Final Report Summary - SPONGE SIGNALING (Using sponges as a model to examine the evolution of the Wnt/beta-catenin signaling pathway)
The central concept of the field of evolutionary developmental biology (evo-devo) is that changes in development give rise to changes in form. While recent advances in both comparative genomics and experimental biology have greatly accelerated the rate of discovery in evo-devo by facilitating the study of non-traditional experimental models, we have only begun to understand how developmental mechanisms themselves first evolved, and how these ancient evolutionary events shaped the morphological evolution of lineages that persists today. The focus of this project is on the evolution of the Wnt/beta-catenin developmental signaling pathway and its role in modern sponges, one of the earliest branching metazoan lineages. In bilaterians, the Wnt/beta-catenin pathway plays a critical role during animal development, as it controls cell differentiation, proliferation and apoptosis by regulating the expression of a high number of target genes. In addition to its normal functions, misregulation of this pathway can lead to serious diseases in humans, such as colon cancer. The discovery of conserved homologs of the Wnt/beta-catenin pathway in sponges raises questions about whether a functional Wnt/beta-catenin pathway is present in sponges and what its role may be in organisms of such simplicity – they lack many features of bilaterians, such as muscles, nerves and a gut.
The specific aims of this study are to (1) test whether the sponge homologs of Wnt/beta-catenin signaling pathway interact by Co-Immunoprecipitation (2) identify the tissue-specific and subcellular localization patterns of beta-catenin by immunostaining (3) identify which target genes are regulated by the beta-catenin/TCF transcriptional complex by ChIP-sequencing and (4) develop techniques for studying gene function in vivo in sponges. The overall goal is to learn more about how metazoan morphology evolves, and about how signaling pathways themselves evolve.
The first three aims of this project required a specific antibody against beta-catenin from sponges. I have been working on obtaining a specific polyclonal antibody against beta-catenin from Ephydatia muelleri. E. muelleri is a freshwater demosponge, distributed worldwide and is also present in lakes in Colorado. This sponge produces ‘gemmules’, which are overwintering spores with a package of stem cells and yolk reserves, which can grow into juvenile sponges in the lab. To obtain a specific antibody against beta-catenin, I took two antigen approaches; (I) peptides and (II) recombinant protein. Peptides were designed and synthesized commercially, whereas I personally cloned, expressed and purified a recombinant His-/Sumo- tagged beta-catenin fusion protein, in-house. Upon validation and affinity purification of these antibodies, I found that the antibody generated from recombinant protein yielded the highest specificity and activity.
Using this antibody I was able to identify tissue-specific and subcellular localization patterns of beta -catenin by immunostaining (Aim 2). Using this antibody I was also able to immunoprecipitate endogenous beta-catenin from sponge cell lysates and analyze the pulled-down proteins by mass spectrometry to identify binding partners (Aim 1). However, I have not been able to use this antibody for ChIP-seq (Aim 3), as the sequencing of the E. muelleri genome is still in progress and is not available yet. I also worked on developing RNAi methods for sponges (Aim 4). I took several approaches, such as dsRNA, siRNA, (vivo-) morpholinos, and different delivery methods, such as soaking, lipofection, endoporter and cell-penetrating peptides. Unfortunately we do not have access to embryos, so we cannot use injections for macromolecule delivery. I have analyzed knockdown efficiency by both qRT-PCR and by Western Blot. I have found that none of the investigative methods result in a significant decrease in transcript or protein levels. Hence, I used an alternative method and took a pharmacological approach by treating sponges with GSK3b inhibitors (LiCl, Alsterpaullone and BIO) to activate the Wnt/beta-catenin pathway.
The main achievements of this project are first of all the development of a specific antibody against sponge beta-catenin, which can be used for Co-IP, immunostaining and ChIP-sequencing. The Co-IP results validated the beta-catenin antibody, but also confirmed the presence of the binding partners classical cadherin and alpha-catenin. This result is the first proof (besides Electron microscopy images) that sponges contain adherens junctions. Another important result is the localization of beta-catenin subcellular and tissue-specific in the sponge E. muelleri. Immunostaining results with beta-catenin show nuclear staining in the pinacoderm (i.e epithelial cells) as well as motile cells (e.g. archaeocytes) in the mesohyl indicating a role as a (co)-transcription factor, possibly in the Wnt/beta-catenin signaling pathway. Also localization of beta-catenin was found in cell boundaries of the exopinacoderm and in actin plaques in the endopinacoderm, indicating a role for cell-cell adhesion. Remarkably, the choanoderm (feeding cells with collars and flagella), does not seem to utilize beta-catenin, as staining is absent from nuclei as well as cell boundaries. Finally, we were able to upregulate the Wnt/beta-catenin pathway in sponges by treating them with GSK3b inhibitors. This resulted in some distinct phenotypes, e.g. canals were mostly absent and the choanoderm was either absent or malformed. These results indicate that when the Wnt/beta-catenin pathway is activated in sponge, the formation of the aquiferous system is being inhibited.
