Abstract
Hydra head regeneration consists of hypostome/organizer and tentacle development, and involves Notch and Wnt/β-catenin signaling. Notch inhibition blocks hypostome/organizer regeneration, but not the appearance of the tentacle tissue. β-Catenin inhibition blocks tentacle, but not hypostome/organizer regeneration. Gene expression analyses during head regeneration revealed the Notch-promoting expression of HyWnt3, HyBMP2/4, and the transcriptional repressor genes CnGsc, Sp5, and HyHes, while blocking HyBMP5/8b and the c-fos–related gene HyKayak. β-Catenin promotes the expression of the tentacle specification factor HyAlx, but not of HyWnt3. This suggests HyWnt3 and HyBMP4 as parts of a hypostome/organizer gene module, and BMP5/8, HyAlx, and β-catenin as parts of a tentacle gene module. Notch then functions as an inhibitor of tentacle production to allow regeneration of a hypostome/head organizer. HyKayak is a candidate target gene for HvNotch-induced repressor genes. Inhibiting HyKayak attenuated the expression of HyWnt3. Polyps of Craspedacusta do not have tentacles and thus after head removal only regenerate a hypostome structure. Notch signaling was not needed for head regeneration in Craspedacusta, corroborating the idea of its requirement during Hydra head regeneration to harmonize two co-operating pattern-forming processes.
Introduction
The small freshwater polyp Hydra belongs to the pre-bilaterian phylum of Cnidaria and consists of a foot, a body column, and a head with a hypostome and a ring of tentacles. Asexual reproduction occurs by budding. Sexual reproduction takes place from fertilized eggs when male and female gametes are formed on the Hydra body column (reviewed by Steele [2012]).
Hydra polyps have the capacity for complete regeneration. After being cut into small tissue parts, they will regenerate a head and a foot accurately at the same position as before. This indicates that whole-body pattern information is conserved in the body column during the adult life of Hydra polyps (reviewed by Bode [2003]). Moreover, as observed in 1909 by Ethel Browne, specific Hydra tissues, after transplantation into a host polyp, have the capacity to recruit host tissue to form an ectopic head growing out into a whole new hydranth (Browne, 1909; MacWilliams, 1983). These tissues included “peristome at the base of tentacles,” regenerating tips and early buds (according to Ethel Browne). By hypostome-contact grafts, it could be shown later that the tip of the hypostome had the same capability. Less “inductive” capacity was found in the tissue of the tentacle zone (Mutz, 1930; Broun & Bode, 2002). Embryonic amphibian tissue with such inductive capacity had been given the name “organizer” by Hans Spemann, and the region where this tissue was taken from was called “center of organization” (Spemann, 1924; Hamburger, 1969). The Hydra transplantation phenomena were then related to the “organizing” property of the transplanted embryonic tissue (Goetsch, 1926). The “organizer effect” entails a “harmonious interlocking of separate processes that makes up development,” or a side-by-side development of structures independently of each other (Spemann, 1935). In addition to inducing the formation of such structures, the organizer must ensure their patterning (Anderson & Stern, 2016). Formation of new hydranths after transplantation of “organizer” tissue involves the side-by-side induction of the hypostome tissue and tentacle tissue. Moreover, it includes the establishment of a regularly organized ring of tentacles with the hypostome doming up in the middle. The function of the Hydra “center of organization” would then be to pattern hypostome/body column and tentacles and to allow for their harmonious re-formation after head removal.
There is an intriguing similarity in gene expression between the amphibian Spemann organizer and the Hydra head organizer (Ding et al, 2017). Spemann organizers induce a Wnt3-dependent anterior–posterior axis and a BMP-dependent dorsal–ventral axis (Anderson & Stern, 2016). The Hydra gene HyWnt3 is strongly expressed at the hypostome, at the tip of regenerates after head removal, and at the tip of developing buds, all regions that had been indicated to possess inductive capacity in organizer experiments (Browne, 1909; Mutz, 1930; Broun & Bode, 2002). In addition, the transcriptional repressor goosecoid is expressed in dorsal blastopore lip cells of frog embryos and had originally been considered a universal organizer gene (Anderson & Stern, 2016). In the Hydra head, CnGsc, a goosecoid homolog, is prominently (not solely) expressed in head cells between the hypostome and the tentacle zone (Broun et al, 1999), and thus in the organizer tissue as defined by Ethel Browne.
Hydra has 11 identified Wnt genes, all of which are expressed in the head and/or tentacles. Of those, most are suggested to induce canonical Wnt signaling through nuclear translocation of β-catenin, whereas HyWnt5 and HyWnt8 have been shown to be associated with non-canonical Wnt signaling in the planar cell polarity pathway. In addition, most known mammalian BMP pathway genes have homologs in Hydra. These include Smad, HyBMP5/8b, and HyBMP2/4 (Hobmayer et al, 2001; Reinhardt et al, 2004; Lengfeld et al, 2009; Philipp et al, 2009; Watanabe et al, 2014). Wnt and BMP pathways have been demonstrated to play a role in Hydra regeneration ([Reddy et al, 2019; Reddy et al, 2020] and citations above). After head removal, the expression of Hyβ-catenin and HyTcf is up-regulated earliest, followed by local activation of Wnt genes. Among these, HyWnt3 and HyWnt11 appeared within 1.5 h after head removal, followed by HyWnt1, HyWnt9/10c, HyWnt16, and HyWnt7 (Hobmayer et al, 2001; Lengfeld et al, 2009; Philipp et al, 2009; Gufler et al, 2018; Tursch et al, 2022). Thus, HyWnt3 and HyWnt11 are swiftly induced by injuries. When their activity is sustained, organizers can be formed, which induce ectopic heads when the original organizer tissue (the head) is removed (Cazet et al, 2021; Tursch et al, 2022). Recently, a Wnt3/β-catenin/Sp5 feedback loop was suggested to be involved in Hydra head patterning (Münder et al, 2013; Vogg et al, 2019; Moneer et al, 2021).
The expression patterns of Wnt and BMP genes can be interpreted as an indication of tentacles, buds, and the main body axis of the polyps being repetitive structures expressing Wnt genes at the apical end and BMP5/8b at the basal end (Meinhardt, 2012; Pan et al, 2024). These could set up opposing signaling gradients to pattern the Hydra body axis and possibly also the bud and tentacle axes. The bud expresses HyWnt2 and later HyWnt3 at the tip and BMP5/8b at the base. The tentacles also express HyBMP5/8b at the base and HyWnt5 at the tip. As Hans Meinhard pointed out, in evolutionary terms the tentacles may therefore be considered as colonialized buds (Meinhardt, 2012). In any case, tentacles and hypostome can be interpreted as independent structures.
Our previous investigations had revealed that the Notch pathway was instrumental for head regeneration and organizer formation by supporting the expression of a strong HyWnt3 signal in regenerating the head tissue. Notch inhibition with the presenilin inhibitor DAPT or the NICD inhibitor SAHM-1 prevented head regeneration and blocked HyWnt3 expression in regenerates, while not preventing the expression of the tentacle boundary gene HyAlx and the tentacle metalloprotease gene HMMP. However, the latter did not obtain their correct expression patterns, and thus, proper tentacles were not formed. Similar experiments using a transgenic Hydra strain expressing an HvNotch–hairpin RNA confirmed the regeneration phenotypes seen with pharmacological inhibitors (Pan et al, 2024). Strikingly, transplantation experiments had revealed that the DAPT-treated regenerating head tissue had lost the capacity to form an organizer (Münder et al, 2010; Münder et al, 2013).
Here, we have further investigated the role of Notch signaling during apical head regeneration. We compared the effect of the Notch inhibitor DAPT with the effect of the β-catenin inhibitor iCRT14 (Gonsalves et al, 2011; Gufler et al, 2018). Although, similar to DAPT, iCRT14-treated animals did not regenerate complete heads, HyWnt3 expression was not blocked and a normal hypostome was regenerated. Accordingly, iCRT14-treated—in contrast to DAPT-treated—regenerating tips retained the ability to form a second axis when transplanted into the body column of an untreated host animal. We also investigated the effect of these inhibitors on the gene expression dynamics of HyWnt and HyBMP genes and transcriptional regulators Hydra Sp5, HyAlx, HyHes, and CnGsc during Hydra head regeneration by qRT–PCR. Our results clearly reveal that the sustained expression of HyWnt3 and hypostome/organizer formation after head removal are controlled by Notch signaling, and not by β-catenin activity. In contrast, the expression of the tentacle specification gene HyAlx and formation of tentacles are dependent on β-catenin activity. In addition, we noted that Notch inhibition increased the expression of HyBMP5/8b, a gene primarily expressed at tentacle boundaries, while blocking the expression of HyBMP2/4, a gene expressed in the head and body column. Moreover, Notch was required for inhibition of the c-fos homolog HyKayak, which we suggest to be a negative regulator of HyWnt3 and a likely candidate for a target of Notch-regulated transcriptional repressors.
We conclude that Notch activity functions in head regeneration to mediate between two independent patterning systems comprising hypostome and tentacle regeneration. In apical regenerates, this probably works through inhibition of the tentacle system in a spatially and temporarily regulated manner. It involves Notch-mediated inhibition of HyBMP5/8b and direct or indirect activation of HyWnt3 and HyBMP2/4 expression.
