SDB Ethel Browne Harvey Postdoctoral Seminar Series - Yuchuan Miao and Kari Price

Summary

Highlights

• Yuchuan Miao discussed human somite segmentation using stem cell-based systems to model developmental processes 🎓.
• Miao's innovative approach provides insight into the segmentation clock and somitogenesis in human biology 🕰️.
• Kari Price examined intracellular bridges, crucial for connectivity during cell division, using Drosophila models 🔬.
• Price's findings on midbody to ring canal transition provide new perspectives on cyto kinesis and cell communication 🔄.
• Both researchers emphasized the importance of modeling techniques in studying developmental processes and diseases 📊.

Key Takeaways

• Yuchuan Miao elucidated the process of somitogenesis using stem cell-based organoid models, enhancing our understanding of human development 🌱.
• Kari Price's research on intracellular bridges sheds light on cell division processes and their impact on fertility 🔍.
• The seminar highlights the significance of foundational research in understanding complex biological systems 🌟.
• Innovative models and methods are crucial for advancing developmental biology and translational medicine 🚀.
• Interdisciplinary collaboration and novel techniques are at the forefront of uncovering developmental biology mysteries 🧩.

Overview

Chapters

• 00:00 - 00:30: Introduction and Welcome The chapter introduces the seminar series organized by the Elal Brown Harvey Postdoctoral community. Casey Griffin, a postdoc at New York University, and Martha Etaria, a postdoc at the University of Wisconsin Madison, are moderating the event. The focus is to highlight the contributions of postdoctoral members, specifically Uan Meow from Harvard Medical School.
• 00:30 - 01:00: Introduction of Speakers The chapter titled 'Introduction of Speakers' focuses on welcoming and providing information about the speakers from Brigham and Women's Hospital and Yale University School of Medicine, who are presenting their research. Each speaker is scheduled to give a 20-minute talk followed by a 10-minute Q&A session. The audience is encouraged to submit their questions via the Zoom Q&A box. The session begins with Yuan Mow, who holds a Bachelor of Science in Chemistry from Nanai University in Tianjin, China.
• 01:00 - 02:00: Yuan Miao's Academic Background Yuan Miao completed his PhD in Biochemistry and Cell Biology from John Hopkins University School of Medicine in 2011. In 2019, he began his postdoctoral training at Harvard Medical School and Brigham and Women's Hospital in the lab of Olivier Porier. He has received numerous honors and awards, including the Top Three Trainee Award from Harvard Stem Cell Institute and the Rising Stars in Engineering and Health from Columbia.
• 02:00 - 08:00: Yuan Miao's Research on Human Somitogenesis In this chapter, the focus is on Yuan Miao's research on human somitogenesis, particularly its reconstruction and deconstruction in vitro. Yuan, who is affiliated with Johns Hopkins University and has received a K99 Pathway to Independence Award from the NIH, introduces the topic and expresses gratitude for the opportunity to present the work.
• 08:00 - 10:00: Somitoid and Segmentoid Models The chapter titled 'Somitoid and Segmentoid Models' delves into the research focuses of a scientist named Tron, who is engaged in studying the processes involved in pattern and shape formation during human development. Tron's work, grounded in cell biology, emphasizes the mechanisms of pattern information at the cellular level. The chapter highlights Tron's postdoctoral focus on the process of somitogenesis, or somit formation, in early embryonic development, clarifying the significance of somites as repeated structures.
• 10:00 - 13:00: Anterior and Posterior Patterning The chapter titled 'Anterior and Posterior Patterning' discusses the role of somites in the development of the vertebrate body plan. Somites, which flank the neural tube, give rise to various structures including the axial skeleton and skeletal muscles. These structures are crucial for establishing the segmental features of the body. Interestingly, the formation of somites is not simultaneous but occurs sequentially from head to tail, with each somite being formed separately.
• 13:00 - 20:00: Cell Sorting and Pattern Formation The chapter discusses a region known as the PSM (presomitic mesoderm) involved in cell sorting and pattern formation. It describes how this region functions during the body's elongation process. The periodicity of somite (sui) formation is regulated by a conserved gene regulatory network oscillator known as the segmentation clock. This clock's activity, often represented as a wave of blue, begins at the posterior end and moves anteriorly.
• 20:00 - 28:00: Questions for Yuan Miao The chapter discusses genetic studies focusing on segmentation in the development of somite formation. It highlights the importance of the 'seven' gene, considered a core gene in the segmentation clock in both mice and humans. Additionally, the expression of the 'mass P2' gene in the anterior region is marked as the beginning of somite formation.
• 28:00 - 30:00: Introduction of Kari Price The chapter introduces Kari Price and begins with a discussion of somites, which are described as intricately patent. Each somite is divided into an anterior and posterior half, characterized by specific gene transcriptomes and developmental trajectories. The discussion focuses on a gene known as 'anks' that labels the posterior half of each somite. The 'anks' RNA expression is illustrated in mouse embryos, where it forms beautiful periodic patterns.
• 30:00 - 35:00: Kari Price's Research on Intracellular Bridges The chapter discusses Kari Price's research on intracellular bridges, focusing on the concept of somite anterior-posterior (AP) polarity patterning, also known as Rose trado patterning. This patterning is critical to the proper development of the body plan, including the formation of vertebrae in the spine. Additionally, only the anterior half of the somite allows for axon migration, thereby influencing the segmental organization of the peripheral nervous system.
• 35:00 - 40:00: Ring Canal Formation This chapter titled 'Ring Canal Formation' delves into the subject of somitogenesis, which is a process crucial in developmental biology. It highlights the significant amount of knowledge we have acquired about this process through the study of animal embryos. Despite the advancements in understanding, the text points out that challenges remain in comprehending the complete scope of somitogenesis. While the segmentation clock controls and the resistive somit formation have been well-characterized, the subsequent processes of somite patterning and morphogenesis are not yet fully understood, indicating areas where further research and investigation are needed.
