Wnt is one of the earliest signals in the embryo and it induces the formation of the primary dorsal–ventral and head-to-tail axes (Loh et al., 2016; Niehrs, 2012). It is known that β-catenin is required for Wnt signaling activation on the dorsal side during early cleavage. Recently, we reported that inhibition of macropinocytosis with EIPA, or of lysosomal acidification with Baf, ventralized endogenous early dorsal axis formation, leading in particular to reduced head and brain structures (Tejeda-Muñoz and De Robertis, 2022a). Activation of lysosomes on the dorsal side of the embryo was detectable as early as the 64-cell stage (Tejeda-Muñoz and De Robertis, 2022a).

The tumor promoter PMA is known to increase macropinocytosis in macrophages (Swanson, 1989). To test its activity during early development, we used a sensitized system in which a small amount of the GSK3 inhibitor LiCl (4 nl at 300 mM in a single ventral injection at 4-cell) causes only a small dorsalization with slightly enlarged heads (Kao et al., 1986; Tejeda-Muñoz and De Robertis, 2022a). The dorsalizing effects of PMA were most striking when phenotypes were analyzed at the early tailbud stage (Figure 1A–H). LiCl alone expanded head structures only slightly and PMA alone was without effect (Figure 1A, B, E). However, the combination of PMA and LiCl lead to radially dorsalized embryos consisting of head structures lacking trunks (compare Figure 1B–F). This striking phenotype was eliminated by co-injection of EIPA or Baf, suggesting that macropinocytosis and lysosomal acidification are required for the effect of PMA (Figure 1G, H). Injection of Baf alone decreased head and neural development, supporting a requirement for lysosomes for endogenous dorsal axis formation (Figure 1D). Target genes of the early Wnt signal can be detected at blastula (stage 9.5, just after the start of zygotic transcription) by measuring transcripts of Siamois and Xnr3 by qRT-PCR (Tejeda-Muñoz and De Robertis, 2022a). As shown in Figure 1I, PMA alone (4 nl at 500 nM) was without any effect, LiCl increased the Wnt target genes, and the combination of PMA and LiCl synergized significantly elevated Siamois and Xnr3 transcript levels, while EIPA and Baf blocked the increase in Siamois and Xnr3 caused by PMA/LiCl (Figure 1I).

Using the BAR (β-catenin activity reporter) assay system (Biechele and Moon, 2008) in a cultured HEK293 permanent BAR/Renilla cell line, PMA synergized with LiCl when assayed by transcriptional BAR luciferase activity (Figure 1J). PMA on its own had no detectable effect on BAR reporter activity (Figure 1—figure supplement 1J). In this cell line, PMA cooperated with LiCl to increase β-catenin levels (Figure 1—figure supplement 1A–H). PMA alone caused a slight increase in β-catenin protein staining, but this lacked statistical significance (Figure 1—figure supplement 1C, I), presumably due to a small amount of endogenous Wnt secreted by this cell line (Colozza et al., 2020). Importantly, the effect of PMA on the activation of the Wnt pathway by GSK3 inhibition was abrogated by the inhibition of V-ATPase with Baf (Figure 1—figure supplement 1H, I).

To test the effect of PMA on Wnt-induced macropinocytosis we used an uptake assay in which 3T3 cells were treated with dextran for 1 hr (Figure 1K–P). The macropinocytosis marker TMR-dextran 70 kDa has a hydrated diameter of more than 200 nM and is diagnostic of large endocytic cups, in particular when sensitive to the macropinocytosis inhibitor EIPA (Commisso et al., 2013; Commisso et al., 2014). In the presence of Wnt3a, but not in the absence, PMA strongly induced dextran uptake and this effect was blocked by EIPA (Figure 1K–P, quantitated in Figure 1Q). PMA also increased dextran uptake in the SW480 CRC cell line that has constitutive Wnt signaling, even in the absence of added Wnt (Figure 1—figure supplement 1J, K). In time-lapse video microscopy of SW480 cells with the plasma membrane tagged by myristoylated-GFP (mGFP), PMA addition triggered the abundant formation of macropinosome cups within minutes of addition when compared to DMSO (Dimethylsulfoxide) controls (Video 1).