The main conclusions we can draw from this study is that beta-catenin in sponges, one of the earliest branching lineages of metazoans, plays a role in adherens junction as well as a role in the Wnt/beta-catenin pathway. These results suggest that this dual role for beta-catenin was already present in the last common ancestor of sponges and all other animals.
Besides these results having an impact on our understanding of metazoan morphological evolution as well as the origin of multicellularity, this study will also give us more insight in basic gene regulatory mechanisms, which will directly benefit research towards human health and disease. For example, cancer is directly related to failure in multicellularity. Understanding the origin of metazoan multicellularity will provide more insight in the origin of cancer.
The specific aims of this study are to (1) test whether the sponge homologs of Wnt/beta-catenin signaling pathway interact by Co-Immunoprecipitation (2) identify the tissue-specific and subcellular localization patterns of beta-catenin by immunostaining (3) identify which target genes are regulated by the beta-catenin/TCF transcriptional complex by ChIP-sequencing and (4) develop techniques for studying gene function in vivo in sponges. The overall goal is to learn more about how metazoan morphology evolves, and about how signaling pathways themselves evolve.
The first three aims of this project required a specific antibody against beta-catenin from sponges. I have been working on obtaining a specific polyclonal antibody against beta-catenin from Ephydatia muelleri. E. muelleri is a freshwater demosponge, distributed worldwide and is also present in lakes in Colorado. This sponge produces ‘gemmules’, which are overwintering spores with a package of stem cells and yolk reserves, which can grow into juvenile sponges in the lab. To obtain a specific antibody against beta-catenin, I took two antigen approaches; (I) peptides and (II) recombinant protein. Peptides were designed and synthesized commercially, whereas I personally cloned, expressed and purified a recombinant His-/Sumo- tagged beta-catenin fusion protein, in-house. Upon validation and affinity purification of these antibodies, I found that the antibody generated from recombinant protein yielded the highest specificity and activity.
Using this antibody I was able to identify tissue-specific and subcellular localization patterns of beta -catenin by immunostaining (Aim 2). Using this antibody I was also able to immunoprecipitate endogenous beta-catenin from sponge cell lysates and analyze the pulled-down proteins by mass spectrometry to identify binding partners (Aim 1). However, I have not been able to use this antibody for ChIP-seq (Aim 3), as the sequencing of the E. muelleri genome is still in progress and is not available yet. I also worked on developing RNAi methods for sponges (Aim 4). I took several approaches, such as dsRNA, siRNA, (vivo-) morpholinos, and different delivery methods, such as soaking, lipofection, endoporter and cell-penetrating peptides. Unfortunately we do not have access to embryos, so we cannot use injections for macromolecule delivery. I have analyzed knockdown efficiency by both qRT-PCR and by Western Blot. I have found that none of the investigative methods result in a significant decrease in transcript or protein levels. Hence, I used an alternative method and took a pharmacological approach by treating sponges with GSK3b inhibitors (LiCl, Alsterpaullone and BIO) to activate the Wnt/beta-catenin pathway.
The main achievements of this project are first of all the development of a specific antibody against sponge beta-catenin, which can be used for Co-IP, immunostaining and ChIP-sequencing. The Co-IP results validated the beta-catenin antibody, but also confirmed the presence of the binding partners classical cadherin and alpha-catenin. This result is the first proof (besides Electron microscopy images) that sponges contain adherens junctions. Another important result is the localization of beta-catenin subcellular and tissue-specific in the sponge E. muelleri. Immunostaining results with beta-catenin show nuclear staining in the pinacoderm (i.e epithelial cells) as well as motile cells (e.g. archaeocytes) in the mesohyl indicating a role as a (co)-transcription factor, possibly in the Wnt/beta-catenin signaling pathway. Also localization of beta-catenin was found in cell boundaries of the exopinacoderm and in actin plaques in the endopinacoderm, indicating a role for cell-cell adhesion. Remarkably, the choanoderm (feeding cells with collars and flagella), does not seem to utilize beta-catenin, as staining is absent from nuclei as well as cell boundaries. Finally, we were able to upregulate the Wnt/beta-catenin pathway in sponges by treating them with GSK3b inhibitors. This resulted in some distinct phenotypes, e.g. canals were mostly absent and the choanoderm was either absent or malformed. These results indicate that when the Wnt/beta-catenin pathway is activated in sponge, the formation of the aquiferous system is being inhibited.
The main conclusions we can draw from this study is that beta-catenin in sponges, one of the earliest branching lineages of metazoans, plays a role in adherens junction as well as a role in the Wnt/beta-catenin pathway. These results suggest that this dual role for beta-catenin was already present in the last common ancestor of sponges and all other animals.
Besides these results having an impact on our understanding of metazoan morphological evolution as well as the origin of multicellularity, this study will also give us more insight in basic gene regulatory mechanisms, which will directly benefit research towards human health and disease. For example, cancer is directly related to failure in multicellularity. Understanding the origin of metazoan multicellularity will provide more insight in the origin of cancer.