Lessons on transparency from the glass frog has potential implications for human blood clotting
Transparency in glass frogs has potential implications for human blood clotting
INTRODUCTION
When cells differentiate during embryonic development or adult regeneration, they acquire specific characteristics, a specialization compensated by a loss of plasticity (1). The protection of the identity of terminally differentiated cells relies on mechanisms that are poorly understood. Therefore, model systems that readily reactivate developmental processes, such as the freshwater polyp Hydra, help decipher the mechanisms underlying the balance between plastic cell fates and stable cell identities. Hydra are simple animals whose body is organized along a single oral/aboral axis. On the oral end, the head structure includes the mouth surrounded by tentacles, while on the aboral end a foot structure is used that allows the animal to attach to solid substrates. The tip of the head, named the hypostome, harbors a head organizer required to maintain apical patterning in intact animals. In line with predicted auto- and cross-catalytic regulations (2), the head organizer activity is positively regulated by Wnt3/β-catenin signaling (3–5) and restricted by the transcription factor Sp5 (6). When Sp5 expression is knocked down, Wnt3/β-catenin signaling is depressed along the body column and ectopic head formation occurs (6). During apical regeneration, the head organizer is reestablished from somatic gastric tissue within 10 to 12 hours (7); this de novo organizer instructs the formation of the complete head, including hypostome and tentacles, a role also under the control of Wnt3/β-catenin signaling and restricted by the transcription factor Sp5. Besides Wnt3/β-catenin signaling, signaling via protein kinase C (8) and Notch (9) positively modulate tentacle formation, while casein kinase 1 activity acts negatively (10). Some additional regulators contribute to tentacle formation like the orphan protein ks1 (11, 12), the peptide Hym-301 (13, 14), and the homeodomain transcription factors HyAlx, possibly via protein kinase C (PKC) signaling (15), and Margin, downstream of Wnt signaling (16). However, it is still unclear how cells at the base of the hypostome acquire a tentacle identity.
Hydra polyps are composed of two epithelial cell layers, the epidermis and the gastrodermis, which in the body column are populated with three distinct stem cell populations, the epidermal and gastrodermis epithelial stem cells (ESCs), and the multipotent interstitial stem cells (ISCs) (17). The ESC populations are multifunctional differentiated epithelial cells that proliferate continuously in the body column and terminally differentiate when they reach the apical (head) or basal (foot) regions (18). When epidermal epithelial cells located in the upper body column reach the tentacle zone, they become committed to the tentacle battery cell (TBC) fate and differentiate as TBCs once they enter the tentacle roots (19–21). This process is characterized by a cell cycle arrest in G2, the change of epidermal epithelial cell shape, and the incorporation by each TBC of 10 to 20 mechanosensory cells called nematocytes (or stinging cells), one large located centrally named stenotele surrounded by numerous smaller ones (22, 23). Each nematocyte houses a venom capsule named nematocyst, whose content is discharged when a specialized cilium, the cnidocil, is stimulated (24). More distally, the epidermal layer of tentacles is exclusively made of fully differentiated TBCs that get passively displaced toward the tentacle tips and are ultimately sloughed off after several days (19, 25). At the basal extremity, the epidermal epithelial cells of the basal disc (foot) terminally differentiate as basal disc cells (BDCs), which produce acid mucopolysaccharide (MPS) droplets that facilitate the attachment to substrates (26). Thus, both the tentacles and the foot are made of highly differentiated cells.
Cellular plasticity in Hydra is well documented for cells of the interstitial lineage, namely, gland cells and nerve cells (27–29). Nevertheless, Hydra depleted of their interstitial cell lineage can fully regenerate (30, 31), highlighting the key role of epithelial cells in the reactivation of developmental processes. We actually showed that in such animals, epithelial cells modify their genetic program within a week following depletion of the interstitial cells, indicating their high potential for plasticity (32). However, the ability of terminally differentiated epithelial cells to acquire an alternative identity in Hydra is unknown. Here, we identified a zinc finger transcription factor, Zic4, expressed under the control of Wnt3/β-catenin signaling and Sp5, which contributes to induce tentacle cell fate. When Zic4 is knocked down, the maintenance and proper development of tentacles is impaired and, unexpectedly, the TBCs transdifferentiate into BDCs. The transformed tentacles are able to attach to substrates, thus adopting a functional foot behavior. This marked change in cell identity, because of a decrease in the expression level of a single transcription factor, provides insight into the gene regulatory networks governing the choice between two epithelial cell fates in Hydra. Given the broad conservation of the genetic module that regulates Zic4, this network might also operate in other metazoans.
