# The Shifting Shape and Functional Specializations of the Cell Cycle During Lineage Development

November 19, 2020## Information

Merav Socolovsky, MBBS, PhD

Professor, Department of Molecular, Cell and Cancer Biology

University of Massachusetts Medical School

YCCEH Invited Speaker

November 12, 2020

ID5910

To CiteDCA Citation Guide

- 00:00Everyone Jeannie Hendrickson I'm one
- 00:02of the Co directors of the enrichment
- 00:05program of the Yale Cooperative
- 00:07Center of Excellence in Hematology.
- 00:09Very excited to have our first virtual
- 00:11speaker sponsored by the Yale Cooperative
- 00:14Center of Excellence in Hematology,
- 00:16but hopefully broadcast to a lot of
- 00:18the other cooperative centers of
- 00:20excellence in hematology across the US.
- 00:23Whether you're looking at us Live
- 00:25Today or virtually on the video that
- 00:28we will eventually post of Mirage.
- 00:31Talk, we're happy that you could join
- 00:33us today and we're extremely grateful
- 00:35to Doctor Moore of Sokolovsky for
- 00:37being our kind of inaugural speaker,
- 00:40virtually for the YCCEH she's a
- 00:41professor at the University of
- 00:43Massachusetts Medical Center,
- 00:45she has had quite a distinguished career,
- 00:47really discovering fundamental insights
- 00:48into Areth row poesis in the process thereof.
- 00:51So again, we're very grateful.
- 00:53But she's our inaugural speaker this year.
- 00:56The way the web and R is going to work.
- 00:59She's going to give her talk.
- 01:01Then at the end of her talk,
- 01:04if you have questions.
- 01:05The questions in the chat or in
- 01:07the question and answer box.
- 01:09I also think I have the option
- 01:10to allow you to talk,
- 01:12so if you put in the chat you
- 01:14would like to talk in person.
- 01:15I can unmute your microphone
- 01:17and you can talk.
- 01:18So without further ado we will turn
- 01:19it over to Rob and thanks again Rob.
- 01:22We're excited to have you.
- 01:25Great, thank you so much.
- 01:27I'm truly honored to be invited to this
- 01:31forum and excited to tell you about
- 01:35the work that we've been doing so.
- 01:39I will attempt to start straightaway.
- 01:42Here we go so.
- 01:44When we think about the cell cycle,
- 01:48we think about a generic program
- 01:50whose purpose is to generate more
- 01:53cells to increase cell number.
- 01:56But when we look at a tissue,
- 01:59the cells that are cycling
- 02:02I usually transient cell.
- 02:04So long term teacher residents
- 02:07like terminally differentiated
- 02:09cells or stem cells.
- 02:11Do not cycle.
- 02:13So what does it mean that only transient
- 02:17cell states are undergoing cell cycle?
- 02:21These cells are continuously
- 02:23changing the genes that they express
- 02:26and our work suggests that they
- 02:28are also continuously changing
- 02:30the kind of cell cycle program
- 02:33that is expressed by these cells.
- 02:36It's likely that linear development.
- 02:39Had really coevolved with
- 02:41modifications in the cell cycle,
- 02:44so cell cycle programs probably
- 02:47adapted to specific tissue and cell
- 02:50cycles and developmental stage,
- 02:53and it is possible that in order
- 02:56to truly understand how a
- 02:59developmental process is regulated,
- 03:02we need to understand the
- 03:05specifics of its cell cycle.
- 03:08So here we're looking at
- 03:11the erythroid linear edge.
- 03:13The erythroid developmental trajectory
- 03:15has two phases earlier through
- 03:18pieces and terminal differentiation.
- 03:21What I'll show you today is that
- 03:24the cell cycle throughout this
- 03:26process varies in logs lockstep
- 03:30with differentiation stage both in
- 03:32terms of the cell cycle length,
- 03:35which here is represented vertically.
- 03:39And in terms of the ratio of
- 03:42S phase to the gap phases.
- 03:44So how did we set out on this question?
- 03:49We started out by asking.
- 03:54One question and that was how is a recruit
- 03:57terminal differentiation activated?
- 03:59We know that in earlier it releases
- 04:02we have progenitors that are already
- 04:04committed to their way through drainage,
- 04:07but that are not expressing any of the
- 04:10jeans that are present in red cells.
- 04:13At some point these progenitors undergo
- 04:16a selfhacked decision that switches on
- 04:19this specific program of red cell genes,
- 04:21and so the question is how does that happen?
- 04:26Of course, many labs are addressing
- 04:28this question, and the angle that
- 04:32we had was to ask.
- 04:35Can we identify the cell in
- 04:38which this activation happens?
- 04:40Our model system is the mouse fetal liver.
- 04:45And and that was,
- 04:46we also have experiments that show you
- 04:49later on in the mouse adult bone marrow.
- 04:52The fetal liver is a great system for
- 04:55working in original places because over 90%
- 04:57of the cells of the erythroid Lenny Edge.
