foxd Ets1/2 Foxa.a Foxd Ets1/2
Fgf9/16/20
Zic-r.b Zic-r.b
Zic-r.b Zic-r.b
Vegetal hemisphere (A5.1, A5.2, B5.1) Animal hemisphere
marginal cells
(A6.2, A6.4, B6.2) endoderm cells
(A6.1, A6.3, B6.1)
16-cell embryo 32-cell embryo
Fgf9/16/20
Off Off
Off On
cell division
Fig. 6.8 Schematic illustrations for regulation of Zic-r.b at the 16-cell and 32-cell stages. Four indicated factors are required for activation of Zic-r.b, and these four factors are active only in the marginal cells of the 32-cell embryo. Foxa.a is present in the anterior animal cells (an asterisk) but not in the posterior animal cells. Note that Ets1/2 is the effector of Fgf9/16/20 signaling at the 32-cell stage
6 Ascidian Zic Genes
mechanism are likely to cooperatively and redundantly regulate Zic-r.b expression in B6.2 (Yagi et al. 2005; Imai et al. 2016; Anno et al. 2006).
Intriguingly, the expression of Zic-r.b in B6.4, which are sister cells of the most posterior germ line cells, is regulated differently. First, the upstream transcription factors Foxd and Foxa.a are not expressed in B6.4 or its parental cells (Imai et al.
2002b, 2004; Shimauchi et al. 1997). Second, knockdown or suppression of either β-catenin, Foxd, Fgf9/16/20, or Gata.a does not impair Zic-r.b expression in B6.4 (Imai et al. 2002c; Hudson et al. 2013, 2016). However, it has not been revealed how this expression is regulated.
6.3.1.2 Functions in the Anterior Marginal Cells
The notochord and nerve cord are differentiated from the anterior marginal cells of 32-cell embryos (Fig. 6.7). After a cell division between the 32-cell and 64-cell stages, the developmental fate of the anterior descendants is restricted to the nerve cord, and the developmental fate of the posterior descendants is restricted to the notochord. Brachyury is then expressed exclusively in the notochord lineage and plays an essential role in specification of notochord fate (Yasuo and Satoh 1993, 1998; Corbo et al. 1998; Chiba et al. 2009). Zic-r.b binds to the upstream regulatory region of this gene, and this binding is essential for the activation of Brachyury (Imai et al. 2002c; Yagi et al. 2004b; Kumano et al. 2006). Consequently, no notochord cells are differentiated in Zic-r.b morphants. Among the daughter cells of cells that express Zic-r.b at the 32-cell stage, the posterior cells (A7.3 and A7.7) abut endoder- mal cells and express Brachyury under the control of Fgf9/16/20 signaling (Fig. 6.9) (Imai et al. 2002a; Nakatani and Nishida 1994). Fgf signaling is weakened in the
Ant.
Post.
64-cell embryo (vegetal view)
A7.7A7.3 A7.8A7.4
notochord nerve cord
nerve cord lineage
notochord lineage Fgf9/16/20
Efna.d
Ets1/2 Zic-r.b Brachyury
Fgf9/16/20 Efna.d
Ets1/2 Zic-r.b Brachyury Zic-r.b
Mrf
Fos muscle structural genes
Tbx6.b
Twist-r.a Zic-r.b B7.8
B7.7 B7.7 mesenchyme lineage
muscleB7.4
mesenchyme muscle lineage
Fgf9/16/20 Ets1/2 Otx
Fig. 6.9 Zic-r.b activates different downstream factors in different mesodermal lineages
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anterior daughter cells (A7.4 and A7.8) by Ephrin signaling. Ephrina.d (Efna.d) is expressed in the animal hemisphere and encodes a membrane-anchored ligand.
Because the contact surface area of A7.4 and A7.8 to cells expressing Efna.d is much larger than that of A7.3 and A7.7, A7.4 and A7.8 are expected to receive a stronger Efna.d signal. The activated receptor suppresses Fgf signaling through p120Ras- GAP (Picco et al. 2007; Haupaix et al. 2013). Thus, Brachyury is activated only in A7.3 and A7.7 by a combinatorial action of Zic-r.b and Fgf signaling. Foxa.a, which activates Zic-r.b, also acts cooperatively with these factors for activation of Brachyury expression (Imai et al. 2006; Kumano et al. 2006). In addition, Foxb is expressed in A7.4 and A7.8 cells of Halocynthia embryos, and Foxb ensures that Brachyury is not activated in A7.4 and A7.8. Zic-r.b is required for this Foxb expression (Hashimoto et al. 2011).
In addition to Brachyury, Zic-r.b activates Chordin, Lhx3, and Mnx in A7.3 and A7.7, which was revealed by an observation that Zic-r.b knockdown resulted in loss of expression of these genes in A7.3 and A7.7 (Imai et al. 2006). A chromatin immu- noprecipitation assay using a Gfp-tagged Zic-r.b suggested that Zic-r.b binds to the upstream regions of Chordin, Lhx3, and Mnx (Kubo et al. 2010).
6.3.1.3 Functions in the Posterior Marginal Cells
In the posterior marginal cells, Zic-r.b contributes to specification of muscle and mesenchyme (Imai et al. 2002c, 2006; Kubo et al. 2010), although knockdown of Zic-r.b does not completely suppress differentiation of muscle or mesenchyme.
In the muscle lineage, Zic-r.b contributes to maintenance of Tbx6.b expression in later embryos (Imai et al. 2006). Consequently, Tbx6.b expression continues until the neurula stage in Ciona and the tailbud stage in Halocynthia, although Zic-r.b expression in the muscle lineage disappears before the late gastrula stage (Imai et al.
2002c, 2004; Wada and Saiga 2002; Yasuo et al. 1996).
Zic-r.b and Tbx6.b activate Mrf (the sole ortholog for vertebrate MyoD, Myf5, Myogenin, and Mrf4, as described above) (Imai et al. 2006). Chromatin immuno- precipitation studies have shown that both Zic-r.b and Tbx6.b are bound directly to the upstream region of Mrf. Muscle structural genes are expressed under the control of Zic-r.b, Tbx6.b, and Mrf. ChIP assays indicated that most of them are regulated directly by Tbx6.b and Mrf but not by Zic-r.b (Kubo et al. 2010). This observation is consistent with the temporal expression pattern of Zic-r.b, because Zic-r.b expres- sion disappears earlier than Mrf and Tbx6.b expression.
Zic-r.b also contributes to expression of Twist-r.a, which is a key regulatory gene for specification of mesenchyme fate, because knockdown of Zic-r.b reduces the expression of Twist-r.a (Imai et al. 2003, 2006). Two distinct cell lineages contribute to the mesenchyme, and Zic-r.b is especially important for specification of the mes- enchyme that is derived from the posterior pairs of cells (B7.7). Knockdown of Zic-r.b resulted in loss of Fos expression in the B7.7-lineage mesenchyme (Imai et al. 2006). Zic-r.b is suggested to bind to the upstream region of Twist-r.a, and a
6 Ascidian Zic Genes
deletion of the region where Zic-r.b binds impairs expression in the posterior lin- eage (Kubo et al. 2010).