• 40:00 - 45:00: Dynamics of Cytokinesis Proteins The chapter titled 'Dynamics of Cytokinesis Proteins' delves into the relatively underexplored area of cellular mechanisms controlling cytokinesis and their link with the cellular clock. While classical studies have predominantly concentrated on gene expression at the tissue level, it's emphasized that it is the individual cells that create complex patterns and shapes. The chapter highlights the gaps in understanding at the cellular level, especially concerning the intricate processes that regulate cytokinesis, pointing out the limited knowledge available in this domain.
• 45:00 - 50:00: Citron Kinase Role in Ring Canal Transition The chapter focuses on the role of citron kinase in the transition of ring canals. It draws a parallel with the human species, specifically discussing the formation of somites within the early weeks of development, between three to four weeks post-fertilization. This period is notably when most women are unaware of their pregnancy, making direct study of human embryos at this stage challenging. Due to the limited access to human embryos at this early stage, the chapter introduces stem cell-based approaches as an alternative method for studying these developmental processes.
• 50:00 - 60:00: Sticky Protein Structure Function Analysis The chapter discusses the use of human pluripotent stem cells to create 3D organoid models. These models are designed to replicate the processes of patterning and morphogenesis, specifically somite formation. The organoid models enable direct study and visualization of human developmental processes, offering improved methods for observing cellular activities in these contexts.
• 60:00 - 70:00: Further Research and Conclusions This chapter discusses the construction and application of two models designed to recapture specific patterning information. The first model, referred to as the 'somit toid' model, is particularly focused on recapturing the formation of somites in the embryo over time. The insights gained from these models are crucial for understanding periodic differentiation and morphogenesis within these groups of cells.
• 70:00 - 89:00: Questions for Kari Price The chapter titled 'Questions for Kari Price' describes the synchronous differentiation trajectory of cells. Initially, the cells undergo an oscillation phase as marked by the 'has s' phase. They then collectively transition to the segmental determining phase, labeled as mp2, before starting to form structures similar to somites, referred to as 'somi-like structures'. Eventually, these structures form a field of somi. The description is enhanced with a video presentation using reported cell lines.
• 89:00 - 95:00: Concluding Remarks The chapter discusses two different models related to embryonic development: 'somit toid' and 'segment to.' 'Somit toid' is explained as a model that mimics the characteristics of somites, structures found in developing animals. It includes key features such as absil blocks and is surrounded by a laminine sheet. A notable advantage of this model is its simplicity and the synchronous differentiation of all its cells, making it a simplified version of somitogenesis.

SDB Ethel Browne Harvey Postdoctoral Seminar Series - Yuchuan Miao and Kari Price Transcription

• 00:00 - 00:30 welcome to the elal brown Harvey postoral seminar series my name is Casey Griffin and I'm a postto at New York University and we'll be moderating today with Martha etaria a postto at University of Wisconsin Madison we are excited to highlight the work of our outstanding postdoctoral members today uan meow from Harvard medical school and
• 00:30 - 01:00 Brigham and Women's Hospital and Carrie Price from Yale University School of Medicine will share their research each speaker will give a 20-minute talk followed by 10 minutes of Q&A please enter your questions in the zoom Q&A box we will begin with Yuan mow Yuan got his Bachelor of Science and chemistry from nanai University in t tanin China
• 01:00 - 01:30 in 2011 he then completed his PhD in Biochemistry and cell biology from John Hopkins University School of Medicine in 2019 uton started his postto at Harvard Medical School and brigam and Women's Hospital in 2019 in the lab of Olivier porier he has received numerous honors and awards including the top three trainy award from Harvard stem cell Institute the rising stars in engineering and health from Columbia and
• 01:30 - 02:00 Johns Hopkins University and a k99 pathway to Independence award from the NIH Yuan will be talking today about reconstruction and deconstruction of human somitogenesis in vitro thank you Yan yeah thank you so much for the kind introduction um and thank you for the opportunity for me to share my work here um let me get it started
• 02:00 - 02:30 okay my name is Tron and I'm interested in how multicellular system make patterns and shapes during human development as a cell biologist by training I'm especially interested in cell cell biological level mechanism of pattern information in my postto I focusing on um I was focusing on the fascinating process of so mitogenesis or somit formation in the early embryo so somites are the repeated
• 02:30 - 03:00 iite blocks flanking our NE tube they give rise to our axal um skeleton and skeletal muscle and so on they're instrumental to establish the segmental features of our body plan um in our vertebrates uh Som Genesis share like several conserved features very in exciting interestingly somites are not formed all at once they are formed sequentially from H to tail uh each somite will will B off from a
• 03:00 - 03:30 unsegmented region called PSM uh one by one in coordination with the elongation of the body axis um and the rity of sui formation is controlled by a conserved oscillator of Gene regulatory Network called segmentation clock in each cycle um the clock activities denoted as blue in the movie cartoon here which start from the posterior end and propagating anteriorly as a wave and when they reach the
• 03:30 - 04:00 anterior region they together with signal ingredients specify a forming a newly forming somites um now extensive studies us extensive genetic Studies have identified that has seven um is a core Gene of the segmentation clock in Mouse and human while the mass P2 Gene in the expression of mass P2 in anterior region uh labels the onset of somi formation
• 04:00 - 04:30 uh in addition somites are also intricately patent each somite is divided into an anterior and posterior half uh characterized with um specific Gene transcriptome as well as developmental trajectories one of the Gen anks labels the posterior half of each somate if we look at the anx RNA expression in Mouse embryo you can see that they form these beautiful periodic
• 04:30 - 05:00 Stripes uh like flank along our body axis now this somite AP polarity pattering also called Rose trado pattering is extremely important to the development of our proper development our body body plan the instruct the formation our vertebraes of our spine and uh also because only anterior half the somite permits the migration of axons they coners the segmental organization to our periphery nervous