Results
Hypostome formation in iCRT14-treated, but not in DAPT-treated, regenerates
Hydra polyps treated either with iCRT14, as described by Gufler et al [2018]; Cazet et al [2021], or with the Notch inhibitor DAPT, as described by Münder et al [2013], fail to regenerate a complete head after decapitation. DAPT blocks Notch intramembrane proteolysis regulated by presenilin and prevents NICD translocation to the nucleus, thus phenocopying loss of Notch function in several organisms including Hydra (Dovey et al, 2001; Geling et al, 2002; Micchelli et al, 2003; Käsbauer et al, 2007; Pan et al, 2024). iCRT14 inhibits the interaction of nuclear β-catenin with TCF in mammalian cell lines and in Hydra (Gonsalves et al, 2011; Gufler et al, 2018).
First, we treated Hydra polyps with 5 μM iCRT14 for 48 h after head removal, and observed that they did not regenerate their heads during the time of treatment, whereas control animals, treated with 1% DMSO (the solvent for iCRT14 and DAPT), clearly showed regularly spaced tentacle buds at this time point (Fig 1A). iCRT14 and DMSO were then replaced with normal Hydra medium. Control animals regenerated heads with long tentacles 24 h later (72 h); however, iCRT14-treated animals did still not show tentacle buds up to 48 h after iCRT14 removal (96 h). For comparison, treatment of head regenerates with DAPT had revealed in our previous study that proper heads could also not be regenerated during the time of treatment. When DAPT was then removed from the medium, irregular heads, dominated by the tentacle tissue, developed in 20% of regenerates (Münder et al, 2013).
(A) Head regeneration progress of Hydra polyps at indicated time points after head removal in control or iCRT14 medium. (B) Semithin sections covering the hypostome region of Hydra polyps after the Richardson staining: untreated polyp (uncut head); polyps 48 h after head removal and regeneration in Hydra medium (HM); and HM with DMSO (control), DAPT, or iCRT14. The mesoglea appears as a dark blue line, and red lines are added to highlight the mesoglea at the hypostome region. Scale bar: 20 μm. (C) Confocal stack images covering the regenerated hypostome region of Hydra polyps of strain AEP “watermelon” 48 h after head removal and regeneration in HM with DMSO (control), DAPT, or iCRT14; GFP present in ectodermal cells, dsRed present in endodermal cells, and DAPI-DNA stain are imaged as indicated. Right-hand panels show merged images. White arrows indicate hypostomal opening in iCRT14-treated and control animals, but not in DAPT-treated polyps. Scale bar: 10 μm. (D) Fluorescence in situ hybridization for HyWnt3 (green) expression in polyps 48 h after head removal and regeneration in iCRT14 or in DMSO control, as indicated. Scale bar: 100 μm, DAPI in blue. (E) Quantification of regeneration of Hydra head structures: mouth and tentacles, 48 h after head removal and regeneration in DMSO control, DAPT, and iCRT14. Data are shown as the mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001.
To further inspect the morphology of head regenerates treated with DAPT or iCRT14, semithin sections were prepared 48 h after head removal and histologically stained with the Richardson tissue stain. Among other structures, this dye stained the mesoglea dark blue. Fig 1B shows middle sections of polyps. The mesoglea is emphasized by red lines. The hypostome of the polyp can be recognized by a “gap” in the mesoglea. After head removal, the hypostome is regenerated in polyps treated with DMSO and iCRT14, but not with DAPT. Head regeneration of the “watermelon” AEP strain of Hydra vulgaris polyps showed a similar result (Fig 1C). These polyps express GFP in the whole of the ectoderm and red fluorescent protein (dsRed) in the whole of the endoderm (polyps were a kind gift from Rob Steele, UC Irvine). Fig 1C shows optical middle sections obtained by laser scanning microscopy clearly representing a mouth opening. Again, hypostome morphology is recovered in animals after regeneration in DMSO and iCRT14, but not in DAPT. Quantification of regenerated hypostomes and tentacles in DAPT- and iCRT14-treated regenerates in comparison with control animals revealed that 70% of iCRT14-treated animals regenerated an intact hypostome with a detectable mouth opening, whereas tentacles were not formed (Fig 1E). In contrast, DAPT-treated animals did not regenerate a mouth opening, and in 25% of regenerates, aberrant tentacles were observed at the tips of regenerates, as previously described (Münder et al, 2013). The apparent regeneration of a hypostomal mouth opening in iCRT14-treated polyps prompted us to perform fluorescence in situ hybridization for HyWnt3 in such regenerates. As shown in Fig 1D, hypostomal HyWnt3 expression was evident in control regenerates and showed a very similar pattern in regenerates treated with iCRT14. This was different from DAPT-treated regenerates, which do not express HyWnt3 (Münder et al, 2013).
Organizer formation observed in iCRT14-treated regenerates
Previously, we had shown that DAPT-treated regenerating Hydra heads lacked organizer activity, as they did not induce the formation of ectopic hydranths when transplanted into the body column of a host animal (Münder et al, 2013). This was in accordance with the loss of HyWnt3 expression in Notch-inhibited regenerates. We now asked the question whether iCRT14-treated head regenerates would retain organizer properties, as they do express HyWnt3. We therefore transplanted regenerating Hydra heads (upper 20% of polyps) 24 h after head removal and treatment with iCRT14 or DMSO (for control) into the body column of Evans blue–stained host animals (Fig 2A). Fig 2B shows that 80% of control regenerates formed ectopic hydranths after transplantation into the body column of the host. Notably, 80% of iCRT14-treated regenerates were also able to form ectopic hydranths and most of them recruited host tissue, indicating organizer activity. This is in accordance with their expression of HyWnt3. From these and previous data, we conclude (1) organizer activity correlates with the presence of HyWnt3 expression; (2) activation of HyWnt3 during the regeneration process is not dependent on β-catenin transcriptional activity; and (3) HyWnt3 must signal via a non-canonical Wnt signaling pathway in iCRT14-treated regenerates.
The tissue from the regenerating tip of polyps 24 h after head removal in HM with DMSO (control), or iCRT14 was transplanted into the middle of blue host polyps stained with Evans blue. (A) Microscopic images were taken 48 h after transplantation. Newly formed secondary axes are indicated by yellow arrows. The transplanted tissue appears orange, the host tissue appears blue, and feet are indicated by F. (B) Percentage of transplants forming new axes in HM with DMSO (control), or iCRT14; differences are not significant (n.s.).
Comparison of gene expression dynamics during Hydra head regeneration in DAPT-treated and iCRT14-treated animals
In order to follow the recovery of head-specific gene expression after head removal, we conducted qRT–PCR analyses from tissue that was left to regenerate. We compared gene expression in regenerates treated with DAPT or with iCRT14, both compounds were administered with 1% DMSO in Hydra- medium (HM). For control, the polyps were treated with 1% DMSO in HM without additional compounds.
Effect of Notch inhibition on gene expression dynamics during head regeneration in Hydra
In a previous transcriptome analysis of DAPT-treated Hydra polyps, besides HyHes, the tentacle boundary gene HyAlx, the “organizer” gene CnGsc, and the Hydra Sp5 gene had been suggested to be potential direct Notch target genes (Moneer et al, 2021). The same analysis had revealed that the fos-related transcription factor gene HyKayak was up-regulated when Notch signaling was blocked.
Here, we performed qRT–PCR analysis to compare gene expression dynamics of these genes during head regeneration 0, 8, 24, 36, and 48 h after head removal. Animals were either treated with 30 μM DAPT in 1% DMSO, or with 1% DMSO as a control. Time point 0 was measured immediately after head removal. The results of these analyses revealed that HyHes expression was clearly inhibited by DAPT during the first 36 h after head removal (Fig 3A), confirming previously published data that had indicated HyHes as a direct target for NICD (Münder et al, 2010). HyAlx expression levels were slightly up-regulated after 24 h, but later partially inhibited by DAPT (Fig 3B). CnGsc expression under DAPT treatment initially (8 h) was comparable to control levels, but then, it was strongly inhibited (Fig 3C). This corresponds to the observed absence of organizer activity in regenerating Hydra tips (Münder et al, 2013). Interestingly, a similar result was seen for HySp5 expression, which was also normal at 8 h but was then inhibited by DAPT at later time points (Fig 3D). HyKayak, while not affected after 8 h, was strongly overexpressed between 24 and 36 h of regeneration in DAPT-treated polyps in comparison with control regenerates (Fig 3E). However, at the 48-h time point expression appeared normal.
(A, B, C, D, E, F, G) qRT–PCR measurements quantifying the gene expression of (A) HyHes, (B) HyAlx, (C) goosecoid, (D) Sp5, (E) HyKayak, (F) BMP2/4, and (G) BMP5/8b. (H, I, J, K, L, M) HyWnt3, HyWnt1, HyWnt7, HyWnt9/10c, HyWnt11, and HyWnt16 during 48 h of Hydra head regeneration in HM with DAPT (orange) or DMSO control (blue). Hydra cartoons indicate gene expression patterns according to published in situ hybridization data and single-cell sequencing atlas (Broun et al, 1999; Hobmayer et al, 2000; Smith et al, 2000; Reinhardt et al, 2004; Lengfeld et al, 2009; Münder et al, 2010; Watanabe et al, 2014; Siebert et al, 2019; Vogg et al, 2019); Relative normalized expression was calculated against the housekeeping genes GAPDH, RPL13, EF1alpha, and PPIB. Regeneration time is shown on x-axes; t = 0 refers to animals immediately after the head was removed. Data are shown as the mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001.