These results in embryos and cultured cells indicate that the tumor promoter PMA potentiates Wnt signaling and that this cooperation requires macropinocytosis and lysosomal acidification.

In previous work, we had shown that immersion of embryos in the V-ATPase inhibitor Baf at 5 µM for only 7 min at the 32-cell stage resulted in ventralized microcephalic embryos lacking cement glands (Tejeda-Muñoz and De Robertis, 2022a). To test whether this ventralization is caused by inhibition of the endogenous Wnt pathway signal, we now carried out order-of-addition experiments. When an initial Baf incubation of 7 min was followed by a second brief incubation in LiCl while still at the 32-cell stage, the ventralization by Baf was rescued, and the resulting tailbud embryos had a LiCl-like dorsalized phenotype (Figure 2A–D). The initial effect of GSK3 by LiCl is to trigger macropinocytosis (Albrecht et al., 2020). The phenotypes are supported by the activation of Wnt signaling at the blastula stage, as indicated by qRT-PCR of direct Wnt target genes (Figure 2E). In the converse experiment, when embryos were first briefly treated with LiCl, washed, and subsequently incubated for 7 min in Baf, the effect of lysosomal inhibition was dominant, resulting in ventralized tailbud embryos (Figure 2F–J). These order-of-addition experiments strongly support the proposal that membrane trafficking and endogenous lysosomal activity are required to generate the initial endogenous Wnt signal that takes place at the 32-cell stage of development (Tejeda-Muñoz and De Robertis, 2022a).

Next, we tested the requirement for macropinocytosis of different components of the Wnt pathway using the axis duplication assay (McMahon and Moon, 1989). Microinjection of Wnt-mimic dominant-negative GSK3 (DN-GSK3-β) mRNA, a catalytically inactive form of GSK3, induced complete axis duplications, which were blocked by the co-injection of the macropinocytosis inhibitor EIPA (1 mM, 4 nl) (Figure 3A–D). Secondary axis induction by β-catenin mRNA was also abrogated by EIPA, as well as the membrane trafficking inhibitor dominant-negative Rab7 (DN-Rab7) (Figure 3E–H). β-Catenin is a transcriptional activator, and one might have not expected a requirement for membrane trafficking. However, axis formation is a multistep process and appears to require macropinocytosis and Rab7 activity in addition to transcriptional activation of Wnt target genes.

Macropinocytosis is an actin-driven process that is orchestrated by Pak1 kinase, which in turn is activated by Rac1. Rac1 activity is essential for the formation of lamellipodia and macropinocytic cups (Hall, 1998). EHT1864 (EHT) is a specific inhibitor of Rac1 that has the practical advantage of being water soluble (Hampsch et al., 2017). When embryos were immersed in EHT at the critical 32-cell stage for 7 min (10 mM), head structures were greatly reduced, and ventral posterior structures expanded (Figure 4A, B). This was accompanied by a significant increase of transcripts of the ventral markers Sizzled and Vent1 at the blastula stage (Figure 4C).

Microinjection of a small amount of EHT (1 mM, 4 nl, 1x ventral at 4-cell) was without effect on its own but was able to block the axis duplication effect of microinjecting Wnt8 mRNA (Figure 4D–G). Quantitative RT-PCR analyses at the blastula stage confirmed that inhibiting Rac1 strongly reduced the induction of the Wnt target genes Siamois and Xnr3 caused by xWnt8 mRNA microinjection at the late blastula stage (Figure 4H).

In SW480 CRC cells, which have constitutively activated Wnt due to mutation of APC (Leibovitz et al., 1976; Faux et al., 2004), EHT inhibited the size of cell spheroids in hanging drop cultures (Figure 4—figure supplement 1A–C) and, importantly, significantly inhibited macropinocytosis uptake of TMR-dextran in these cancer cells (Figure 4—figure supplement 1D–F).