- 05:01So we have two cells of his markers cities.
- 05:04Have anyone until 119 with just
- 05:07these two markers we can divide the
- 05:10fetal liver into a number of subsets
- 05:13that form a developmental sequence.
- 05:15So we find that in the S node
- 05:19subset and S1 subset,
- 05:21that's where we see colony
- 05:23forming progenitors.
- 05:24So this is where we see earlier with
- 05:28paresis and then in subsets as 22S5,
- 05:31we see progressively more
- 05:34differentiated erythroid precursors.
- 05:36And you can see this also here.
- 05:38The colonies that are formed by the S note
- 05:41cells and S1 cells are pretty similar.
- 05:43We wouldn't be able to tell them apart.
- 05:47But two important findings
- 05:48suggested to us over 10 years ago.
- 05:51Now that the S not see if you,
- 05:55we are really quite different
- 05:57to the those in S1.
- 05:59So the first finding was when
- 06:01we examined the fetal livers
- 06:03of a preceptor knockout cells.
- 06:05So here we're looking at two litter mates.
- 06:09This was published.
- 06:10The knockout was first published by Home
- 06:14Grow in the Lodish lab back in 1995 and.
- 06:17When we did flow cytometry on the
- 06:20fetal livers of embryos from a.
- 06:23Some this knockout we found that whilst
- 06:26in the wild type number we already see
- 06:30cells populating most of these subsets.
- 06:32This is Embedded Day 12.5.
- 06:35In the perception knockout,
- 06:37we see an absolute block at the
- 06:40transition from S No 2 S one,
- 06:42so that tells us that the transition
- 06:44is dependent on a preceptor signaling.
- 06:47These few cells that you see over here
- 06:50that tagline positive in the IP receptor
- 06:53knockout belong to the yolk SAC limit,
- 06:56and they're not part of
- 06:58definitive erythropoiesis.
- 06:58Now the second kind of experiment
- 07:01that we did that told us there's
- 07:03something different about S1 and S
- 07:06Note was a cell cycle experiment,
- 07:08and here we have a cartoon that illustrates
- 07:11typical cell cycle experiment that I'll
- 07:13show you a number of times during my talk,
- 07:17so.
- 07:17We take a mouse.
- 07:19In this case it's a pregnant female and
- 07:24injected with beyond you and then harvest
- 07:28hematopoietic tissue 30 minutes later.
- 07:31What we find is 2 things.
- 07:33First, we find which cells are positive.
- 07:36For Bru, these cells have
- 07:38incorporated be audio,
- 07:39which is a finding analog into their DNA.
- 07:43So these cells are in S phase
- 07:46during the 30 minutes of the pulse.
- 07:50A second finding. Is.
- 07:52Allows us to determine the speed of
- 07:55aspects and so we can compare the
- 07:58level of the audio incorporation in
- 08:02the cells of different populations.
- 08:05Cells that have incorporated less beardi.
- 08:08You must be synthesizing.
- 08:10DNA slower and have a longer
- 08:13essays than cells that incorporate
- 08:15the audio at a faster pace,
- 08:19and we've confirmed that with
- 08:21direct experiments that look that
- 08:24use a double family impulse to
- 08:27Measure S phase duration directly.
- 08:30And so when we did this experiment
- 08:32in the mouse fetal liver here we're
- 08:34looking at cells from each of the
- 08:37subsets and at the cell cycle status.
- 08:40We found 2 interesting things first.
- 08:44Whilst about 60% of the cells
- 08:47in S nought so these cells are.
- 08:52In S phase, at any one time.
- 08:53So this is a highly replicative tissue.
- 08:56When we look at S1 over here,
- 08:58nearly all of the cells, 90% in this case.
- 09:03Are in S phase of the cycle.
- 09:05So that's interesting finding number one.
- 09:09And the second finding was that
- 09:11the speed of S phase is about
- 09:1450% faster in S phase.
- 09:17Cells that are in S1 compared with S phase
- 09:21cells in the preceding as node subset.
- 09:25And so a number of.
- 09:27We've done a lot of work on this
- 09:30and I'll give you the summary.
- 09:33The way that we've interpreted our data.
- 09:37Is that the transition from S note to S1
- 09:40can only happen in S phase of the cycle.
- 09:44And this is based on a number of
- 09:47experiments where we have arrested as space,
- 09:50either genetically or using drugs
- 09:52that inhibit DNA polymerases.
- 09:53And when we do that,
- 09:55we totally prevent the transition
- 09:58from S note to S1.
- 10:00And.
- 10:00What we prevent isn't simply the
- 10:02upregulating I population of city 71,
- 10:04which is the marker of this transition,
- 10:06but all of the events that
- 10:08are associated with it,
- 10:10which is the induction activation of
- 10:13the array through transcriptional.
- 10:15Program their research differentiation
- 10:17program.
- 10:20The second thing that we found and I
- 10:23will talk about that a bit more later,
- 10:25running the talk is that S phase at the
- 10:29time of this transition is much shorter