In addition, we tested the expression dynamics of the two BMP homologs described in Hydra, HyBMP5/8b and HyBMP2/4. They have mutually exclusive expression patterns in the head. BMP2/4 is expressed in endodermal and ectodermal epithelial cells of the head, whereas BMP5/8b expression is restricted to the base of tentacles and is not found in apical head cells (Reinhardt et al, 2004; Watanabe et al, 2014; Siebert et al, 2019). Interestingly, the two BMP genes were conversely affected by Notch inhibition. HyBMP2/4 expression was blocked with DAPT beginning at 24 h of regeneration (Fig 3F). In contrast, HyBMP5/8b expression was drastically increased (Fig 3G).
We had previously shown by in situ hybridization that HyWnt3 is not expressed in DAPT-treated head regenerates (Münder et al, 2013). This was confirmed now by qRT–PCR measurements, which revealed that HyWnt3 expression was comparable to the control group 8 h after head removal. However, after this time point, its expression was strongly inhibited by DAPT and almost completely lost after 36 and 48 h (Fig 3H). Eventually, we analyzed most of the Wnt genes suggested to engage in canonical Wnt signaling, including HyWnt1, HyWnt7, HyWnt9/10c, HyWnt11, and HyWnt16 (Lengfeld et al, 2009). In the presence of DAPT, these genes all exhibited similar expression levels to the control group 8 h after head removal, but between 24 and 48 h, the expression of HyWnt1, HyWnt7, HyWnt9/10, and HyWnt16 declined to almost zero (Fig 3I–M). As an exception, HyWnt11 was only partially inhibited and even appeared up-regulated after 48 h (Fig 3L).
In summary, qRT–PCR analyses showed that Notch signaling during Hydra head regeneration is necessary for activating all HyWnt genes, which are expressed in the Hydra head region and implicated in canonical Wnt signaling. Notch is also necessary for activation of the expression of BMP2/4, a gene expressed in the Hydra head and body column. Moreover, Notch is contributing to the expression of transcriptional repressor genes, HyHes and CnGsc. In contrast, HyBMP5/8b and HyKayak seem to be subject to inhibition by Notch signaling. HyAlx, although previously identified as a Notch target gene, is only partially inhibited by DAPT during head regeneration.
Effect of β-catenin inhibition on gene expression dynamics during head regeneration in Hydra
Next, following the same procedure as described for DAPT, we compared the gene expression dynamics of iCRT14-treated regenerates with control regenerates. We found that the expression of the Notch target gene HyHes remained similar to control regenerates up to 24 h, but then was attenuated (Fig 4A), possibly because of the failure of tentacle boundary formation, the tissue where HyHes is strongly expressed. HyAlx expression was completely abolished by iCRT14, consistent with the observation that iCRT14-treated head regenerates did not regenerate any tentacles (Fig 4B). Furthermore, we found that CnGsc levels in iCRT14-treated regenerates remained similar to control regenerates up to 24 h, but reached only half of the control levels after 48 h, similar to HyHes (Fig 4C). Sp5 did not significantly respond to iCRT14 treatment (Fig 4D). The expression of HyKayak was decreased at 8 h after head removal in the presence of iCRT14, came back to normal after 36 h, and was suddenly increased after 48 h (Fig 4E), correlating with inhibition of the HyHes repressor. There were no significant changes in the expression dynamics of HyBMP2/4 and HyBMP5/8b between iCRT14-treated regenerates and controls (Fig 4F and G).
(A, B, C, D, E, F, G) qRT–PCR measurements quantifying the gene expression of (A) HyHes, (B) HyAlx, (C) goosecoid, (D) Sp5, (E) HyKayak, (F) BMP2/4, and (G) BMP5/8b. (H, I, J, K, L, M) HyWnt3, HyWnt1, HyWnt7, HyWnt9/10c, HyWnt11, and HyWnt16 during 48 h of Hydra head regeneration in iCRT14 (green) or DMSO for control (blue). Hydra cartoons indicate gene expression patterns according to published in situ hybridization data and single-cell sequencing atlas (Broun et al, 1999; Hobmayer et al, 2000; Smith et al, 2000; Reinhardt et al, 2004; Lengfeld et al, 2009; Münder et al, 2010; Watanabe et al, 2014; Siebert et al, 2019; Vogg et al, 2019); relative normalized expression was related to the housekeeping genes GAPDH, RPL13, EF1alpha, and PPIB. Regeneration time is shown on x-axes; t = 0 refers to animals immediately after the head was removed. Data are shown as the mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001.
Confirming FISH images shown in Fig 1D, HyWnt3 was not inhibited by iCRT14 during head regeneration; it even appeared slightly up-regulated at the 8-h time point (Fig 4H). In contrast to HyWnt3, the expression of all other canonical HyWnt genes was inhibited by iCRT14 during head regeneration. HyWnt1, HyWnt7, and HyWnt16 were inhibited throughout the whole regeneration period (Fig 4I, J, and M). HyWnt9/10c and HyWnt11 were blocked up to 36 h, but their expression levels returned to control values at 48 h (Fig 4K and L).
In summary, qRT–PCR analyses show that β-catenin transcriptional activity is not required for the expression of HyWnt3 during head regeneration. However, it is involved in up-regulating the canonical Wnt genes HyWnt1, HyWnt7, HyWnt9/10, HyWnt11, and HyWnt16. Moreover, HyAlx expression strongly depends on β-catenin activity. The expression of both HyHes and CnGsc seems strengthened by β-catenin during later regeneration stages, when β-catenin also seems to inhibit HyKayak expression. These effects on gene expression may be due to the failure of tentacle development in iCRT14-treated animals. In contrast, BMP2/4, BMP5/8, and Sp5 do not appear to be regulated by β-catenin during head regeneration.
From these analyses, we conclude (1) Notch signaling is responsible for the sustained expression of HyWnt3 and all canonical HyWnt genes during head regeneration. In addition, it is required for the expression of BMP2/4 (Broun et al, 1999) and the suggested Hydra organizer gene CnGsc, supporting our previous experiments where DAPT-treated regenerating head tissue did not develop organizer activity (Münder et al, 2013). (2) Notch activity is required for inhibiting HyKayak and HyBMP5/8b gene expression during regeneration, which coincides with DAPT causing down-regulation of the transcriptional repressor and Notch target gene HyHes. (3) β-Catenin transcriptional activity is not necessary to express HyWnt3, acquire organizer activity, and form a new hypostome after head removal. However, β-catenin–dependent transcription is indispensable to express HyAlx and form tentacles.
HyKayak
HyWnt3, albeit inhibited by DAPT specifically during head regeneration, had so far not been indicated as a potential target for Notch-mediated gene activation in Hydra (Münder et al, 2013; Moneer et al, 2021). By analyzing the HyWnt3 promoter region, Nakamura et al found proximal elements similar to Drosophila Su(H) and RBPJ sites (−155 to −143 [Nakamura et al, 2011]). Notch could therefore directly activate Wnt3 expression. However, several repressor genes are Notch-regulated (Moneer et al, 2021). We thus considered the possibility that a repressor of HyWnt3 could be inhibited by Notch signaling, especially at the tip of regenerating heads.
According to our previous report, the Hydra fos homolog HyKayak (t5966aep) was up-regulated after Notch inhibition with DAPT (Moneer et al, 2021). This suggests that HyKayak may serve as a potential target gene for Notch-regulated repressors including HyHes and CnGsc, and in this way, HyKayak may be inhibited when these repressors are activated by Notch signaling.
Analysis of the domain structure and sequence comparison of HyKayak with fos and jun sequences from Aurelia aurita, Stylophora pistillata, Caenorhabditis, Drosophila, mouse, and human revealed a strong conservation of the bZIP domain (basic leucine zipper domain), which is responsible for DNA binding and dimerization (Fig S1A and B). Phylogenetic analysis showed that HyKayak is related to c-fos sequences of various species including Hydra (Fig S1C). HyKayak is expressed in ectodermal cells of the Hydra head, tentacles, and body column, excluding the basal disk (Fig S1D) (Siebert et al, 2019). A second fos gene described by Cazet et al [2021] is expressed in epithelial cells and gland cells (referred to as fos_Cazet). In addition, we identified two transcripts encoding Jun-related proteins, HyJun_nem (t17964aep) expressed in nematoblasts 3–5 and HyJun_epi (t19405aep) expressed in all cells, with especially high levels in epithelial cells (Fig S1D). By SDS–PAGE of Hydra lysates and staining with anti-HyKayak antibody, we found that the HyKayak protein remained in the pellet fraction (Fig S1E-a) and only a small percentage could be solubilized after treatment with DNase (Fig S1E-b), suggesting that HyKayak is strongly associated with DNA and lending support to its suggested role as a DNA binding protein.