Rac1 itself was stabilized in 3T3 cells by Wnt signaling, as indicated by experiments showing (1) that LiCl treatment increased levels of Rac1 protein (Figure 4I–J’’), (2) that cells transfected with constitutively active β-catenin-GFP had higher levels of Rac1 than control untransfected cells (Figure 4K–K’’), and (3) that Rac1 immunostaining was stabilized in cells transfected with CA-Lrp6-GFP when compared to untransfected cells (Figure 4L–L’’).

The results show that signaling by the Wnt/β-catenin pathway requires macropinocytosis, Rac1, and membrane trafficking in Xenopus embryos and cancer cells.

CRC is particularly favorable for the analysis of tumor progression because it gradually accumulates mutations, and in over 85% of the cases the initial driver mutation is in the APC tumor suppressor (Kinzler and Vogelstein, 1996; Weinberg and Weinberg, 2007). This offered an opportunity to test our proposal that membrane trafficking and its molecular components play an important role in Wnt-driven cancers (Albrecht et al., 2021). Commercially available arrays of paraffin-sectioned histological sections containing 90 cases of adenocarcinoma of various grades I–IV and 90 samples of corresponding adjacent normal tissues (from TissueArray) were used for immunostaining. Arrays were double stained for β-catenin and antibodies against proteins involved in the macropinocytosis/MVB/lysosome/GSK3 pathway. Each individual section was evaluated, and normal colon, adenocarcinoma I, and adenocarcinoma IV were compared to assess the effects of cancer progression.

As shown in Figure 5, β-catenin levels consistently correlated with grade IV malignancy when compared to normal colon or grade I cancers. Pak1, a kinase required for macropinocytosis and Wnt signaling (Redelman-Sidi et al., 2018; Albrecht et al., 2020) was significantly elevated with increased malignancy (Figure 5A–C’’). CD63, a tetraspan protein that marks MVB intraluminal vesicles (Escola et al., 1998), reached high levels in adenocarcinoma IV (Figure 5D–F’’), particularly in cells with the highest β-catenin expression (compare Figure 5F–F’). This increase in MVBs is in agreement with the GSK3 sequestration model of Wnt signaling (Taelman et al., 2010). The V-ATPase subunit V0a3, a marker for acidic lysosomes (Ramirez et al., 2019), also increased strongly with the CRC malignancy (compare Figure 5G–I). Importantly, the cellular levels of total GSK3 decreased with malignancy (Figure 5J–L’’, compare Figure 5J–L), as predicted by the sequestration model (Taelman et al., 2010). During Wnt signaling, GSK3 is sequestered into MVBs which are trafficked into lysosomes and a decrease was to be expected after sustained Wnt activation. Previously, we had been unable to demonstrate a decrease in total cellular GSK3 levels during acute Wnt signaling experiments (Taelman et al., 2010). The co-localization of β-catenin with Pak1, CD63, V0a3, or GSK3 in human colorectal adenocarcinomas was quantified in Figure 5—figure supplement 1A–D.

These results were confirmed by examining immunostaining levels of Pak1, V0a3, and GSK3 compared to β-catenin protein in mouse xenografts in a CD1 NU/NU nude mouse model injected with SW480 human CRC cells (in which Wnt activation is known to be caused by APC mutation, Faux et al., 2004). In this case, tumor cells were compared to normal colon mouse tissue sections. The results showed that CRC cells had high levels of β-catenin, Pak1, and lysosomes (Figure 5—figure supplement 1E–J), while GSK3 levels were reduced (compare Figure 5—figure supplement 1K–L) in agreement with the results in human tissue arrays.

These in vivo observations in human tumors are consistent with the view that macropinocytosis components, MVBs, and lysosomes increase with CRC malignancy. Further, the decrease in total GSK3 levels suggests a role in the trafficking and degradation of this key enzyme in cancer.