• 05:00 - 05:30 system as well so extensive study using animal embryos have gathered have allowed us to gain lots of knowledge about soog Genesis but there are still few challenges in further understanding somitogenesis um so although the uh much the the the segmentation clock control the ResMed resistive somit formation has begun to be well characterized but the subsequent somite patterning and morphogenesis and
• 05:30 - 06:00 their connection with the clock has are less understood and among our all of our knowledge that we have a a much less understanding on the cellular level mechanisms of controlling this process most of a classical studies focusing on tissue level gene expression after all but after all it's the cells that build these beautiful patterns and shapes and furthermore we have extremely little about to un about the knowledge of this
• 06:00 - 06:30 important process in our own species in humans somites are formed um are curs very early during development between three to four weeks after fertilization it's a Time window uh when most women haven't realized their pregnancy yet th we have extremely little limited access to human embryos at this stage so to address these challenges today I'm showing you some of my work using stem cell based approaches um to
• 06:30 - 07:00 address these challenges I I will use human plop poent stem cells to build 3D organoid models that Rec capsulate the patterning and morphogenesis process somis formation these will allow us to directly study human development and also they allow direct visualization much easier visualization and the a sophisticated paration of cellular activities in in this processes
• 07:00 - 07:30 and then I will show you some of the insights we have gain by using these indidual models uh to study so my patterning okay so long story is short I have built two models to recapture sua information the first model we call them somit toid this is model recalculate that Rec capsulate somi formation in time um in the embryo so my a group of s are per will periodically undergo differentiation and morphogenesis and in the model sum suid model other
• 07:30 - 08:00 cells undergo this differentiation trajectory synchronously as shown by this video here uh with the reported cell lines you can see that the first the cells going to this oscillation phase of has s and then they all together go into this segmental determining phase labeled by mp2 and then starting to go to forming these r that or somi like structures and eventually for forming a field of somi
• 08:00 - 08:30 like structures so as and each of these RADS are um has the characteristics of somit including the absil blocks as well as um surrounded by laminine sheet so somit toid is a model that only have one AIS time that all cells synchronously um differentiate so it's a simple simplified model of semog Genesis now the second model I have built called segment to so this is model um Rec
• 08:30 - 09:00 capture both time and space during somi information so if I play this video you will see that oscillat expression of High s will occur at the posterior end uh as the structure Yong Gates while the segmental gsp2 follows suit and eventually they give a rise to these Rod shap shape like structures with so my like rosettes as well as
• 09:00 - 09:30 recapitulate anterior posterior um patterning of each row of somites as labeled by the stripes expression of anare yeah so together with several other models built by uh other labs we provide a platform to to like St to allow us to study human somitogenesis between the two models I have built they each have their own uh advantages Som toid for Som toid it's a ation where other cells are because other cells are
• 09:30 - 10:00 precisely synchronized so we have unlimited amount of cells at each time point to for in-depth characterization their configuration is also amenable for higher resolution imaging to look at a cellular activities on the other hand segment toid is embedded in matrial and but they display all these important Hallmarks of somitogenesis can be used as a proxy for to test these insights we
• 10:00 - 10:30 have gained from using somid doid um so what as I mentioned earlier I'm really interested in the patterning and morphogenesis process so the the question I have used my starting to use my system to address is some my anterior posterior polarity patterning in this uh in this process um much of our understanding about this process comes from um the extensive work by yumo Saga and it is observe that mp2 the segmental
• 10:30 - 11:00 gene is first inducted uniformly across the whole lens of the full newly form the segments and then mp2 expression is is restricted to the anterior portion and because mb2 uh is a repressor of Notch signaling so in the anterior half of the somite we're experiencing a lower Notch signaling well on the posterior half in addition with um app further applications it experience higher Notch
• 11:00 - 11:30 signaling that's this differential Notch signaling really determines anterior or posterior fate maturation however what is not known is how is the initial symmetry breaking process established how is mb2 transitioned from this Broadband expression to the only half band expression it is perceived that this process is a process of a gene tissue L Gene remodeling process that somehow um the posterior part Mass P expression is
• 11:30 - 12:00 suppressed however there are no concrete models for for this perception then I decided to use my models to look at in detail how this process established I first turned to my Som toid model this is really interesting because Som toid is a simplified model and it challenges this whole system in complete new context that does not exist n naturally if you
• 12:00 - 12:30 think about this model doesn't really have anterior posterior information how would the patent be like in this model and much to our surprise we found that not only can anterior posterior fade cells uh faded cells come up um but also they form into a patterns that in this model this formed mostly entire some somewh like structures with entirely anterior or posterior face labeled by ANS and MP
• 12:30 - 13:00 here this is extremely interesting so I decided to use Li Imaging to look at how this process is uh is occurred um so this movie is going to show showing you the the time window labeled as the uh the blue box here as the green box here um so it's the time window when mp2 is transiently expressed on the left is the mp2 reporter you will see is that the signaling comes up but then at the end of time like 72 hours
• 13:00 - 13:30 when M P2 intensity is not increasing anymore they ended up in this s and peer s and peer fashion that not all cells Express that the cells Express like different have the difference in Mass P2 expression uh as big as up to tfold this directly contracts our traditional notion of a uniform mp2 expression in a forming segment and if you follow this movie a little bit longer now is is uh