(A) Schematic representation of the HyKayak protein structure (191 amino acids). The bZIP domain with DNA binding and dimerization function is located between amino acids 120 and 183. (B) Alignment of the protein sequences of Hydra-Kayak, fos-Cazet, Jun-epi, and Jun-nem from Hydra vulgaris (HYDVU), FOS and JUN from human, mouse, Caenorhabditis elegans (CAEEL), Aurelia aurita (AURAU), Stylophora pistillata (STYPI), and Drosophila melanogaster (DROME); the bZIP domain is underlined, green background indicates amino acids involved in DNA binding, and violet background indicates amino acids of the dimerization interface. (C) Phylogenetic tree based on the alignment of 15 full-length protein sequences affiliated to the FOS and JUN families using MEGA software. Species code: Aurelia aurita (AURAU), Hydra vulgaris (HYDVU), human, mouse, Caenorhabditis elegans (CAEEL), Drosophila melanogaster (DROME), Stylophora pistillata (STYPI). (D) Dot-plot of the expression patterns for the genes HyKayak (t5966aep), fos_Cazet (t25302aep), HyJun_nem (t17964aep), and HyJun-epi (t19405aep) from the single-cell portal (Siebert et al, 2019). (E) Western blot was stained with anti-kayak antibody (in-house). Lysates from Hydra polyps indicating cytoplasmic proteins (CP), nuclear proteins (NP), pellets after centrifugation at 14,000g (Pellet1), supernatant after treatment of pellet with DNase, supernatant after treatment of pellet fraction with 2 M NaCl, and pellet fraction after both treatments (Pellet2).
Fos proteins interact with Jun proteins (also bZIP domain proteins) to form the transcriptional regulation complex AP-1 (activator protein 1) (Karin et al, 1997). To test such interactions for the Hydra proteins, we performed immunoprecipitation of HyKayak and HyJun_epi-proteins expressed in HEK293T cells. This revealed that HyKayak did not interact with itself, but strongly interacted with the HyJun_epi protein (Fig S2). To investigate the function of HyKayak/AP-1 in Hydra head regeneration, we used the Fos/jun inhibitor T5224 to block DNA binding activities of Fos/Jun complexes (Xiong et al, 2022), and analyzed gene expression and phenotypes during Hydra head regeneration. This revealed a mild inhibition of HyKayak expression in contrast to a strong up-regulation of HyJun (Fig 5A and B). In addition, we discovered that HyWnt3 expression was strongly up-regulated by T5224 (Fig 5C).
(A) EGFP-tagged Jun-epi was immunoprecipitated with GFP-Trap agarose beads and detected on Western blot using an anti-GFP antibody. Co-precipitation of HA-tagged Kayak was detected on Western blot using an anti-HA antibody in input, flow-through, and beads fractions. 4K + 2J: 4 μM Kayak-HA plus 2 μM Jun-epi-EGFP; 4K + 4J: 4 μM Kayak-HA plus 4 μM Jun-epi-EGFP; negative control: 4 μM Kayak-HA plus 2 μM empty pEGFP-C1 vector. I: input; F: flow-through; B: beads. (B) EGFP-tagged Kayak or Jun-epi were immunoprecipitated by GFP-Trap agarose beads and detected on Western blot using an anti-GFP antibody. Co-precipitation of HA-tagged Kayak was detected on Western blot using an anti-HA antibody in input, flow-through, and beads fractions. 4K + 2K: 4 μM Kayak-HA plus 2 μM Kayak-EGFP; 4K + 2J: 4 μM Kayak-HA plus 2 μM Jun-epi-EGFP, which was used for a positive control; the empty plasmid pEGFP-C1 was used for a negative control.
(A, B, C) qRT–PCR measurements quantifying the expression of (A) HyKayak, (B) HyJun_epi, and (C) HyWnt3 for 48 h after head removal in HM with DMSO serving as a control (blue) and HM with 7.5 μM T5224 for inhibition of Fos/AP1 activity (pink). (D, E, F) qRT–PCR measurements quantifying the expression of (D) HyKayak, (E) HyJun-epi, and (F) HyWnt3 after knockdown of HyKayak by shRNA (pink), compared to control knockdown with scramble shRNA (dark blue) and mock knockdown control (light blue). Relative normalized expression was related to the housekeeping genes GAPDH, EF1alpha, and PPIB. Regeneration time is shown on x-axes; t = 0 refers to animals immediately after the head was removed. Data are shown as the mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001.
To confirm the specificity of the T5224 effect, we knocked down HyKayak using shRNA directed against HyKayak. We achieved HyKayak knockdown by ca. 80% in comparison with control polyps either mock-treated or treated with scrambled control shRNA (Fig 5D). Moreover, Kayak knockdown led to the up-regulation of HyJun, consistent with the effects of T5224 treatment (Fig 5E). Importantly, knockdown of HyKayak induced an up-regulation of HyWnt3 (Fig 5F). From these data, we conclude that (1) HyKayak attenuates the expression of HyWnt3; (2) HyKayak may work within the AP-1 complex together with Jun-epi; and (3) Notch signaling may block the inhibitory activity of HyKayak on HyWnt3 by activating repressor genes. With DAPT, HyKayak remains active and inhibits the sustained expression of HyWnt3 at later stages of head regeneration.
Regeneration of Craspedacusta polyps
Our data dissect the regeneration of Hydra heads into two processes, formation of the hypostome and head and formation of tentacles. For hypostome formation, HyWnt3 is needed, but βcatenin transcriptional activity is dispensable. Notch signaling then appears to be responsible to “organize” these two morphogenetic processes. To test this hypothesis, we asked how the inhibition of Notch signaling might affect regeneration of polyps with a simpler, one-component head. We used polyps of the freshwater hydrozoan Craspedacusta sowerbii. They have a mouth opening that is surrounded by epithelial cells carrying nematocytes, but they do not possess tentacles (Ramos et al, 2017).
Craspedacusta polyps are shown in Fig 6A. They often occur as mini-colonies with one foot carrying two polyps. Actin fibers are running along the polyp’s body column and form a ring where the two polyps separate just above the foot. Actin cushions carrying nematocysts are visible and indicate the positions of capsules along the body column and in a ring surrounding the mouth opening (Fig 6A and B). Additional capsule staining with DAPI (Szczepanek et al, 2002) very clearly reveals the pattern of nematocysts in the head (Fig 6B). When we removed the heads of the polyps, most of them fully regenerated within 96 h (Fig 6B). Some retracted into a podocyst (the “dauerstadium”) (Fig 6D). Polyps treated with DMSO or DAPT also completed head regeneration after 96 h (Fig 6C). Quantification of Craspedacusta development after head removal revealed that the similar numbers of proper head regeneration and podocyst formation occurred (Fig 6D). This indicated that Notch signaling was not required for head regeneration in Craspedacusta polyps.
(A) Craspedacusta colony with two animals sharing one foot. Scale bar: 100 μm. Magnification of the polyp head with nematocytes surrounding the hypostome. Scale bar: 50 μm. Nematocyte capsules stained with DAPI (green). (B) Regeneration of Craspedacusta polyps at 24, 48, 72, and 96 h after head removal with reappearing nematocytes and actin fibers. Scale bar: 50 μm. (C) Regenerated heads 96 h after head removal in HM with DMSO (control) or DAPT as indicated. Scale bar: 50 μm. Actin fibers are shown in green after staining with FITC-phalloidin, and nuclei are shown in blue after staining with DAPI. (D) Percentage of Craspedacusta polyps showing normal regeneration with 1, 2, or 3 heads (black) or “podocyst” form (green) at indicated time points after head removal. Representative images of Craspedacusta polyps showing a colony with two heads (upper panel) and a podocyst (lower panel). Scale bar: 200 µm.
To confirm that DAPT was taken up by the polyps even in the absence of a visible regeneration phenotype, we investigated the effect of the drug under regeneration conditions on the expression of some possible Notch target genes. We choose homologs of HyAlx and HySp5, both genes had been identified as Notch target genes in Hydra (Moneer et al, 2021), and a homolog of NOWA, a gene encoding a protein of the outer nematocyte capsule wall (Figs S3, S4, S5, and S6). In Hydra, NOWA is down-regulated by DAPT because of the defect in nematocyte differentiation, which occurs when Notch signaling is blocked (Käsbauer et al, 2007; Moneer et al, 2021). The results are shown in Fig 7. DAPT inhibits the expression of CsAlx and of CsSp5 during head regeneration. It also inhibits the expression of CsNOWA. This effect of DAPT on the expression of homologs of suggested Hydra Notch target genes confirms that the drug must have entered the cells in Craspedacusta polyps.
(A) Schematic representation of the CsAlx protein structure (243 amino acids). The HOX domain with DNA binding function is located between amino acids 71 and 133. (B) Alignment of the protein sequences of Alx homologs from Craspedacusta sowerbii (CraSo), Stylophora pistillata (StyPi)—Acc#: PFX33415.1; Hydractinia symbiolongicarpus (HdrSy)—Acc#: XP_057291727.1; Danio rerio (DanRe)—Acc#: XP_001340966.1; Drosophila melanogaster (DroMe)—Acc#: NP_788420.1; Hydra vulgaris (HydVu)—Acc#: AAG03082.1; Nematostella vectensis (NemVe)—Acc#: XP_001634166.2; Actinia tenebrosa (ActTe)—Acc#: XP_031560466.1; human—Acc#: NP_620689.1; and mouse—Acc#: NP_001292869.1. The HOX domain is underlined, the pink background indicates conserved amino acids involved in DNA binding. (C) Phylogenetic tree based on the alignment of the 10 protein sequences affiliated to the aristaless family using MEGA software.