RESULTS
The Zic4 transcription factor gene is an Sp5 target gene
Exploiting RNA-sequencing (RNA-seq) and chromatin immunoprecipitation–sequencing (ChIP-seq) data in human embryonic kidney (HEK) 293T cells overexpressing the Hydra and zebrafish Sp5 transcription factors, we have previously identified 83 putative Sp5 direct target genes (6). From the 80 targets detected in the Hydra transcriptome, we identified the zinc finger transcription factor Zic4 as the unique gene displaying an expression pattern that is graded from apical toward the basal end of the animal (Fig. 1, A and B; fig. S1; and dataset S1) (33). We thus focused on this transcription factor to characterize its role and mechanism of action. We found Zic4 predominantly expressed in the tentacle zone of intact animals, reexpressed between 4 and 8 hours post-amputation (hpa) in apical-regenerating tissues (Fig. 1C and fig. S2). Among the four Zic-related genes expressed in Hydra, Zic4 is the only one to display these features (figs. S1 and S2 and table S1).
Zic4 appears positively regulated by Sp5 and Wnt/β-catenin signaling in homeostatic conditions, as Zic4 expression is notably reduced in the tentacle zone after Sp5 or β-catenin knockdown by RNA interference (RNAi) (Fig. 1, D and E, and fig. S3A). To test Zic4 regulation in conditions where organizer activity gets reestablished in multiple spots (34), we performed reaggregation experiments from tissues knocked down for β-catenin (Fig. 1F and fig. S3B). In control conditions, Hydra reaggregates can regenerate and present axes that form tentacles emerging 5 days after the second electroporation (EP2). When β-catenin is down-regulated, the levels of Zic4 are reduced about twofold and the aggregates cannot form tentacles in the manner control animals do. The Wnt/β-catenin signaling pathway can also be ubiquitously activated by incubating the animals in alsterpaullone (ALP), a treatment that efficiently promotes stabilization of β-catenin in Hydra (35). After a 2-day treatment, we observed a dual regulation for Zic4, first a transient down-regulation in the apical region accompanied by an overall up-regulation along the body column, followed 2 days later by the appearance of an “Octopus” phenotype, characterized by multiple Zic4-expressing rings, each ring corresponding to the base of an ectopic tentacle (Fig. 1G and fig. S4). This dynamic expression pattern of Zic4 both in the tentacle zone and along the body column when Wnt/β-catenin signaling is up-regulated indicates that Zic4 is downstream of Wnt/β-catenin signaling.
The Zic4 promoter is positively regulated by Wnt/β-catenin and Sp5
To test whether Wnt/β-catenin regulates Zic4 directly, we characterized a 3505–base pair (bp) fragment upstream to the Zic4 transcription start site and produced a Zic4-3505:GFP transgenic line that drives green fluorescent protein (GFP) fluorescence in the tentacle zone mimicking the Zic4 expression pattern (Fig. 1H). Upon ALP treatment, the expression of GFP is enhanced in the upper body column, indicating a positive regulation of the Zic4-3505 promoter via canonical Wnt signaling (Fig. 1H). This regulation is possibly direct as the 3505-bp Zic4 upstream sequences contain four consensus T cell factor/lymphoid enhancer factor (TCF/LEF) binding sites (Fig. 2A). When expressed in HEK293T cells, the Zic4-3505:luciferase construct is up-regulated when human β-catenin, Wnt3, LRP6, or full-length Sp5 are overexpressed (Fig. 2, B and C). We produced a series of deletion mutants of the Zic4-3505 promoter (Fig. 2D) and noted a two-step reduction in the levels of luciferase in HEK293T cells in the presence of active β-catenin when the most upstream TCF-BS1 is deleted and then when the most proximal TCF-BS4 is additionally removed (Fig. 2E). In the presence of Hydra Sp5, we recorded the most significant reductions of luciferase activity when three regions are removed: (i) the most upstream 500 bp (−3505 to −2999), (ii) the −1985 to −1478 region, and (iii) the −978 to −463 region that contains TCF-BS4 and Sp5-BS5 (Fig. 2F). These data demonstrate a positive regulation by Wnt/β-catenin signaling and Sp5 on Zic4 expression, likely direct in the mammalian HEK293T cells. Overall, we concluded that Zic4 is an excellent candidate for a transcription factor directly regulated by Wnt/β-catenin and Sp5, during both homeostasis and regeneration in Hydra.
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