Lophotrochozoan Zic Genes
5.5 Roles of Zic Genes in Planarian Head Regeneration
Planarians are flatworms belonging to the Turbellaria class of phylum Platyhelminthes. Biologists have been fascinated by planarians’ enormous regen- erative abilities. Following pioneering studies to establish freshwater planarian Dugesia species (e.g., S. mediterranea [also called Dugesia mediterranea], Dugesia tigrina, and Dugesia japonica) as an experimental model system for current molec- ular genetic analyses (Garcia-Fernandez et al. 1993; Umesono et al. 1997), impor- tant findings have been accumulating to explain the molecular mechanisms underlying planarian regeneration. Planarian adult pluripotent stem cells, called neoblasts, produce all differentiated cell types needed for whole-body regeneration (Wagner et al. 2011). Neoblasts are the only known proliferating cells in planarians (Newmark and Sanchez Alvarado 2000) and are instructed to proliferate, migrate, and differentiate into the required cell types in response to injury. Recent technical advances such as global gene expression analysis, RNAi, and small molecule treat- ment have enabled researchers to identify the signaling cascades involved in the regeneration processes. These include Wnt, BMP, and hedgehog (Hh), all of which are known to be involved in Zic-mediated developmental controls in other animal models and in other developmental contexts. However, neither the involvement of the Zic protein in planarian regeneration nor its relationship to known biological signals has been explained. Here we review the role of planarian Zic in the head regeneration process in two recent independent reports (Vasquez-Doorman and Petersen 2014; Vogg et al. 2014).
There are two planarian Zic genes, zicA and zicB, in both S. mediterranea and D.
japonica (Aruga et al. 2006). S. mediterranea zicA and zicB are known as Smed- zic- 1 and Smed-zic-2 (hereafter zic1 and zic2) (Vasquez-Doorman and Petersen 2014), but zic1 is well characterized because of its functional redundancy and clear phenotype of zic1 loss-of-function analysis (Vasquez-Doorman and Petersen 2014).
zic1 is expressed in the anterior pole of intact planarians (Fig. 5.6a) (Vogg et al.
2014; Vasquez-Doorman and Petersen 2014). When the planarians were cut into three pieces to generate anterior (head), middle (trunk), and posterior (tail) pieces, zic1 expression was enhanced in the anterior-facing side of the head and tail pieces 18 h after amputation, and by 72 h, zic1 was strongly expressed in the anterior pole and in the surrounding region (Fig. 5.6b). Most zic1-expressing cells in the anterior pole were identified as neoblasts at 24 h after amputation. The zic1-expressing cells at the anterior pole also expressed notum (secreted Wnt inhibitor), foxD (transcrip- tion factor), or follistatin (secreted BMP inhibitor). Double labeling with neoblast markers indicated that some zic1-expressing neoblasts form zic1-expressing ante- rior pole cells.
zic1 knockdown during regeneration resulted in head defects, including cyclopia, absence of the eyes, and head regeneration failure (Fig. 5.6c) (Vasquez-Doorman and Petersen 2014; Vogg et al. 2014). In contrast, tail regeneration was not affected. In the marker analysis, head-defective zic1-suppressed planarians showed the absence of markers for the brain (gpas and chat), head tip (sFRP-1), head region (prep), and
Fig. 5.6 Role of Zic in planarian head regeneration. (a) Expression of zic1 in the head region and head tip in uninjured animals (arrow). (b) Expression of zic-1 in regenerating animals. Zic1 is expressed preferentially near anterior-facing versus posterior-facing amputation (arrows). (c) Animals 8 days after amputation of heads and tails after zic1 knockdown [zic-1 (RNAi)] and in the control [control (RNAi)]. (d) Zic-1 is required for anterior pole formation at 72 h and not for early wound- induced notum expression at 18 h of regeneration. (e) Programs underlying zic1-mediated anterior pole cell generation and maintenance. (f) Single and double RNAi as indicated to examine interac- tions between zic-1 and β-catenin (a–d, f Reprinted from Vasquez-Doorman and Petersen 2014)
anterior pole (notum, follistatin, and foxD). The normal tail regeneration and intact neoblast-specific marker expression suggested that zic1 may be required not for the maintenance of neoblasts but for the specification of anterior pole cell types.
Concerning the zic1 downstream targets during head regeneration, ovo, notum, and distalless (dlx) were reduced in the X1 subpopulation of neoblasts from zic1- knockdown planarians (Vasquez-Doorman and Petersen 2014). Ovo is a zinc- finger- type transcription factor required for eye regeneration in planarians and is known to be required for germline development in both mouse and Drosophila (Hayashi et al.
2017). Distalless is also required for planarian eye regeneration (Lapan and Reddien 2011). Notum is required for head regeneration and is expressed in the anterior pole (Petersen and Reddien 2011). Accordingly, Vasquez-Doorman and Petersen (2014) found that notum-expressing neoblasts are reduced in zic1-inhibited regenerating heads (Fig. 5.6d), and Vogg et al. (2014) found that notum-expressing anterior pole cells and follistatin-expressing anterior pole cells were reduced in zic1-knockdown regenerating heads.