(A) Schematic representation of the CsSp5 protein structure (367 amino acids). At the N-terminus, the signal peptide (SP) is shown in red from amino acids 1 to 46. The three zinc-finger domains with DNA binding function are located at the C-terminus between amino acids 268 and 292, 298 and 322, and 328 and 350 shown in blue. (B) Alignment of the protein sequences of Sp5 homologs from Craspedacusta sowerbii (CraSo), Drosophila melanogaster (DroMe)—Acc#: NP_727360.1, NP_651232.1; Stylophora pistillata (StyPi)—Acc#: PFX28957.1; Hydra vulgaris (HydVu)—Acc#: AXP19710.1; Hydractinia symbiolongicarpus (HdrSy)—Acc#: XP_057304028.1; and Clytia hemisphaerica (ClyHe)—Acc#: XP_057304028.1. The signal peptide is underlined, the red background indicates conserved amino acids; zinc-finger domains are underlined, the blue background indicates conserved amino acids involved in DNA binding. (C) Phylogenetic tree based on the alignment of the 15 protein sequences affiliated to the transcription factor Sp5 family using MEGA software. Human—Acc#: NP_001003845.1; Rattus norvegicus (RatNo)—Acc#: NP_001100022.1; Danio rerio (DanRe)—Acc#: NP_919352.1, NP_851304.2; and mouse—Acc#: XP_036013171.1, XP_036013172.1.
(A) Schematic representation of the CsNOWA protein structure (679 amino acids). At the N-terminus, the signal peptide (SP) is shown in red from amino acids (aa) 1 to 20, followed by a CAP domain from 46 to 183 aa in violet, a carbohydrate recognition LectinC domain from 194 to 183 aa shown in green, and a SEA domain for membrane interaction shown in blue from 321 to 420 aa. The three internal repeats at the C-terminus are shown in orange between amino acids 424 and 457, 470 and 493, and 618 and 656. (B) Alignment of the protein sequences of NOWA homologs from Craspedacusta sowerbii (CraSo), Hydra vulgaris (HydVu)—Acc#: AAN52336.1; Hydractinia symbiolongicarpus (HdrSy)—Acc#: XP_057312482.1; Clytia hemisphaerica (ClyHe)—Acc#: XP_066935203.1 and precursor Acc#: ABY71251.1; and Sarsia lovenii (SarLo)—Acc#: WVX52206.1. The signal peptide is underlined, the red background indicates conserved amino acids; CAP domains are underlined, violet background indicates conserved amino acids; LectinC domains are underlined, green background indicates conserved amino acids; SEA domains are underlined, the blue background indicates conserved amino acids; internal repeats are underlined, orange background indicates conserved amino acids. (C) Phylogenetic tree based on the alignment of the 6 protein sequences affiliated to the nematocyte-producing antigen family using MEGA software.
(A) Schematic representation of the CsWnt3 protein domain structure (341 amino acids). The N-terminal signal peptide (SP) is shown in red from amino acids (aa) 1 to 21, followed by the Wnt domain from 45 to 333 aa in green. (B) Alignment of the protein sequences of Wnt3 homologs from Craspedacusta sowerbii (CraSo), Danio rerio (DanRe)—Acc#: XP_005163717.1; mouse—Acc#: NP_033547.1; human—Acc#: NP_110380.1; Hydra vulgaris (HydVu)—Acc#: CDG70667.1; Clytia hemisphaerica (ClyHe)—Acc#: XP_066919214.1; Hydractinia symbiolongicarpus (HdrSy)—Acc#: XP_057304029.1; and Hydractinia echinata (HdrEc)—Acc#: CAK50826.1. The signal peptide is underlined, the red background indicates conserved amino acids; Wnt domains are underlined, green background indicates conserved amino acids. (C) Phylogenetic tree based on the alignment of the 24 protein sequences affiliated to the Wnt3 family using MEGA software. Hydra vulgaris: Wnt1—Acc#: BAH23782.1; Wnt7—Acc#: BAH23781.1; Wnt11—Acc#: BAH23776.1; Clytia hemisphaerica (ClyHe)—Acc#: XP_066919469.1, AFI99119.1, AFI99118.1; Dynamena pumila (DynPu)—Acc#: QBC65507.1; Hydractinia symbiolongicarpus (HdrSy)—Acc#: AIA10263.1; Hydractinia echinata (HdrEc)—Acc#: AIU99839.1; mouse—Acc#: NP_067254.1; human—Acc#: NP_110388.2, KAI4085194.1; and Danio rerio (DanRe)—Acc#: NP_001188327.1, XP_005162280.1, NP_878296.1.
(A, B, C, D) qRT–PCR measurements quantifying the expression of Craspedacusta genes, (A) CsAlx, (B) CsSp5, (C) CsNOWA, and (D) CsWnt3 during 96 h of head regeneration in DAPT (orange) or DMSO for control (blue). Relative normalized expression related to the housekeeping genes GAPDH, actin, and PPIB. Regeneration time is shown on x-axes; t = 0 refers to animals immediately after the head was removed. Data are shown as the mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001.
Finally, we investigated the expression of the Craspedacusta Wnt3 gene (Fig 7) and its response to DAPT treatment during head regeneration. We observed a low expression level of CsWnt3 immediately after head removal (t = 0), which dramatically increased as the head regenerated, suggesting that Wnt3 is expressed in the head of Craspedacusta polyps like its expression in the heads of other cnidarians, including Hydra, Hydractinia, and Nematostella (Hobmayer et al, 2000; Kusserow et al, 2005; Plickert et al, 2006). Consistent with its lack of effect on head regeneration, DAPT also did not inhibit CsWnt3 expression during this process in Craspedacusta. This is opposite to the situation in Hydra. If CsWnt3 would be involved in Craspedacusta head regeneration, this could explain the failure of DAPT in disrupting this process.
Discussion
Head regeneration in Hydra can be divided into two processes, re-formation of a hypostome–body column axis and re-formation of tentacles. We show here that tentacle formation requires β-catenin transcriptional activity, but hypostome regeneration does not. Conversely, hypostome regeneration requires Notch signaling, whereas tentacle tissue does not. By qRT–PCR gene expression analysis, we investigated the expression dynamics of selected genes in response to inhibition of β-catenin transcriptional activity, or of Notch signaling over a regeneration time of 48 h in polyps after heads had been removed at an apical position, just underneath the tentacles.
The results of these gene expression analyses are schematically displayed in Table 1. We distinguish two phases of regeneration, the first 8 h and the time thereafter. With the exception of the direct Notch target gene HyHes (Münder et al, 2013; Moneer et al, 2021), the expression of our selected genes is not affected by DAPT 8 h after head removal. This time is allocated to wound healing, and this process appears independent of Notch signals (Cazet et al, 2021). However, over the following time course, expression levels of HyWnt1, HyWnt3, HyWnt7, HyWnt9/10, HyWnt11, and HyWnt16, all implied in canonical Wnt signaling, declined to almost zero in DAPT-treated polyps. In addition, the potential “organizer” gene CnGsc was inhibited with DAPT corresponding to the observation that Notch-treated regenerates do not acquire organizer activity. Sp5, which was suggested to be part of an inhibition loop for HyWnt3/β-catenin (Vogg et al, 2019) and a direct Notch target gene (Moneer et al, 2021), was also blocked by DAPT during head regeneration. HyAlx, which has repeatedly been shown to induce differentiation of tentacle tissue (Smith et al, 2000; Broun & Bode, 2002; Broun et al, 2005; Gee et al, 2010; Münder et al, 2013), was only slightly affected by DAPT, corresponding to the detection of irregular tentacles in some regenerates (Münder et al, 2013). However, the lack of organizer activity in such regenerates may be responsible for their failure to produce correct tentacle patterns. We also observed that the expression of HyBMP2/4 is strongly dependent on Notch signaling. Together, these results suggest that Hydra head regeneration requires canonical Wnt and BMP2/4 signaling to produce an organizer and a hypostome, both of which depend on the presence of Notch signaling. In contrast, HyBMP5/8b and HyKayak were up-regulated by DAPT, suggesting that Notch was required to inhibit these genes.
Summary of changes in gene expression during Hydra head regeneration in medium with Notch and β-catenin inhibitors.
We also found that tentacle tissue formation, especially the expression of HyAlx in apical regenerates, was completely blocked with iCRT14. On the contrary, it is known that increasing nuclear β-catenin (and thus its transcriptional activity) by alsterpaullone induces formation of ectopic tentacles, but not hypostomes or even complete heads (Broun et al, 2005). Therefore, the phenotype observed with iCRT14 is obviously caused by a lack of tentacle activation, whereas ectopic activation of β-catenin induces tentacle formation through activation of HyAlx.