We have previously reported a crosstalk between the Wnt and FA signaling pathways in which Wnt3a treatment rapidly led to the endocytosis of Integrin β1 and of multiple FA proteins into MVBs (Tejeda-Muñoz et al., 2022c). FAs link the actin cytoskeleton with the extracellular matrix (Figure 6A), and we now investigated whether FA activity is affected by Wnt signaling, PMA treatment, or CRC progression. FA components containing LIM domains can shuttle between the nucleus and cytoplasm (Nix and Beckerle, 1997; Kadrmas and Beckerle, 2004; Anderson et al., 2021). We examined whether constitutive Wnt signaling regulates FA trafficking using the SW480 and SW480APC system, in which the stable reconstitution with full-length APC restores these CRC cells to a non-malignant phenotype (Faux et al., 2004), which is accompanied by a decrease in β-catenin levels (Figure 6B). As shown in Figure 6C–D’’, the PET-LIM domain FA protein Tes (also known as Testin) (Garvalov et al., 2003) was cytoplasmic in SW480APC, but was nuclear in SW480 cells in which Wnt is activated by APC mutation. The nuclear localization of Tes in SW480 cells required macropinocytosis, as suggested by treatment with EIPA which resulted in its re-localization to the cytoplasm (Figure 6E–F’’). In the nude mouse xenograft model, Tes was abundant in the cytoplasm of normal colon epielium, while in transplanted SW480 cells it colocalized with β-catenin in the nucleus of those cells with the highest β-catenin levels (Figure 6G–I’’). The function of TES is poorly understood, but it has been proposed to act as a tumor suppressor (Tatarelli et al., 2000; Tobias et al., 2001).

Focal adhesion kinase (FAK) is a key regulator of FA maintenance and signaling, known to interact with the Wnt pathway in various ways (Chuang et al., 2022). In HEK293 BAR/Renilla cells, treatment with the FAK inhibitor PF-00562271 strongly decreased β-catenin signaling by LiCl inhibition of GSK3 (Figure 6J; see also Figure 6—figure supplement 1A–B’’). In the context on the Xenopus embryo, treatment with the FAK inhibitor (100 µM, 7 min at 32-cell stage) resulted in ventralized embryos with reduced dorsal-anterior structures such as central nervous system, in particular brain structures stained by the pan-neural marker Sox2 (Figure 6K’–L’’). The addition of the tumor promoter PMA to SW480 cells (in which the Wnt pathway is constitutively activated) strongly increased protein levels of β-catenin and FAK protein (Figure 6—figure supplement 1C–D’’). The effect of PMA on β-catenin and FAK levels was abrogated by EIPA or Baf treatment (Figure 6—figure supplement 1C–H’’), suggesting that it requires macropinocytosis and lysosome activity.

Taken together, the results suggest a possible involvement of FA components and FAK in Wnt signaling, in particular during the endogenous early Wnt signal in Xenopus embryos. The results also suggest that nuclear Tes could serve as a Wnt-regulated marker in CRC. The regulated shuttling of FA components between nucleus and cytoplasm is probably just the tip of the iceberg of the role of membrane trafficking in Wnt-driven cancers.

Wnt signaling triggers macropinocytosis and lysosomal acidification (Albrecht et al., 2020). In cultured cells, Wnt3a-induced macropinocytosis was increased by PMA and blocked by EIPA. In embryo microinjections, Baf blocked the synergy between PMA and the Wnt-mimic LiCl. Baf also blocked development of the endogenous axis, particularly the head region when embryos were briefly immersed in Baf at the 32-cell stage, as previously reported (Tejeda-Muñoz and De Robertis, 2022a). Using order-of-addition experiments in which embryos were treated with 7 min pulses of Baf or the GSK3 inhibitor LiCl, we showed that the requirement for lysosome acidification was due to inhibition of the Wnt pathway at the crucial 32-cell stage (Figure 2). Co-injection experiments of DN-GSK3 or β-catenin mRNAs with DN-Rab7 or EIPA supported the view that membrane trafficking and macropinocytosis are essential for the induction of secondary axes in Xenopus (Figure 3).