• 13:30 - 14:00 labeled by this blue box you will see is that there are extensive C movement and then mass mass two high sell starting to aggregate while Mass two low Sal starting to come together eventually forming these different clusters M to high and mp2 low regions uh now here's the movie with the further labeling and to see this process the Orange Line outlines the future mesu low region while the the color cells
• 14:00 - 14:30 labels Mass to high cells you can see that the cells starting randomly but then they gradually are have moved out of this region and in the end they form um starting to form mass2 low regions after all these M High cells have moved out th it says that the cells um it seems that this cell Phat are first determined before any pattern has formed so to further to to confirm this um we
• 14:30 - 15:00 decided to using dissociation and reaggregation assets so we take cells at 72 hours remember this is a time when Mass B2 has finished its expression it's ended up in a Sal and peer fashion there are no pattern formed yet and then we dissociate the somit toys completely into single cells um and then using facts to separate the top 10 mes to higher cell and bottom 10 m cells and re
• 15:00 - 15:30 aggregate them just C centrifugation and we continue culture them and by 120 hours we want to see their expression profile of ons the mature Gene that labels the posterior Fates now interestingly you can see is that the mass two high cells isolated at the S peer no pattern stage they will continue to um Express lower level of xks while Mass to low cells at this earlier stage will Express much more on
• 15:30 - 16:00 expression so this suggesting that indeed the cell identities are already determined at the salt and pepper stage before any patterns has formed so all of these together with some other quantifications led to a sequence of events that we think what's happened in somit toid is that the mp2 is first is inducted in s pepper version and importantly at this stage the anterior posterior fade in each cells is more or
• 16:00 - 16:30 less already determined and then through cell sorting they quickly form differential clusters and as time goes on each each uh roset emerge from each clusters as you can see see see that is the cluster can be much bigger than the typical somi lens so that's why in the end we end up with somi of entirely anterior or posterior face so what does this mean for um for for what happens in
• 16:30 - 17:00 in in embryos could it be that the conventionally sought Broadband narrow band expression actually be a result of self sorting so we further test this using the other model segment toy to used as embryo proxy so in this uh uh video with label with the red box label o forming segment in the middle you can see that in this forming segment Mass P2 is already inducted and in a certain peer fashion and then as time goes on uh
• 17:00 - 17:30 you can see there extensive cell movement and eventually the M two high cells starting to uh claster and low cells starting to claster eventually forming differential compartments of anterior or posterior Fates and then here here's a video forther zoom in video to look at this process on the left is unlabeled on the right is labeled um in this forming segment region uh you can see we can start looking at this blue cells like or
• 17:30 - 18:00 the cells corresponding to here you can see that it inducted in the posterior half but then as this somit develops is gradually starting to move anteriorly towards the anterior half so indeed this this suggesting that the conventionally S Gene remodeling tissue L Gene modeling process might actually is a result of a cell sorting or cell cell rearrangement
• 18:00 - 18:30 and furthermore when we using uh when we over in exploit overexpress TM one which rack one G which um perturbs the cell sceletal properties we can complete block cell sorting and then you can see that there's still oscillation of the clock but then eventually you can there will there will be no organized Stripes expression formed in the end so all of these together leads us to
• 18:30 - 19:00 a um General framework uh integrating cell sorting cell act Dynamics with the setion clock and um fade specific fade specification I don't have time to uh to to elaborate this model but if you're interested this you can check out our published work for this General framework now in the end I just want to say that um the I I I just hope I convince you that the power of invidual
• 19:00 - 19:30 systems in uncovering novel mechanism of development these models are not perfect and and but they they will but they can we can use them to dissect molecular especially cellular mechanism in detail and and the goal of the field is not to build embryos the goal is to understand biology and I'm extra particular interested in using further using the system I have bu to understand the
• 19:30 - 20:00 cellular mechanism of the pattering and morphogenesis during Soo Genesis and on other hand I'm also interested in continue building uh 3D models of modeling different aspects of human development so with this I like to thank uh my mentor rier and other collaborators and the whole poy lab and our collaborator collaborator Stefano ditalia whose work I I did not have time to show today and also thank my founding source and thank you so much for your
• 20:00 - 20:30 attention and I'll be out for questions thank you ywan um while we wait for questions to open um and come in I will start off um what do you think is the mechanism behind this cell sorting yeah so that is a continuing work um but
• 20:30 - 21:00 we did do a RNC to compare the trans Crypton between mass to high and Mass to low cells and we found quite a lot of interesting um differences uh involving several adhesion proteins such as PCD protoc cateran 8 um and also e seever several eering proteins and and and a lot of cell skeleton regulatory proteins so this is is Contin exciting work going on but I I think it's just that they are
• 21:00 - 21:30 sorted Parts through the differential adhesion as well as well as differential um cell skeleton properties great thank you so we have a couple questions coming in first off um chitra prabhakara says an excellent talk Yuan I have a question are patterns formed in suids with just mes 2p mes P2 high as as well as M P2 low
• 21:30 - 22:00 cells um yes so at this stage so the somite anterior posterior fate is the first L specify m 2 expression levels at this stage m 2 hea will specify future anterior pH M to low cell with specify um future low cells and then as the Contin maturation uh further um mature genes such as anks will come up to um to label more posterior fate yeah so at
• 22:00 - 22:30 this stage is solely separated through mp2 differential expression great how yangang way says very cool talk I wonder if you sort the mesp2 high and low cells and recede them in a certain spatial pattern that is different from the normal somite organization will the predetermined fate be preserved and cell sorting happening um thank you for the question um let me
• 22:30 - 23:00 try to understand reeding certain spatial pattern they different from the normal so yeah so this is a very interesting point um we I haven't explored this too much yet but right now what I did is simply sort them part and reag them um together I assumed that by playing with the density or geome geometry we can potentially um introduce more parations um actually might be