Most intriguingly, induction of HyWnt3 expression in apical regenerates was not blocked in the absence of β-catenin transcriptional activity, indicating that HyWnt3 is not up-regulated via β-catenin–dependent autoactivation after head removal, as had been suggested to occur in undisturbed polyps (Nakamura et al, 2011). In contrast to HyWnt3, all other canonical Wnt genes were down-regulated by iCRT14, at least to some extent, indicating that they were β-catenin–dependent. In the presence of iCRT14, HyWnt3 must perform its function during head regeneration by signaling through a β-catenin–independent pathway. Remarkably, iCRT-treated tissue regenerated perfect hypostomes with the normal HyWnt3 expression pattern.
The effect of iCRT14 had also been analyzed in previous studies (Gufler et al, 2018; Cazet et al, 2021; Tursch et al, 2022). All studies showed β-catenin dependency for the down-regulation of head-specific genes in foot regenerates at time points up to 12 h after head removal, including HyWnt3. They also stated a failure of head regeneration in the presence of iCRT14 but, in accordance with our study, did not reveal that HyWnt3 expression at future heads depended on β-catenin. None of these studies analyzed the regeneration of tentacles and hypostomes separately, and they did not report whether the regeneration of hypostomes 48 h after head removal occurred normally upon iCRT14 treatment.
Although the tissue left after head removal has the capacity to form both tentacles and hypostome/head, final patterning of the new head involves emergence of hypostome and tentacle structures at distinct locations. A model proposing two independent patterning systems, each comprising an activator and an inhibitor for head and tentacle formation, had been introduced before, when HyAlx was discovered (Smith et al, 2000). After cutting off the head at apical positions, HyAlx first appeared at the tip. This was explained with high tentacle activation potential in this region, leading to a fast establishment of the tentacle system with HyAlx expression and tentacle markers (like HMMP) covering the whole regenerating tip. Tentacle activation is then inhibited by a tentacle inhibitor. Head activation takes over, and the expression of canonical Wnt genes becomes stronger. HyAlx shifts to the emerging tentacle region and finally appears in rings from which tentacles emerge (see Fig 8).
Schematic representation of DAPT and iCRT14 effects on Hydra head regeneration. The hypostome is labeled in green; the tentacle boundary is in red. Animals treated with DAPT regenerate tentacle boundary gene expression in irregular patterns and show irregular tentacle morphology. Animals treated with iCRT14 regenerate regular expression patterns of hypostomal genes (HyWnt3) and show normal hypostome morphology. They do not regenerate any HyAlx expression and do not show tentacles. Model for the course of Hydra head regeneration in the presence of Notch signaling (control, upper panel), the absence of Notch signaling (middle panel), and the absence of Wnt/β-catenin signaling (lower panel) are shown. After head removal, the potential to re-form a head (green circles) and tentacles (red triangles) arises in the regenerating tip of the polyp. Notch signaling then mediates inhibition of the tentacle fate in the upper part of the regenerate by repressing HyBMP5/8 and allowing the expression of HyBMP2/4 and HyWnt3 (hypothetically by repressing inhibitors of these genes as indicated by a dotted line). This allows re-establishment of the hypostome and organizer tissue, while confining tentacle development to the lower part of the regenerate (control). With inhibition of Notch (middle panel), tentacle fate is not inhibited in the tip of the regenerate preventing the expression of hypostomal genes (HyWnt3 and HyBMP2/4) and allowing tentacle tissue development in the whole regenerating tip. However, as an organizer does not develop, this tissue cannot be patterned properly (red triangles). Without transcriptional activity of β-catenin, hypostomal genes (Wnt3 and HyBMP2/4) are expressed, whereas tentacle tissue is not induced (no HyAlx). Created with BioRender.
In contrast, budding starts with head activation being established and HyAlx is expressed later, always excluding the apical part of the bud. This was attributed to higher head activation potential in the budding region in comparison with tentacle activation activity. Moreover, older regeneration experiments had revealed that apical and basal regenerates differed in the order of appearance of the head and tentacle tissue. The tentacle tissue appeared first in apical regenerates and later in basal ones (Technau & Holstein, 1995).
Here, we have only considered apical regenerates where the heads of the polyps were cut off just underneath the tentacles. We suggest that Notch signaling fulfills a role in tentacle inhibition in this case. Without this inhibition, head activation with the expression of all canonical Wnt genes does not occur. However, Notch also affects head regeneration at basal cuts, as we have recently shown by analyzing transgenic Hydra with inhibited Notch function. Here, a substantial part of the animals regenerated two heads (Pan et al, 2024). This again confirms the idea that head formation and tentacle formation use two independent patterning systems, and Notch is required to mediate between them. When the tentacle system is activated first, Notch inhibits it to allow emergence of the head system. When the head system emerges first, Notch blocks it to prevent the formation of multiple heads.
How does tentacle inhibition work? It is well established that Notch activates transcriptional repressors, including HyHes genes, and thereby suppresses specific cell fates in signal-receiving cells, but allowing those fates in signal-sending cells (Bray, 2006). Our data show that DAPT inhibits the expression of two established transcriptional repressor genes, HyHes and CnGsc. This poses the question for targets of these repressors, which should be up-regulated when Notch signaling is inhibited. We observed this behavior for BMP5/8b and HyKayak. On the basis of the published BMP5/8b expression patterns (Reinhardt et al, 2004), this gene is probably part of the tentacle patterning system.
HyKayak encodes a homolog of Fos proteins, which are components of the AP1 transcriptional complex, as we show by sequence comparison and phylogenetic analysis of the bZIP domain. Moreover, HyKayak interacted with HyJun, but not with itself, similar to the behavior of human c-Fos, which does not form homodimers but instead heterodimerizes with Jun proteins (Kouzarides & Ziff, 1988). Fos is suggested to be a negative regulator of its own promoter (Sassone-Corsi et al, 1988), and Fos can function as a repressor on cellular immediate–early genes, such as Egr genes (Gius et al, 1990). Both repressions are mediated by the C-terminus of Fos and are independent of Jun (Gius et al, 1990; Ofir et al, 1990). However, the C-terminus of fos is not required for the repression of cardiac transcription and muscle creatine kinase enhancer (Lassar et al, 1989; Li et al, 1992; McBride et al, 1993). Our hypothesis that HyKayak could repress the HyWnt3 gene was confirmed by shRNA-mediated HyKayak knockdown, which resulted in the up-regulation of HyWnt3 expression. In addition, HyJun-epi was also up-regulated. This is in accordance with previously published observations in human prostate cell lines where fos loss of function has resulted in an up-regulation of jun expression (Riedel et al, 2021). Moreover, experiments with pharmacological inhibition of the AP1 complex with T5224 during head regeneration revealed that HyWnt3 and HyJun-epi were strongly up-regulated. We therefore suggest that the Hydra fos homolog HyKayak inhibits HyWnt3 expression and can be a target for a Notch-induced transcriptional repressor (such as HyHes) in the regenerating Hydra head. Nevertheless, we were not able to rescue the DAPT phenotype by inhibiting HyKayak, neither by the inhibitor nor by shRNA treatment, probably because of the strength of the DAPT effect. Therefore, we cannot exclude the possibility that Notch activates HyWnt3 directly, or that it represses unidentified Wnt inhibitors through activation of HyHes or CnGsc.
Different bZIP transcriptional factors (TFs) may have different effects on the expression of Wnt genes, and these effects are context-dependent. In previous research, Cazet et al identified another Hydra fos gene (here referred to as fos_cazet), and bZIP TF binding sites in the putative regulatory sequences of HyWnt3 and HyWnt9/10c. They also showed that bZIP TF genes, including jun and fos, were transiently up-regulated 3 h after amputation and hypothesized that bZIP TFs could induce TCF-independent up-regulation of HyWnt3 during the early generic wound response (Cazet et al, 2021). In contrast, HyKayak expression continuously increased throughout the entire head regeneration process (Figs 3E and 4E) including the morphogenesis stages (24–48 h post-amputation). Another study reported that inhibition of the JNK pathway (which disrupts the formation of the AP-1 complex) resulted in up-regulation of HyWnt3 expression in both head and foot regenerates (Tursch et al, 2022). This result might support our hypothesis, but it only included the first 6 h after amputation. Therefore, it appears that HyKayak and fos_Cazet may have opposing roles in the regulation of Wnt gene expression and are possibly activated by different signaling pathways depending on the stages of regeneration.
The requirement for Notch activity is dependent on the regeneration time. At early time points, it is apparently not required, but between 8 and 48 h after head removal, loss of Notch activity severely impairs the regeneration process (Figs 3 and 8). In addition, the gene expression dynamics for many of the analyzed genes appears in wave-like patterns in some experiments (see Figs S7 and S8). As we have only measured four time points, we cannot draw strong conclusions from these observations, except that some of the deviations in our data points (e.g., 48-h HyHes) might be due to oscillations. It is tempting to speculate that the gene expression patterns over the time course of regeneration occur in waves. Hes genes, the best-studied Notch target genes, can produce waves of gene expression, for example, during segmentation and as part of the circadian clock (Kageyama et al, 2007). This property is due to the capability of Hes proteins to inhibit their own promoter. Future models for head regeneration in Hydra should consider this potential of the Notch/Hes system. Oscillations in gene expression could explain how the observed local changes in the expression of some genes within the 48 h of head regeneration come about. Examples are HyHes itself and BMP5/8b, both at the beginning strongly expressed at the tip of the regenerate, and later apparently “moving” to the bases of tentacles (Reinhardt et al, 2004; Münder et al, 2013).