Rac1 is an upstream regulator of the Pak1 kinase required for the actin machinery that drives macropinocytosis (Redelman-Sidi et al., 2018). Rac1 is a member of the family of small Rho GTPases that drives the formation of lamellipodia, membrane ruffles, and macropinocytosis (Hall, 1998; Egami et al., 2014; Fujii et al., 2013). The Rac1 inhibitor EHT1864 (Hampsch et al., 2017) strongly ventralized the endogenous axis of embryos after brief incubation at the critical 32-cell stage (Figure 4). In co-injection experiments EHT blocked the axis-inducing effect of microinjected xWnt8 mRNA. Rac1 is a regulator of multiple signaling cascades and has been reported to increase the nuclear translocation of β-catenin (Wu et al., 2008). Other work has shown that Rac1 binds to β-catenin and promotes the formation of nuclear β-catenin/LEF1 complexes (Jamieson et al., 2015). In the context of the present study, Rac1 protein levels were increased by treatments that enhanced Wnt/β-catenin signaling. The results suggest that Rac1 may crosstalk with Wnt through macropinocytosis in early development.

The lysosome is considered a promising target for the treatment of cancer (Fehrenbacher and Jäättelä, 2005; Lawrence and Zoncu, 2019). We used human CRC tissue arrays to analyze whether lysosomes and membrane trafficking correlate with cancer progression. Most colorectal tumors are initiated by mutation of APC, which drives Wnt signaling (Kinzler and Vogelstein, 1996; Segditsas and Tomlinson, 2006). It was found that advanced grades of colon carcinoma correlated with increased immunostaining for Pak1 (a driver of macropinocytosis), CD63 (a MVB/late endosome marker), and V0a3 (a lysosomal V-ATPase marker) (Figure 5). Importantly, cancer malignancy correlated with low levels of GSK3 staining, both in human samples and nude mouse SW480 xenografts. Cancer cells require lysosome function and have changes in lysosomal volume and subcellular localization during oncogenic transformation (Kirkegaard and Jäättelä, 2009; Kirkegaard and Jäättelä, 2009; Gocheva and Joyce, 2007; Kolwijck et al., 2010). V-ATPase has been associated with acidification of the extracellular milieu in tumors (Capecci and Forgac, 2013; Hinton et al., 2009; Perona and Serrano, 1988). Extracellular tumor acidification is probably due to increased numbers of lysosomes which are exocytosed, since V0a3 was located within the cytoplasm in advanced cancer or xenografts in mice (Figure 5I and Figure 5—figure supplement 1I). The results reported here support the view (Taelman et al., 2010; Tejeda-Muñoz and De Robertis, 2022a) that Wnt-driven lysosomal acidification and GSK3 sequestration may play a role in cancer. In addition, MVB/lysosome components could serve as markers for advanced Wnt-driven cancers.

The cytoskeleton interacts with proteins of the extracellular matrix though FAs (Geiger et al., 2001). We previously reported that Wnt causes the endocytosis of FA proteins and depletion of Integrin β1 from the cell surface (Tejeda-Muñoz et al., 2022c). We now report that Tes, a member of the PET-LIM family of FA proteins, changed subcellular localization after Wnt activation in SW480 cells. Other FA proteins shuttle between the nucleus, cytoplasm, and FA sites, and may play important roles in cancer (Chaturvedi et al., 2012; Nix and Beckerle, 1997). FAK is a key signaling kinase that regulates FA signaling. In SW480 cells (which have constitutive Wnt signaling), FAK protein levels were increased by PMA, and this was inhibited by EIPA or Baf. The FAK inhibitor PF-00562271 inhibited β-catenin signaling in Luciferase assays, and reduced dorsal-anterior structures such as CNS in embryos after incubation for 7 min at 32-cell stage that is critical for the early Wnt signaling. These results suggest a possible crosstalk between FAs, Wnt signaling and tumor promoters in cancer progression and embryonic development.