Interestingly, the regulatory relationship among the zic1, notum, and follistatin is not limited to the regeneration process. This idea comes from the analysis of ante- rior pole cells in intact planarians. The anterior pole cells in intact head tissue expressed notum, foxD, follistatin, and zic1. Prolonged zic1 RNAi treatments resulted in the disappearance of notum expression in the anterior pole cells, whereas notum RNAi did not affect zic1 expression. These results indicate that zic1 acts upstream of the secreted signaling molecules and that zic1 plays a role in the main- tenance of the anterior pole cells. These results suggest the presence of a “zic1→ notum/follistatin” regulatory relationship in both injury-induced regeneration and cell maintenance in intact tissue (Fig. 5.6e).
The relationship between foxD and zic1 is an intriguing issue addressed by the two studies mentioned above. Forkhead transcription factor, foxD, also promotes the specification of midline neoblasts for anterior pole regeneration. Vogg et al.
(2014) identified zic1 as a gene that is downregulated in the foxD1-suppressed regenerating head at 72 h after amputation, and foxD/zic1 suppression showed addi- tive effects on the severity of eye defects. The co-expression of zic1 and foxD was small at an early stage of regeneration, but was extensive in anterior pole cells at 72 h after amputation (Vasquez-Doorman and Petersen 2014). The co-expression profiles of notum-zic1 and follistatin-zic1 were similar to that of foxD-zic1.
Therefore, zic1, notum, follistatin, and foxD are not co-expressed early after head amputation, but are later co-expressed at the regenerating anterior pole.
The underlying mechanisms of zic1 induction after wounding have also been reported. Wnt1 and notum are expressed prior to zic1 induction in response to injury.
In functional terms, RNAi-mediated notum knockdown reduced zic1 expression on the anterior side, and wnt1 knockdown resulted in ectopic zic1 expression on the posterior side. The involvement of Wnt signaling in the activation of zic1 expression was further confirmed by the knockdown of Wnt signaling downstream components (Vasquez-Doorman and Petersen 2014). This study concluded that Wnt signaling inhibition by early injury-induced notum is necessary and sufficient to activate early zic1 expression by 24 h (Fig. 5.6e) (Vasquez-Doorman and Petersen 2014).
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In previous studies, Wnt signaling was shown to play a key role in head regenera- tion. Functional interaction between zic1 and wnt signaling was further investigated by combining β-catenin (wnt canonical pathway component) knockdown and zic1 knockdown (Fig. 5.6f). When β-catenin was solely suppressed in the trunk pieces, ectopic head regeneration occurred from the posterior side, consistent with its key role in head regeneration. zic1 suppression causes the loss of head structure and intact tail as above. But the combined knockdown treatment abolished the zic1 effect, identical to β-catenin knockdown animals (Fig. 5.6f). This result indicates that β-catenin inhibition can promote head regeneration in the absence of zic1, sug- gesting a critical role of β-catenin inhibition in zic1-mediated head regeneration (Fig. 5.6e).
Besides the components of Wnt signaling, other candidate genes were examined to determine their requirements for zic1 expression activation after amputation.
Follistatin knockdown resulted in the reduction of the zic1-expressing cells at 24 h after amputation. FoxD knockdown resulted in the reduction of zic1 expression 3 days after amputation (Vogg et al. 2014). In addition, Pbx1 (homeodomain tran- scription factor) knockdown also reduced the number of zic1-expressing cells and zic1 expression levels 24 days after amputation.
Based on these two studies, the role of planarian Zic in head regeneration can be summarized as below (Fig. 5.6e). At the anterior side of the wounds, zic1 expression in the stem cells (neoblast) is induced by the secretory wnt signaling inhibitor (notum).
zic1 in the stem cells is necessary for the wnt signaling inhibitor and TGFβ (activin) signaling antagonist (follistatin). The anterior-most zic1-expressing stem cells are called anterior pole cells and also express the winged helix-type transcription factor foxD. zic1 and foxD cooperate to establish the anterior pole cell properties.
The method of zic1 involvement in planarian head regeneration is intriguing when compared with the involvement of vertebrate Zic family in the repression of Wnt-β-catenin signaling (Fujimi et al. 2012; Pourebrahim et al. 2011). Xenopus Zic3 suppresses β-catenin signaling to control the organizer, which emanates TGFβ (BMP) signaling antagonists (chordin, follistatin, etc.) (Fujimi et al. 2012). On the other hand, the CNS disorganization caused by planarian zic1 RNAi is similar to brain abnormalities in holoprosencephaly patients caused by ZIC2 loss-of-function mutations (Brown et al. 1998) and in Zic2 knockdown mutant mice (Nagai et al.
2000). In either case, the midline region of the “brain” is impaired. Despite such phenotypic similarities, planarian head regeneration may provide a promising experimental system to clarify the evolutionary conserved gene regulatory network for brain establishment.