GAM-based visualization of gene expression as measured by qRT–PCR in DAPT-treated animals relative to control animals (y-axis) depending on the time after head removal (x-axis). Gray points show raw data (quotients of mean values of DAPT-treated relative to DMSO-treated animals), the colored lines show the smooth GAM-based estimates, and color-shaded areas are 95% confidence bands. Gene expression was followed for 48 h after head removal in DMSO control and DAPT. For time point 0, polyps were used immediately after head removal without any exposure to inhibitor or control medium. (A) HyHes, HyAlx, CnGsc, HySp5, HyKayak, HyBMP2/4, and HyBMP5/8. (B) HyWnt3 and HyWnt1, HyWnt7, HyWnt9/10c, HyWnt11, and HyWnt16 during 48 h. Relative normalized expression was related to the housekeeping genes GAPDH, RPL13, EF1α, and PPIB.
GAM-based visualization of gene expression as measured by qRT–PCR in iCRT14-treated animals relative to control animals (y-axis) depending on the time after head removal (x-axis). Gray points show raw data (quotients of mean values of iCRT14-treated relative to DMSO-treated animals), the colored lines show the smooth GAM-based estimates, and color-shaded areas are 95% confidence bands. Gene expression was followed for 48 h after head removal in DMSO control and iCRT14. For time point 0, polyps were used immediately after head removal without any exposure to inhibitor or control medium. (A) HyAlx, CnGsc, HySp5, HyBMP2/4, and HyBMP5/8. (B) HyWnt3 and HyWnt1, HyWnt7, HyWnt9/10c, HyWnt11, and HyWnt16 during 48 h. Relative normalized expression was related to the housekeeping genes GAPDH, RPL13, EF1α, and PPIB.
Is Notch part of the organizer? The organizer is defined as a piece of tissue with inductive and structuring capacity. Notch is expressed in all cells of Hydra polyps (Prexl et al, 2011), and the overexpression of NICD does not induce second axes all over the Hydra body column (Pan et al, 2024), in contrast to the overexpression of stabilized β-catenin (Gee et al, 2010). Moreover, Notch functions differently during regeneration after apical and basal cuts. Phenotypically during head regeneration in Notch-inhibited polyps, we clearly recognize a missing inhibition of tentacle tissue after apical cuts, and a diminished inhibition of head induction after basal cuts (Pan et al, 2024).
We would thus rather suggest that the organizer activity of the Hydra tissue uses Notch signaling as a mediator of inhibition. As our study of transgenic NICD-overexpressing and Notch knockdown polyps had suggested, the localization of Notch signaling cells depends on relative concentrations of Notch and Notch–ligand proteins, which are established by gradients of signaling molecules that define the Hydra body axis (Sprinzak et al, 2010; Pan et al, 2024). This is in very good agreement with the greatly accepted “reaction–diffusion model” provided by Alfred Gierer and Hans Meinhardt (Gierer & Meinhardt, 1972; Meinhardt & Gierer, 1974), which suggests a gradient of positional value across the Hydra body column. This gradient may determine the activities of two activation/inhibition systems, one for tentacles and one for the head. When the polyps regenerate new heads, Notch could provide inhibition for either system, depending on the position of the cut.
Head regeneration also occurs in the colonial seawater hydrozoan Hydractinia. Colonies consist of stolons, covering the substrate, and connecting polyps, including feeding polyps, which have hypostomes and tentacles, and are capable of head regeneration, similar to Hydra polyps. Wnt3 is expressed at the tip of the head, and by RNAi-mediated knockdown, it was shown that this gene is required for head regeneration (Duffy et al, 2010). In the presence of DAPT, proper head regeneration did not occur, similar to Hydra. However, regeneration of the nerve ring around the hypostome was observed, indicating the possibility that hypostomes had been regenerated. Unfortunately, this study did not include gene expression data, and therefore, it is not clear whether Wnt3 expression was affected or not (Gahan et al, 2017).
An interesting question was whether regeneration of cnidarian body parts, which are only composed of one structure, also requires Notch signaling. This is certainly true for the Hydra foot, which regenerates fine in the presence of DAPT (Käsbauer et al, 2007). Moreover, we tested head regeneration in Craspedacusta polyps, which do not have tentacles, and showed that DAPT does not affect this regeneration process. This corroborates our idea that Notch is required for regeneration in cnidarians, when this process involves two pattern-forming processes, which are controlled by different signaling modules. This would be the case for Hydra and for Hydractinia heads, but not for Craspedacusta.
Future studies on expression patterns of the genes that control formation of the Hydra head, including Sp5 and Alx in Craspedacusta, could provide new insights into the evolution of cnidarian body patterns. Sp5 and Alx appear to be conserved targets of Notch signaling in the two cnidarians we have investigated. Wnt3, while being inhibited by Notch inhibition in Hydra head regenerates, is not a general target of Notch signaling. It was not affected by DAPT in our comparative transcriptome analysis (Moneer et al, 2021) on uncut Hydra polyps, and it was also not affected by DAPT in regenerating heads of Craspedacusta.
Materials and Methods
Animal treatment
Hydra polyps were cultured in Hydra medium (HM) (0.29 mM CaCl2, 0.59 mM MgSO4, 0.5 mM NaHCO3, 0.08 mM K2CO3 dissolved in Milli-Q water) at a constant temperature of 18°C. They were fed with freshly hatched Artemia nauplii 2–3 times per week, with the exception of 2 d before conducting the experiments. For regeneration experiments, all animals were decapitated at 80% of their body length and left to regenerate for 2 d in HM containing the respective inhibitors dissolved in 1% DMSO. Control animals were left to regenerate in HM with 1% DMSO. Treatments included 35 μM DAPT/1% DMSO, 5 μM iCRT14/1% DMSO, or 7.5 μM T5224 for 8, 24, 36, and 48 h after head removal. Time point 0 refers to animals immediately after the head was cut off. The inhibitor/DMSO-containing medium was renewed every 12–14 h.
C. sowerbii polyps were grown in modified HM (0.29 mM CaCl2, 0.59 mM MgSO4, 0.5 mM NaHCO3, 0.08 mM K2CO3 dissolved in Milli-Q water) at 19°C. They were fed with Brachionus calyciflorus twice a week. For regeneration experiments, all animals were decapitated at 80% of their body length and left to regenerate for 3–4 d in HM containing the respective inhibitors dissolved in 1% DMSO. Control animals were left to regenerate in HM with 1% DMSO. Treatments included 35 μM DAPT/1% DMSO or 5 μM iCRT14/1% DMSO for 8, 24, 36, 48, 72, or 96 h after head removal. Time point 0 refers to animals immediately after the head was cut off. The inhibitor/DMSO-containing medium was renewed every 12–14 h.
Standardizing conditions for qRT–PCR
For quantitative estimates of gene expression dynamics during Hydra head regeneration over time, we performed real-time quantitative RT–PCR (qRT–PCR) experiments. We used a fluorescence-based qRT–PCR method and adhered to the quality standards of the MIQE guidelines (Bustin et al, 2009). After in silico primer design, each primer pair was empirically validated for (1) specificity defined by a single melt peak corresponding to a unique band of expected size, (2) efficiency defined by doubling of the signal in every cycle, and (3) sensitivity defined by a broad linear range, and reproducibility. Primers and gene accession numbers are listed in Table S1. Total RNA was isolated from Hydra polyps, and RNA quality was tested with the Agilent bioanalyzer. Only RNA with an integrity number higher than 8 was used for cDNA synthesis. During head regeneration, mRNA for qRT–PCR was isolated from whole regenerates collected after 8, 24, 36, and 48 h (t = 8, 24, 36, 48). Immediately after head removal, the sample for t = 0 was obtained. All experiments included three biological replicates with three technical replicates each. Quantitative gene expression for each gene was calculated as the ratio of target gene expression to housekeeping gene average (relative normalized gene expression). We plotted the relative normalized gene expression of analyzed genes against the regeneration time points. Housekeeping genes included GAPDH, PPIB, EF1alpha, and RPL13.
Table S1. Primer list for qRT–PCR.
Regression analysis of comparative expression levels
To visualize temporal changes in expression levels of different genes, we used appropriate regression methods. In particular, we used generalized additive models (Wood, 2017) enabling the visualization of nonlinear dependencies on the time-dependent variables based on appropriate regression splines (Wood, 2017). Here, we used the Tweedie probability distribution (Kokonendji et al, 2004), which is known to describe non-negative (possibly over-dispersed) data well—in particular if mean values are close to zero. Temporal autocorrelation of model residuals has been investigated based on pacf-plots (Wood, 2017) and was not apparent. The optimal amount of smoothness of regression splines has been estimated separately for each temporal expression pattern based on generalized cross-validation methods (Wood, 2017). For the analysis of expression patterns relative to the control (DMSO) type, the response variable in regression analysis has been defined by dividing separately for each experiment/time point the mean value of the repeated measurements of the treatment of interest (DAPT respectively iCRT) by the mean value of the repeated measurements of the corresponding DMSO treatment from the same experiment/time point.