PMA activates PKC signaling by mimicking its physiological second messenger DAG, an activator of PKC. There is previous literature implicating PKC isozymes in the regulation of the Wnt signaling pathway (Fang et al., 2002; Goode et al., 1992) and that different PKC isoforms can have positive or negative effects on Wnt/β-catenin signaling (Ohno and Nishizuka, 2002). However, the molecular role of PKC isoforms in the Wnt signaling pathway remains poorly understood (Bhatia and Spiegelman, 2005; Schwarz et al., 2013). It has been reported that PMA can stabilize CK1ε (Casein Kinase 1 epsilon), enhance its kinase activity, and induce LRP6 phosphorylation at Thr1479 and Ser1490, leading to the activation of the Wnt/β-catenin pathway (Su et al., 2018). In the context of the present study, it is relevant that Wnt signaling rapidly triggers macropinocytosis and trafficking into lysosomes (Tejeda-Muñoz et al., 2019). Macropinocytosis requires the activation of phosphoinositide 3 kinase (PI3K) which leads to the formation of patches of PIP3 in the inner plasma membrane leaflet that initiate the formation of macropinocytic cups (Egami et al., 2014; Yoshida et al., 2018). During macropinosome formation, the PIP₃ lipid is then converted into DAG by phospholipase C gamma 1 (PLCγ1), and DAG activates PKC signaling (Yoshida et al., 2018). It seems likely that PMA may facilitate Wnt/β-catenin signaling by mimicking the DAG lipid in macropinosomes. We have tested this hypothesis by direct addition of DAG to cells and found cooperation with Wnt signaling (Azbazdar et al., 2023).

In the future, it will be interesting to study the role of chronic inflammation on macropinocytosis and tumor promotion. Active PKC phosphorylates many targets, including IκB kinase (IKK), which becomes activated and phosphorylates inhibitor of NF-κB (IκB), targeting it for degradation and triggering inflammation (Weinberg and Weinberg, 2007). Tumor progression is promoted by many chronic inflammatory agents such as turpentine, distilled alcoholic beverages, hepatitis viruses, and Helicobacter pylori that do not cause mutations by themselves but regulate the expansion of previously mutant clones (Rous and Kidd, 1941; Weinberg and Weinberg, 2007). Taken together, the results presented here suggest that macropinocytosis, lysosomal activity, and membrane trafficking are possible therapeutic targets for tumor progression in Wnt-driven cancers.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Edward M. De Robertis.

No new materials were generated in this study. Requests for reagents used should be directed and will be fulfilled by the Lead Contact. No custom code, software, or algorithm central to supporting the main claims of the paper were generated in this manuscript.

HeLa, HEK293T, SW480, and SW480APC cells were plated on glass coverslips and transferred to 2% FBS 6–12 hr before overnight experimental treatments. Coverslips were acid washed and treated with Fibronectin (10 μg/ml for 30 min at 37°C, Sigma F4759) to facilitate cell spreading and adhesion. Cells were fixed with 4% paraformaldehyde (Sigma #P6148) for 15 min, permeabilized with 0.2% Triton X-100 in PBS Gibco for 10 min, and blocked with 5% BSA in PBS for 1 hr.

Primary antibodies were added overnight at 4°C. Cells were washed three times with PBS, and secondary antibodies were applied for 1 hr at room temperature. After three additional washes with PBS, the coverslips were mounted with Fluoroshield Mounting Medium with DAPI (ab104139). Immunofluorescence was analyzed and photographed using a Zeiss Imager Z.1 microscope with Apotome.

Transfections of β-cateninGFP (0.8 mg) and CA-Lrp6GFP (0.8 mg) were performed on 3T3 cells plated on coverslips for 24 hr. Cells were stained following the immunofluorescence protocol described above and stained with Rac-1 antibody.

Cell lysates were prepared using RIPA (Radioimmunoprecipitation Assay) buffer (0.1% NP40, 20 mM Tris/HCl pH 7.5), 10% glycerol, together with protease (Roche #04693132001) and phosphatase inhibitors (Calbiochem #524629), and processed as described (Tejeda-Muñoz et al., 2019).

Experiments were performed with stably transfected HEK293T cells stably expressing BAR and Renilla reporters (Albrecht et al., 2020), treated with LiCl and with or without Bafilomycin for 8 hr, and Luciferase activity measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions, using the Glomax Luminometer (Promega). Luciferase values of each sample were normalized for Renilla activity.