• 23:00 - 23:30 interesting to to show what happens to to for example to test the like requirements for cell sorting in term of density in term of geometry and so on yeah so that would be very interesting point going forward great uh Natalia baso Aras wants uh says very interesting congratulations for your work do you know if the segment toids show signs of left right pattern it um I I don't know right now we
• 23:30 - 24:00 haven't looked at it yet um but I want to point out that in our model so far it's only consists of the peroxy mism uh so some of the patterning such as um I I I presume you asking about uh left to right or or other axis they might need the signaling inputs from other germ layers in so far we don't have it but that does not mean is this AIS ex uh extion could not be a self organized
• 24:00 - 24:30 process uh it's certainly something interesting to further look at thank you way Shu says enjoyed your talk are there any human diseases caused by defects in oscillation of the segmentation clock so your human use your models to examine the underlying mechanisms yes that's actually a exciting direction we're going to um because a lot of the the segmental
• 24:30 - 25:00 defects of human embryo are caused by players involved in the segmentation clock and segmental uh genes so for example has s and and D1 these are all genes involved in human um segmentation clock their mutation always cause um a severe uh segmentation defects our spine so these models can be a very in very interesting way to to further look at the molecular and
• 25:00 - 25:30 cellular details of theology here Martin Aros says great talk and work have you noticed any cells begin to delaminate or migrate out of the SIDS or segment toids yeah so it depends on time if you cure these models like longer time other the cell was starting to migrate out um and also I want to point out for is I think it's especially interesting for somit toids because at the edge where
• 25:30 - 26:00 the cell layer are usually much thinner than the in the center so at Edge they don't form rets um they so there seems to be interesting um interesting to look at the to look at the edge of the how the cells Behavior cells cell Behavior at the edge to tell us more about the r that formation but at this stage um yeah yeah they they don't they really
• 26:00 - 26:30 tend to stay in this Ros that structure as well as there's a certain cell density loic Fort says great talk the mesp2 sorting by expression level reminds me of a similar event during intestine specification and folding driven by pdgfr High versus low in this system they show it is dependent on actomyosin which might be reminiscent with your team one data have you tried
• 26:30 - 27:00 to abgate cell contractility in this system uh yes we have tried to using Rock inhibitor to add it into the cell culture here um with the rocking in the presence of rocking inhibitor they cannot form Rik structures however in the presence of rock inhibitor they can still show some rudimentary um cell like cell clustering process so I I think it's um so my perception is that the
• 27:00 - 27:30 cell contracted here perturbing it with Rocky with rock inhibitor might uh change the um the robustness of P information but it doesn't seem to be essential for the Sorting process great and how young way asks again uh may I ask if the left side and right side of somite are physically separated if so how does the somite formations synchronized between left and
• 27:30 - 28:00 right um the left side and right side is so might physically separated I think what you mean is the each row of the SE each row of somites in the segment toid um yes they are if we do a standing you can see they're indeed separated by uh ECM um and also we've found that that when we SE this when we starting with a bigger cell aggregate we will form a
• 28:00 - 28:30 cell a a segment structure that's much wider you can consists three or four somites so it seems that in in all these cases um the one row perpendicular to the AP AIS are always synchronized um we don't know exactly how but I think it have to do with the CL segmentation clock and how it's a propagation Direction and yeah it will be like investigated further in the future great that's all the questions we have
• 28:30 - 29:00 in the box so with that I think we will move on thank you so much hi everyone it's my pleasure to introduce our next speaker Carrie Price um Carrie completed her training at the University of California Davis in the laboratory of Dr lesie Rose and then she transition it to to a postu at the jail School of Medicine in the laboratory of
• 29:00 - 29:30 Dr lene KY her postductal research focuses on elucidating the mechanism of intracellular Bridge formation in animal germline cells her research has been supported by an NIH F32 and the jail Venture F fund and CNA Foundation post sectoral Fellowship today her presentation is entitled Bridging the Gap connecting cells through intracellular Bridges so Carrie you can
• 29:30 - 30:00 go ahead okay play all right thank you Martha for the introduction and thank you sdb for the opportunity to present my work here today and my research focus is on trying to understand how the canonical program of cyto Kinesis is modifi ified in certain cell types so that following
• 30:00 - 30:30 incomplete cyto canis these cells remain connected by intercellular Bridges and in most animal cells cyto canis is complete and produces two physically separate daughter cells with every cell division however a number of cell types undergo or utilize a highly regulated program of incomplete cyto Kinesis that gives rise to cells connected by intercellular Bridges and it all the formation of these intracellular Bridges allows these cells
• 30:30 - 31:00 to develop in effect as censia and intercellular Bridges have been observed in cells with high developmental potential including embryonic cells and multi-potent stem cells as well as inter terminally differentiated cells colonal and colonal multicellular animals but intercellular bridges are most prominent in the context of the animal germ line where intercellular Bridges um and their formation is absolutely critical to the formation of male and female germ
• 31:00 - 31:30 cells and in The Germ line these intercellular bridges are called ring canals aptly named because they take on this ring-shaped appearance and this ring-shaped cytoskeletal structure maintains the cytoplasmic opening connecting the sibling cells and on the ultr structural level um ring canals can be easily identified by the presence of the electron dense plasma membrane that marks the cytoplasm or the the the
• 31:30 - 32:00 membrane between connecting these two cells and I'm showing you here an example of two spermatocytes that are connected by a single cytoplasmic bridge and marking here with the Red Arrows I'm denoting the the boundary of this electron dense electron density and we've now generated fibsem data sets and we can reconstruct these data sets um to actually visualize the ring canals um in 3D and I'm showing you here just a