Semithin sections with the Richardson staining
Animals were fixed with 4% PFA and prepared for semithin sectioning by re-fixation in 1% osmium tetroxide solution for 2 h. Samples were washed with water and dehydrated four times with serial acetone dilutions (30%, 50%, 70%, and 90%, four times 100%). Finally, they were embedded in Spurr low-viscosity embedding medium standard mix, which was exchanged four times, and dried after each exchange for 24 h at 60°C in a cuboid shape. The resin-embedded probes were sectioned with a semidiamond and stained after Richardson on a microscope slide. One drop of color solution (1% azure in H2O and 1% methylene blue in 1% Na2B4O2 in H2O mixed 1:1) covering the semithin sections was heated to 80°C for 30 s and cleansed with water. After drying, the slides were analyzed with a brightfield microscope.
Histochemistry of polyps
Polyps were relaxed in 2% urethane and fixed with 4% PFA in HM for 1 h. They were permeabilized with ice-cold 100% ethanol and blocked in 0.1% Triton/1% BSA in PBS. For phalloidin staining, they were incubated with Phalloidin-iFluor 488 (ab176753; Abcam) (1:500) for 1 h, followed by DAPI (1:1,000) staining before mounting on slides with Vectashield. Slides were analyzed with a Leica SP5 point scanning laser confocal microscope equipped with oil-immersion HCX PL APO Lambda Blue 20 × 0.7 and 63 × 1.4 objective lenses. Alexa Fluor 488 fluorochromes were visualized with an argon laser at an excitation wavelength of 488 nm and emission filters of 520–540 nm, and a diode laser at an excitation wavelength of 405 nm and with emission filter at 450–470 nm was used for DAPI. The produced light optical serial sections were stacked with the ImageJ plugin StackGroom to produce 3D images of the treated polyps. DAPI staining of nematocyte capsules was done according to Szczepanek et al [2002].
Fluorescence in situ hybridization
This experiment was carried out as previously described (Siebert et al, 2019).
Transplantation experiments
Non-budding Hydra polyps were pre-treated with 5 μM iCRT14/1% DMSO in HM for 24 h. After that, they were bisected at 80% of the body column underneath the head and left to regenerate in iCRT14-treated HM for another 24 h. The newly regenerated head region (top 20%) was grafted onto a blue host animal (treated with Evans blue for two weeks) at about 50% of the body column. After 3 h, the rod was removed and the animals were left in HM for another 48 h. Finally, the animals were classified for the presence of newly formed secondary axes displaying a clear hypostome and tentacles. Tissue recruitment was recognized by the blue/white color distribution within the new axes.
ShRNA knockdown
shRNA design and production were done according to Karabulut’s protocol (Karabulut et al, 2019). For electroporation, 30 budless Hydra polyps were washed five times with Milli-Q water and incubated for 45 min in Milli-Q water. Then, excess water was removed and replaced with 200 μl of a 10 mM Hepes solution at pH 7.0. The suspended animals were then transferred into a 4-mM-gap electroporation cuvette, and 4 μM of purified shRNA or scramble shRNA was added to the cuvette. The mixture was mixed by gently tapping the cuvette five times and incubated for 5 min to let animals relax before electroporation. The electroporation was carried out using BTX Electro Cell Manipulator 600 by setting up the condition to 250 V, 25 ms, 1 pulse, 200 μF capacitance. 500 μl of restoration medium (80% HM and 20% dissociation medium: 3.6 mM KCl, 6 mM CaCl2, 1.2 mM MgSO4, 6 mM sodium citrate, 6 mM sodium pyruvate, 6 mM glucose, 12.5 mM TES, and 50 mg/ml rifampicin, pH 6.9) was added into the cuvette immediately after electroporation. The entire volume of electroporated animals was then transferred into a petri dish. In our experiment, three times of electroporation were done every 2 d to achieve a significant knockdown of HyKayak. And two hairpins of Kayak were used for electroporation at 1:1.
Monoclonal anti-HyKayak antibody
Mice were immunized with fusion protein Hydra_KAYAK-HIS (amino acid of HyKAYAK: 1–111) using a mixture of 50 μg protein, 12 μl Oligo 1,668 (500 pmol/μl), and 150 μl IFA in a total volume of 400 μl. After 6 wk, a single boost was given with the same mixture except for the IFA, which was omitted. Fusion with Ag8 myeloma cells was performed using standard procedures. Candidate selection was based on positive selection using KAYAK-HIS and negative selection using Hydra_HES-HIS. Hybridoma kayak 3C10-1-1 and 13A4-1-1 were cloned using standard procedures and subsequently grown for antibody production.
Multiple sequence alignment and phylogenetic analysis
The multiple sequence alignment was done using Clustal Omega. The conserved domains were identified by PROSITE. The phylogenetic trees were produced by MEGA. The protein sequences for comparison were retrieved from UniProt and NCBI.
Subcellular fractionation and Western blot
500 Hydra were dissociated into single cells with 10 ml dissociation medium by pipetting. After centrifuge at 2,000g for 10 min, the cellular pellet was resuspended in 500 μl RIPA buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 10 ng/ml pepstatin A, 10 ng/ml aprotinin, 10 ng/ml leupeptin, and 0,5 mg/ml Pefabloc) and incubated for 20 min on ice. Subsequently, the mixture was homogenized with a Dounce homogenizer 30 times and then centrifuged for 10 min at 1,000g. The resulting supernatant including cytoplasmic proteins was collected and labeled as CP. The pellet was treated with 500 μl RIPA buffer and then sonicated at 180 W for 3 min (in rounds of 10-s sonication and 50-s rest on ice for each cycle). After centrifuging at 14,000g for 30 min, the supernatant was collected and labeled as nuclear proteins (NP); the pellet was resuspended with the same volume of RIPA buffer and kept for SDS–PAGE analysis.
For DNase treatment, the pellet from the second centrifuge was resuspended with 500 μl RIPA buffer supplemented with 200 U/ml DNase, 10 mM CaCl2, and 10 mM MgCl2, and incubated at room temperature for 15 min. After centrifuging at 1,000g for 10 min, the supernatant was collected, whereas the pellet was resuspended in 500 μl RIPA buffer with 2 M NaCl and incubated on ice for 10 min. Then, the same centrifuge was done, and both the supernatant and pellet were collected for gel analysis. Western blots were stained with the in-house mouse anti-Kayak monoclonal antibody.
Co-immunoprecipitation
HEK293T cells were transferred with C-terminal HA-tagged Kayak and N-terminal GFP-tagged Jun-epi or Kayak using Lipofectamine 2000 (11668030; Thermo Fisher Scientific). The GFP-Trap agarose beads (ABIN509397; ChromoTek) were used for immunoprecipitation as described previously (Webby et al, 2009; Heim et al, 2014). Western blot was stained with the following primary antibodies: mouse anti-GFP antibody (11814460001; Roche) and rabbit anti-HA antibody (H6908; Sigma-Aldrich).
Identification of Craspedacusta genes
Craspedacusta total RNA was extracted from 120 polyps using QIAGEN RNeasy Mini Kit. RNA quality was verified with the Agilent bioanalyzer, the RNA was then reverse-transcribed into cDNA, and cDNA was sequenced with Illumina. The resulting gene sequences were aligned, and by comparison with sequences for HyWnt3, NOWA, HyAlx, and Sp5, the corresponding Craspedacusta cDNA sequences could be identified (CsWnt3, CsNOWA, CsAlx, and CsSp5) and confirmed by sequencing of cDNA clones obtained after qRT–PCR from Craspedacusta total RNA.
Data Availability
All data presented in the main article and supplementary files will be provided by the corresponding author (Angelika Böttger) upon request.
Acknowledgements
We want to express our gratitude to the funding agencies for supporting this work. A Böttger and L Sauermann are funded by the German Research Foundation (DFG) project BO1748, M Mercker by DFG grant SFB1324, Q Pan is funded by a CSC grant (Chinese Scholarship Council), and L Sauermann is funded by Evangelisches Studienwerk Villigst e.V. Special thanks go to Dr. Herwig Stibor, LMU, for sparking our investigation of Craspedacusta head regeneration. We thank Dr. Stefan Krebs, LMU Munich, for sequencing of Craspedacusta cDNA, Dr. Sergio Vargas, LMU Munich, for bioinformatics sequence analysis and Bianca Sammer for carrying out transplantation experiments. The drawings in this document were produced using Affinity Designer 2 (version 2.3.0) by L Sauermann.
Author Contributions
M Steichele: conceptualization, investigation, methodology, and writing—original draft.
L Sauermann: data curation, investigation, methodology, and writing—review and editing.
Q Pan: conceptualization, investigation, methodology, and writing—review and editing.
J Moneer: conceptualization, investigation, methodology, and writing—review and editing.
A de la Porte: investigation and methodology.
M Heß: resources and methodology.
M Mercker: data curation, formal analysis, and methodology.
C Strube: investigation and methodology.
H Flaswinkel: methodology.
M Jenewein: conceptualization and methodology.
A Böttger: conceptualization, supervision, investigation, methodology, project administration, and writing—original draft, review, and editing.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
- Received September 19, 2024.
- Revision received October 30, 2024.
- Accepted October 30, 2024.
- © 2024 Steichele et al.
https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
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