SW480 cells were cultured in a Petri dish using DMEM:F-12 medium with 5% FBS. The top cover was removed from 60 mm tissue culture dishes and 3 ml of PBS placed in the bottom of the dish to act as a hydration chamber. Cells were counted and 500 cells were added as 25 μl drops deposited onto the Petri dish cover, and immediately inverted over the humid chamber. At least 6 drops per condition were plated, keeping enough distance between each other. The inverted drop cultures were incubated at 37°C in 5% CO₂/95% humidity.

The drops were monitored daily; after 4 days, aggregates had been formed and EHT treatment was added to the spheroids. After 4 days, spheroids were incubated with TMR-dextran 70 kDa (1 mg/ml) for 1 hr, and each spheroid was photographed with an Axio Zoom.V16 Stereo Zoom Zeiss microscope with apotome function.

SW480 cells were seeded on glass bottom culture dishes (MatTek Corp). After 4 hr, the cells were transfected with mGFP with Lipofectamine 3000 (Albrecht et al., 2020). The video was taken after 3 days in culture. Control cells had DMSO added for 15 min. Then, cells were treated with PMA (0.3 µM) for 15 min. Filming was with a Zeiss Observer.Z1 microscope and AxioVision 4.8.2 SP3 software was used for imaging.

3T3 or SW480 cells were seeded on cover glass coverslips for 12–18 hr as described in Tejeda-Muñoz et al., 2022c. The cells were incubated with Wnt3a overnight in the presence or absence of PMA and EIPA. After incubation, the cells were treated with 1 mg/ml TMR-dextran 70 kDa for 1 hr, then washed, fixed, and blocked for 1 hr with 5% BSA in PBS to reduce background from nonspecific binding. The coverslips were mounted using Fluoroshield Mounting Medium with DAPI (Abcam, ab104139). Immunofluorescence imagings were performed using a Zeiss Imager Z.1 microscope with Apotome.

We determined experimentally that optimal ventralization with loss of head structures of Xenopus embryos was obtained after incubation with Baf (5 μM) or FAK inhibitor PF-00562271 (100 µM) for 7 min at the 32-cell stage in 0.1× MMR solution. After incubation, embryos were washed two times with 0.1× MMR solution and cultured overnight until early tailbud tadpole stage. Embryos were microinjected once ventrally at the 4-cell stage with any of the following reagents: LiCl (300 mM), PMA (500 nM), EIPA (1 mM), EHT (1 mM), or mRNAs such as DN-GSK3-β (150 pg), DN-Rab-7 (500 pg), and xWnt8 (2 pg), either alone or co-injected with the other reagents. In some experiments, microinjected embryos were incubated in 0.1× MMR solution until the 32-cell stage and transferred to 0.1× MMR solution with or without Baf (5 μM) and incubated for 7 min. Embryos were then washed twice with 0.1× MMR solution and cultured until tadpole stage. In situ hybridization was performed as described at http://www.hhmi.ucla.edu/derobertis.

Whole embryos at the 32-cell stage were incubated with LiCl (300 mM) alone for 7 min. After this treatment, the embryos were washed two times with 0.1× MMR solution and cultured until the tadpole stage, or incubated again for 7 min in 0.1 MMR solution containing 5 μM Baf, and washed as described before. In the converse experiment, embryos were first treated with Baf and then with LiCl. All treatments were done at the 32-cell stage, which plays a key role in the early Wnt signal (Kao et al., 1986; Tejeda-Muñoz et al., 2022c).

Quantitative RT-PCR experiments using Xenopus embryos were performed as previously described (Colozza and De Robertis, 2014). Primer sequences for qRT-PCR were as follows:

Data were expressed as means and standard error of the means (SEMs). Statistical analysis of the data was performed using the Student t-test; a p-value of <0.01** was considered statistically significant for differences between means. Fluorescence was quantified in control versus treated cells using ImageJ software analyses with n > 30 cells or n > 30 images from arrays from humans or mouse sample per condition. Fluorescence intensity was normalized in images compared in each condition and results from three or more independent experiments were presented as the mean ± SEM.