• 32:00 - 32:30 reconstruction of this data set where you can more easily see the ring canals structure um ring Canal formation is an incredibly well-conserved feature of gam Genesis and kinan 6 is an um not only a component of the central spendin complex an important cyto Kinesis regulator but it's also a universal ring Canal component and I should mention that ring canals are absolutely critical to the formation of germ cells as they allow
• 32:30 - 33:00 the transfer of mrnas proteins and even organel between sibling cells and when we look with this Kines in six we could actually see that across um the animal kingdom Kines and six localizes robustly to these rain canals and I'm showing you here just a panel of male germ lines from the respective species where I have taken these germ lines and imuno labeled with an antibody that recognizes the chines and six orthologue in each of these species and so it's this um localization
• 33:00 - 33:30 of this critical cyto inesis component to ring canals that has hinted at this really intimate relationship between uh programs of cyto canis and ring Canal formation and for most of my talk I am going to be referring to the dropo orthologue of kinese in sixs which is a protein that we call pavara or pav typic at the end of the cell cycle cyto canis
• 33:30 - 34:00 terminates with midbody mediated excision which physically separates these nent daughter cells and during cyto Kinesis an atomas and contractile ring assembles at the equatorial cortex between the segregating sister chromatids and does so in response to cues from the central spindle and Central spindal Lin which is a heterotetrameric complex of mgc rat Gap and kinese and six it's obligate finding partner following constriction
• 34:00 - 34:30 of the actomyosin contractile ring a midbody forms which marks this transient intercellular bridge and its architecture can be subdivided into two groups the midbody core shown here in purple which is an accumulation of central spendin protein and the midbody ring which is a comprised of former components of the contractile ring including Anan Citron cyas and the SE and the midbody plays an important role in mediating cyto canis as it serves as
• 34:30 - 35:00 a platform for the recruitment of these obis Machinery which is necessary to sever this transient cytoplasmic Bridge physically separating and permanently separating these uh newly synthesized daughter cells in following division a midbody Remnant forms um and this midbody Remnant depending on the cell context can either be absorbed by one of the daughter cells or extruded into the medium however um in contrast during incomplete
• 35:00 - 35:30 cyto canis um this process is marked by a characteristic absence of obis and there's been much work that has um been done to try and understand the mechanism inhibiting exision um but it remains unclear exactly how cyto nesis is altered in the dividing germ cell in my research and I am particularly interested in understanding how the process of obis inhibition is coordinated with the cytoskeletal
• 35:30 - 36:00 changes that underly ring Canal formation and specifically how components that localize to the central spindle and to the contractile ring come to be localized into these membrane bound stable ring Canal structures and I use the dropo melanogaster test as my model system where spermatogenesis occurs in a spaciotemporal pattern and at the apical tip of the test the germline stem cells and the somatic stem cells surround a
• 36:00 - 36:30 cluster of somatic cells called the Hub the germline stem cell will divide asymmetrically to produce a gonal blast and another germline stem cell the gonio blast then goes on to divide four times mitotically and two times meiotically to generate 64 hpid sperm and with each mitotic and myotic division cyto canis is incomplete resulting in the formation of a single ring canal and in contrast the somatic stem
• 36:30 - 37:00 cell divides completely to generate a somatic stem cell and a sematic cyst cell that will envelop the gonal blast and will surround the developing germ cell throughout the entire process of spermatogenesis another feature that's important to um my work is this structure called the fusome which is shown here in this translucent Gray um and it serves as another form of connectivity between cells a sibling
• 37:00 - 37:30 cells within a cyst and the fusome is this Branch like structure that extends throughout all of the Ring Canal lumens that connect all of the cells in a germine CST and when I first joined the lab I really wanted to um characterize ring Canal formation in real time um with high spatio temporal live Imaging um and the first thing that he did was to generate flies that were Express expressing an endogenously tagged pob IM
• 37:30 - 38:00 Cherry fusion protein and it performed live Imaging and I'm showing you here um an example movie of a dissected dropa tesus that's expressing pav in Cherry to Mark the ring canals and histone gfp to correlate this ring Canal formation with the cell cycle and I'm showing here in the Stills um these are stills from this time- lapse movie highlighting a two cell sis with a single ring canal and following Ana's onset pavara localizes
• 38:00 - 38:30 to the cleavage Furrow and after contractile ring constriction we see that pav forms this bright midbody like intermediate and this persists for about 20 minutes before pav under goes an apparent reorganization uh to form the ring canal and you can see that clearly here in these insets of ring Canal formation and the onos orientation and we call this transition the midbody to ring Canal transition um
• 38:30 - 39:00 and it differs uh greatly from the Dynamics of cell division or cyto canis in the somatic stem cell division where again this is a complete Division and following um anaphase onset pav localizes to the cleavage Furrow and after contractile ring constriction forms these uh forms these midbodies that are very different than the midbodies or the midbody likee structures that we observed in the germ cell divisions and that they are much smaller in diameter than their um germ
• 39:00 - 39:30 cell counterparts and also they do not undergo the same apparent reorganization to form a ring canal and with the observation of these germ cell midbodies I sought to further characterize these intermediate structures and I performed additional live Imaging of other U midbody and cyto canis proteins I'm showing you here um Sills from a time-lapse movie um of testes that were expressing Citron cyas or as in drosophila this protein is
• 39:30 - 40:00 known as sticky which is a major midbody mediating and organizing protein um during canonical cell division and um in contrast to what I've already described to you about the Dynamics of poav during ring Canal formation sticky remains localized to the cleavage Furrow and then the subsequent midbody ring um throughout the entire process of ring Canal formation um and it's only when poav under goes this apparent reorganization do the two proteins then
