Early sea urchin embryos contain cells called micromeres, which play an important role in the formation of three mesodermal cell types: skeletogenic, blastocoelar and pigment cells. When micromeres are removed, the embryo can replace the skeletogenic and blastocoelar cells via a process called ‘transfating’, whereby other cells in the embryo step in to take on new roles. However, the pigment cells do not reappear, and the reasons for this are unclear. A new paper in Development reveals how the timing of developmental signals can affect transfating outcomes. To learn more about the story behind the paper, we caught up with first author Alejandro Berrio and corresponding author David McClay, the Arthur S. Pearse Professor Emeritus of Biology at Duke University, USA.
Alejandro Berrio (left) and David McClay (right)
David, what questions are your lab trying to answer?
DM: I'm interested in early development. Many years ago, I selected the sea urchin as a model for study because it is a deuterostome and a relatively simple model of early development. We ask questions that address specification, lineage divergence, gene regulatory networks and morphogenesis. We participate with the community in identifying gene regulatory networks governing specification of all cell types produced up until gastrulation. We explore morphogenesis, addressing questions such as how early epithelial-to-mesenchymal transitions (EMTs) work, how the archenteron invaginates, and how primordial germ cells home to the coelomic pouches. We are also interested in questions involving transfating and injury response mechanisms.
Alejandro, how did you come to work in the lab and what drives your research today?
AB: I began my postdoctoral research at Duke University under the mentorship of Dr Greg Wray after completing my PhD on the evolution of monogamy and sexual fidelity of prairie voles at the University of Texas at Austin. Initially, my work focused on developing methods to detect positive selection and conducting bioinformatics analyses in primates. Just before the lockdown and during the merger of the McClay and Wray labs, I transitioned to studying sea urchins, analyzing old datasets and performing basic bioinformatics research. During the lockdown, I dedicated my efforts to analyzing the evolutionary patterns of SARS-CoV-2 and assisted the university in reporting the various genetic variants emerging in North Carolina. Around this time, I also initiated my own single-cell RNA sequencing (scRNA-seq) experiments with sea urchins. What drives me is the natural history and developmental biology of sea urchins, which is fascinating; they are excellent organisms for understanding the molecular mechanisms of gene regulatory networks.
Tell us about the background of the field that inspired your work
DM & AB: Sea urchin development has been studied for well over 100 years. Many discoveries at the cellular level and later at a molecular level have provided important contributions to our understanding of how development works. The embryo is easy to manipulate and is readily available at marine labs around the world. As such, it was a favorite of many investigators who went to marine labs during the summer months. I (DM) entered the field initially with an interest in cell adhesion and how adhesive changes participate in morphogenesis. The many tools that had been established by previous investigators allowed us to gain insights into the role of adhesion, both at the cellular and molecular levels. Those early investigations led us deeper into questions that continue at many levels. Current molecular tools now allow us (DM & AB) to use genomics, the datasets, and computational tools to address many questions in development, including a chance to discover details of the transfating mechanisms.
Sea urchin development has been studied for well over 100 years. Many discoveries at the cellular level and later at a molecular level have provided important contributions to our understanding of how development works.
Can you give us the key results of the paper in a paragraph?
DM & AB: With scRNA-seq, we studied three mesodermal lineages during development of control and micromereless embryos. We learned that skeletogenic cells and blastocoelar cells follow previously unappreciated specification pathways then converge toward the control lineage paths. Those temporal atlas comparisons show the sequential movement of transfating from an early endoderm specification state to an endomesoderm state, then to a mesoderm state, and finally the specification matches the control specification trajectory of blastocoelar or skeletogenic cells. We confirmed that pigment cells fail to be specified when micromeres are absent. That prompted us to investigate possible reasons for the missing pigment cells and led to the discovery that the timing of Delta versus Nodal signaling governs the ability of pigment cells to reappear. Further tests strongly supported the hypothesis that Delta signaling must occur prior to Nodal signaling in the mesoderm for pigment cells to emerge.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
AB: Absolutely, we did! We were fascinated when we observed the reappearance of skeletogenic cells in the micromereless single-cell data with an endoderm gene signature, along with the delayed appearance and lack of pigment cells. This aligned perfectly with previous observational evidence. It was truly amazing to see our expectations fulfilled with our dataset.