• 40:00 - 40:30 seem to colocalize and so by Imaging sticky as well as a nyan non-muscle mein um and one of the septins um these data strongly suggest to us that these ring canals are actually the product of a stabilized midbody ring and I sought to further investigate the role of Citron Chinas um during incomplete cyto Kinesis um where performed RNA experiments specific specifically in The Germ cells in the
• 40:30 - 41:00 test and I'm showing you here um a panel of control images where um ring canals are now labeled with pop gfp and I'm also using an antibody to Mark the fusome and in controls ring canals have a uniform D well an average diameter of about one and a half microns and the lumens of these ring canals are really obvious um in contrast uh depletion of sticky RNA or depletion of sticky specifically in The Germ cells results
• 41:00 - 41:30 in the formation of these very small ring canals as you can see Quantified here as well as a high incidence of what we were calling closed ring canals because there was no obvious uh Lumen um but actually appear more like ectopic midbody like fosi and interestingly these midbody fosi um when they're associated with the fusome um the fusome is very thin and elongated and often there are gaps in the fusome suggesting that cyesis was
• 41:30 - 42:00 complete and in fact um further analyses of this phenotype we find that the level of Po um is reduced in these structures we show this with both show this with both quantification of poav gfp fluoresence intensity as well as Western blooding of whole testos lates and so these data suggested that Citron kinase um or sticky facilitates this mid body Terin Canal transition and likely does so indirectly through its role in
• 42:00 - 42:30 scaffolding pav protein in these germ cell midbodies and the functional consequence of having an aberant midbody is that these midbodies impact the communication between these sensial cells and so here I was able to um perform photoactivation experiments of a photoactivatable gfp protein um in intact testes and here I'm showing you that on one end of the tesus I or one end of the of the germ cell
• 42:30 - 43:00 cyst we' photoactivate um gfp and track its movement to the opposite end of the cyst and measure how long it takes for the protein to become equilibrated through all cells in the cyst and in controls this takes an average of 120 seconds however in the sticky RNA condition where you can see these very small midbodies the time that it takes to equilibrate photoactivation able gfp protein is significant significantly delayed on the order of 500 seconds and
• 43:00 - 43:30 you can see here directly comparing these 122nd time points that most of the protein in the sticky RNA condition is still um accumulated around that initial site of photo activation and so these results really highlight the significance of the midbody ter ring Canal transition and mediating cell cell communication and so we published these results
• 43:30 - 44:00 earlier this year and so I want to highlight the major findings and that is that um we with extensive Life Cell Imaging for the first time we've identified that ring canals form through this extensive reorganization of these germ cell midbodies um importantly these midbodies do not recruit any of the components necessary for excision and in fact um undergo this rapid reorganization that's marked by reduction and a concomitant reorganization of this pavati
• 44:00 - 44:30 protein we show that sticky likely scaffolds P protein in the germ cell midbody and mediates this midbody to ring Canal transition and again we highlight um that these aberant midbodies um impact the movement of cytoplasmic gfp and so these results have um changed the way that we think about Ring Canal formation and have provided some really interesting platforms and jumping off points for us to now go in and begin to understand the mechanistic details that mediate this midbody to ring Canal transition and
• 44:30 - 45:00 we're using sticky as our genetic entree to begin to understand this molecular mechanism and so sticky is this multi-domain and very large protein it has an inter terminal kinas domain a coil coil domain that's shown here split into two halves an inter terminal and a c terminal half a C1 membrane Target targeting domain and a cnh or Citron nion homology
• 45:00 - 45:30 domain and working together with Elie Winkler a Yale College undergrad we've now been performing a structure function analysis where we have generated um transgenic constructs or overexpression um and I should say transgenic mutant constructs um with each of these domains deleted and in these studies um so far we've only looked in the context of the wild type Pro um and so we are looking for things that localize normally to the ring Canal um
• 45:30 - 46:00 but have a dominant effect in that um um in terms of sticky function and so we first generated an endogenously tagged sticky gfp we show that sticky gfp localizes to the ring canals into the cytoplasmic puncta um overexpression of sticky full length gfp mirrors this localization as the endogenous protein and also um when we delete the C1 membrane targeting domain we see that U
• 46:00 - 46:30 sticky still localizes to the ring canals into the cytoplasmic puncta um when we delete the C terminal half of the cc2 domain we find that this disrupt uh disrupts sticky localization to the ring canal and instead sticky then localizes predominantly to the fusome um and our most interesting result was when we deleted the cnh domain and we find that sticky localizes
• 46:30 - 47:00 to ring canals but we also see an increase in the number of both cytoplasmic as well as ectopic fosi um that are associated with these fusomes and we looked at this phenotype a little closer and we find again the formation of these ectopic midbody like fosi um as well as a localization of sticky to these ring canals so we were excited by this phenotype because it um mirrored the phenotype that we saw when we depleted sticky um um in our RNA
• 47:00 - 47:30 experiments and this was particularly exciting to us because um it's been shown in completely dividing dropa S2 cells that the cnh domain um plays an important role in binding to the small GTP row a and maintaining its localization at cleavage furrows and so we wanted um this suggested that perhaps one way that sticky is me mediating or contributing to ring Canal formation is by maintaining row a at the ring
• 47:30 - 48:00 canal and so we wanted to investigate the localization of active row a and so we did this using a row a GTP sensor which is the row a GTP binding domain of Anan fused to gfp and here I'm showing you just the tip of the test where the mitotic divisions are but I hope that you can appreciate that um this Anan row binding domain Fus to gfp localizes to the canals and we actually see that this localization um persists um throughout
• 48:00 - 48:30 the entire process of spermatogenesis and so as row GTP levels typically decline at the end of canonical cyto canis this suggests um that one possibility is that row a um is being atypically regulated during incomplete cyto canis and this is a hypothesis that we are now following up on and so um in the future we are really trying to understand and um identify the molecules that facilitate this midbody
• 48:30 - 49:00 terrain Canal transition um and this includes investigating um two candidates from an RNA screen that we carried out that I did not have time to talk about um and so we're now generating transgenic tools to um begin to look at the localization of these proteins um as well as pursuing this hypothesis of atypical regulation of row a um I'm especially interested in understanding whether or not the mechanism of ring Canal formation is conserved across biology um given that ring canals are
• 49:00 - 49:30 such an integral part of gam Genesis and towards this end I have um performed live Imaging of U mid buddies uh or of ring Canal formation in the dropa ovary and find that this midbody to ring Canal transition is present in the dropa ovary and occurs um with the same timing or um similar timing as in the the test and Al I find evid evidence that these germ cell midbodies are um present in the male germ lines in
• 49:30 - 50:00 distantly related species including the mouse tesus shown here um is a midbody intermediate um and in the um nidian and non-bilaterian Hydra Garis tesus where we also find evidence of these midbody intermediates and I'm particularly interested in following up on um the work in Hydra um because it provides a really unique opportunity to begin to ask questions of Evolution um and also um ring canals are readily abundant in
• 50:00 - 50:30 in Hydra and so we find that ring canals are present in not only cells um in the germ line but also in the s in the Soma um and um 70% or more of cells in the in in an adult hydrop poop um are connected by rain canals and so we're now performing comparative analyses just trying to survey the landscape of the Ring canal in Hydra um using our custom uh chesan six antibody to label ring canals as well as commercially available
• 50:30 - 51:00 antibodies and so these are revealed some really interesting um insights um when we compare them to what's known about the drop ring Canal um and that um similarly to dropil ring canals both germine and somatic ring canals in Hydra are enriched in phosphotyrosine epitopes and eatin um but in contrast to what's known about dropa we also find that a subset of sematic Ren canals and Hydra also cabel with Alpha tubulin and so we're using these similarities and
• 51:00 - 51:30 differences to begin to inform hypothesis about the evolutionary modifications um of ring Canal formation and ultimately in my future research program I would really like to understand the functional role that ring canals play in multicellular development and Physiology and I will end by saying thank you to my mentor Lyn kie and the rest of the kolie lab um as well as the Yale um light and electr microscopy
• 51:30 - 52:00 cores um the dropa and Hydra communities for critical reagents at technical advice um as well as our neighbor Labs at Yale who have provided tissue for my investigations into zebra fish and mouse ring canals and of course my funding without which um none of this would be possible and so I will end there and I would be delighted to answer any questions
• 52:00 - 52:30 okay so we have some questions already so Bruce Draper asks great talk Carrie what effect does scky KD have on fertility um the kise dead Mutant um I presume um so uh we have um I have made actually chrisper mutant um deleting the the um K the making a kyes dead Mutant and found that actually those animals
• 52:30 - 53:00 are homozygous lethal and so there's clearly an effect developmentally um and so now we're working to generate clones um in the germ line so that we can begin to understand um the effect um or the the kyes activity and potential role in the formation of of ring canals okay our next question is from Michelle Stars guyo nice work Carrie for your overexpression of the sticky Delta cnh mutants do you see abnormal germline
• 53:00 - 53:30 development it kind of looked like the fusomes were less Branch um so I have not seen that and that would be an interesting thing to go back and look for um and so yeah I I will definitely look for that yeah okay so oh there here's another one so Celesta Berg asks um she says night's work Carrie is TIY also present in
• 53:30 - 54:00 somatic ring canels uh yeah so um that was a bit of a surprise I guess to me because we've shown that the ring canals in the male germ line I in The Germ cells it seems to have this very transient localization to the midbody and and nent ring um but when we've looked at the follicle um ring canals we actually see that sticky localizes there so um yeah we do see it on um sematic green
• 54:00 - 54:30 canals and sorry I see Bruce's knockdown um so yes uh it did affect fertility but because these are RNA experiments um it so it impacted fertility and reduced it but it didn't completely um uh abgate it our next question is from L Port have you look at row a localization and activity in your cyon knockdown strain
• 54:30 - 55:00 because the ring does not open will you expect row a to be overactivated and citon acting as a buffering protein um so I have not looked um at row a localization and activity in the um context of a sticky RNA ey yet um and it's interesting because there are some studies of um complete uh completely dividing
• 55:00 - 55:30 cells in culture where row ectopic row a activity actually results in the formation of bcle cells because they fail to undergo cyto canis or to to finish it and so that was sort of the hypothesis that we were working on was that um perhaps this um ectopic row a activity is is um functioning um in a similar way in the context of The Germ line okay our next question is by how John we hi krie great work I wonder if the
• 55:30 - 56:00 midbody ring Canal transition is synchronized across different cells within the same Cy uh yes definitely so all of the cells that are dividing synchronously all have um synchronous midbody ring Canal transitions yep our next question is by Iris Pano did you know if the ring can panels are present only in specific Hydra cell types is there any difference in their
• 56:00 - 56:30 frequency closer to the head or foot yes so ring canals are present in the interstitial stem cells and all the daughters of that interstitial stem cells so all of the all of the cells that will go on to make the the nytes um and so I haven't seen any difference in the frequency um they really are all over so anywhere you see a nest of of nytes or you know nidol blasts that are dividing to generate um the the neaty
• 56:30 - 57:00 you will see uh that they are connected connected by ring canals okay so it seems we'll give one more minute to see if there's any other questions lesie Rose says just a comment great talk Carrie so thanks
• 57:00 - 57:30 Leslie okay so with that I would like to thank jhuan and Carrie for their excellent talks and this seminar has been recorded and will be available on the SCB website next week and I also want to invite you to please join us for the next month seminar on Friday November 10th where NAA alha from the University of Texas Sou and Jason Smith from the University of Chicago will present thank you thank you
• 57:30 - 58:00 everyone for coming