It was truly amazing to see our expectations fulfilled with our dataset.
Late gastrula stage of the sea urchin Lytechinus variegatus. Skeletogenic cells (green), blastocoelar cells (orange), and pigment cells (red) enter the blastocoel during gastrulation by way of an EMT. If removed early in cleavage, all three cell types can return by the process of transfating from early endoderm cells. Skeletogenic cells and blastocoelar cells return without further experimental intervention but pigment cells reappear only if the timing of two signals (Delta and Nodal) is manipulated. The cells are stained by hybridization chain reaction fluorescence in situ hybridization and all nuclei are stained with Hoechst 33342 dye.
Late gastrula stage of the sea urchin Lytechinus variegatus. Skeletogenic cells (green), blastocoelar cells (orange), and pigment cells (red) enter the blastocoel during gastrulation by way of an EMT. If removed early in cleavage, all three cell types can return by the process of transfating from early endoderm cells. Skeletogenic cells and blastocoelar cells return without further experimental intervention but pigment cells reappear only if the timing of two signals (Delta and Nodal) is manipulated. The cells are stained by hybridization chain reaction fluorescence in situ hybridization and all nuclei are stained with Hoechst 33342 dye.
And what about the flipside: any moments of frustration or despair?
AB: The 10x Genomics single-cell protocol and its sequencing are quite expensive and require a certain number of cells to be successful. We failed in our first two attempts before we were able to obtain sufficient numbers of cells for the protocol.
Why did you choose to submit this paper to Development?
DM & AB: We published our first scRNA-seq paper in Development and found the review process to be constructive in improving that paper. Also, Development reaches the audience we want to reach so it was an easy choice to submit this paper to Development. Both reviewers contributed substantially with their scepticism and suggestions for clarification. To address the scepticism, further experiments allowed us to gain confidence that the timing sequence of Delta and Nodal was crucial for the reappearance of pigment cells. At their suggestions, we also added the graphics that made it easier for the reader, especially those not familiar with sea urchin development, to follow the logic of the experiments.
David, where will this story take your lab next?
DM: The datasets are quite rich with information that will help us further explore how transfating works. They also provide useful information toward other ongoing projects in the lab, especially toward our interest in how the cell biology of EMTs work. All three cell types in this paper – skeletogenic cells, blastocoelar cells and pigment cells – undergo an EMT. These three EMTs are governed by different but partially overlapping groups of transcription factors. They also occur at different times in development, so the goal is to learn how similar they are at a molecular level, and the scRNA-seq datasets provide a rich set of candidate molecules to explore.
Alejandro, what is next for you after this paper?
AB: This dataset is incredibly rich, leading to many new questions. There are particular cell types that were unexpectedly over-represented, and I am currently focused on understanding the molecular and cellular mechanisms behind this finding. In addition, I am working on new datasets that we have obtained from the vegetal versus animal halves of the Mediterranean sea urchin, as well as the lecithotrophic sea urchin in Australia.
Finally, let's move outside the lab – what do you like to do in your spare time?
DM: I've been running for more than 60 years and continue, albeit at a much slower pace. Travel is also a most enjoyable part of my life. I work at marine labs in France, Woods Hole, Friday Harbor, Australia, Japan, Italy, Bermuda and Chile. In addition, travel with family and friends has been a great way to learn about the culture and history of the world.
AB: In my spare time, one of my favorite activities is riding my gravel bicycle around the Research Triangle in North Carolina, or anywhere else I travel. I am part of a wonderful bike community and often get to lead bike rides. I also enjoy going to outdoor events with my friends. When I have time at home, I enjoy cooking, making cheese, playing crapette and word puzzles, and I also make digital art and do woodcarving.
Department of Biology, Duke University, Durham, NC 27708, USA.
E-mail: [email protected]