- 10:32than S phase of preceding cycles.
- 10:35OK. And that the actual length of S
- 10:40phase is really only about four hours,
- 10:43which is pretty short for MA million S phase.
- 10:50Now what else is happening at the
- 10:52time of this suite at the time
- 10:55of transition from S note to S1,
- 10:58it turns out that there is a time
- 11:00when we see reconfiguration of
- 11:02chromating at the beta globin locus.
- 11:05We see a change in the timing of replication
- 11:07of the locals histone tail modifications.
- 11:10We see a loss in DNA methylation beginning
- 11:13at the time of this switch and more
- 11:16recent work from Dark Hexes Lab done by.
- 11:19Rob Bakery and others have shown
- 11:22that eight acsec and using tiled see,
- 11:26we see either changes in chromatin
- 11:29Accessibility promoter enhancer Contacts
- 11:31that begin with this transition,
- 11:33so this is clearly a key
- 11:36developmental switch.
- 11:40I haven't explained a color scheme here.
- 11:44What we think is happening is
- 11:47that CF uer undergoing expansion
- 11:50whilst in the S North subset.
- 11:54And then the very last generation of CFUE
- 11:58starts its life in the S note subset in G1.
- 12:02When it enters this specialized short space,
- 12:06it is at it up. Regulates city 71.
- 12:09And it is at that point that
- 12:12it undergoes commitment.
- 12:14The progeny of DCF.
- 12:15UE will no longer be safe UE.
- 12:17They will be priority through
- 12:19blast in the race for blast that
- 12:22undergo terminal differentiation.
- 12:24So when we asked next is is this
- 12:27CFU E2E TD transition true sweet?
- 12:30So it's all very well and good if
- 12:33we take all of us not cells and all
- 12:37of this one sells and compare them,
- 12:40large differences suggest to switch but
- 12:43is not is a heterogeneous population
- 12:45of cells and by taking all of them
- 12:49together we could be masking incremental
- 12:52changes within this population.
- 12:54The problem with addressing this
- 12:57question was that we really had no
- 13:02reliable way of taking apart this subset.
- 13:05And in fact,
- 13:07the entire trajectory starting
- 13:10with hematopoietic stem cells
- 13:12and ending at this point where
- 13:16terminal differentiation starts,
- 13:18this trajectory was really
- 13:21only partially understood,
- 13:23and so about four years ago the
- 13:27technology of single cell RNA SEQ had
- 13:32advanced massively by microfluidic.
- 13:35Approaches were introduced by
- 13:36a number of labs,
- 13:37including the lab of a long climb.
- 13:40And we were very fortunate in
- 13:42that he agreed to collaborate with
- 13:45us on this question.
- 13:47And so we took kit positive cells
- 13:49from the bone marrow or from fetal
- 13:52liver and we also took kid positive
- 13:54cells from the bone marrow of mice
- 13:57that were injected with Ipoh.
- 13:59Although I will not discuss that here.
- 14:02And we've undertaken single cell RNA
- 14:05sequencing on these projectors and
- 14:08what you're looking at here are two
- 14:11D projections of K nearest neighbor
- 14:14graphs of gene expression in the
- 14:17fetal liver and in the bone marrow.
- 14:21Topologically, these graphs are very similar.
- 14:24Each dot is a single cell,
- 14:27and the proximity of dots suggests
- 14:30a proximity in terms of their
- 14:33transcriptome similarity transcriptomes.
- 14:35And what you can see is that
- 14:38these transcriptomes are form
- 14:40one continuous structure.
- 14:41It's a branching structure.
- 14:43And in the fetal liver and bone marrow,
- 14:46the branches are pretty similar.
- 14:49Except that here we see a very
- 14:52large bulge compared with a much
- 14:54smaller bulge in the marrow.
- 14:55This bulge in fact contains the CFUE,
- 14:58as I will show you.
- 15:02I won't dwell on it today, but.
- 15:06We've used GNU algorithm that was
- 15:09developed by the client laboratory,
- 15:12especially Caleb Weinreb and some
- 15:15Wallach in the client lab called
- 15:18Population Balance analysis.
- 15:20And this algorithm allowed us
- 15:22to assign each cell within this
- 15:25structure with a fate probability.
- 15:28In fact, with a set of seven
- 15:31self check probabilities,
- 15:33which told us what is the probability
- 15:36of each cell to ultimately attain a cell
- 15:40fate in one of these seven branches and that?
- 15:46Really can be the result of our analysis
- 15:48can be collapsed into this structure,
- 15:51which is a hierarchical structure,
- 15:53not unlike the classical
- 15:55structure of hematopoiesis.
- 15:56The main difference is that
- 15:58we don't see discrete stages.
- 16:00We see a continuum which is meant to
- 16:03be represented by this kind of cloud.
- 16:09And one more point that I'd like to
- 16:12make is subsequent subsequent work
- 16:14of the client lab together with Luca
- 16:18Biosca Slab looking at single cell RNA
- 16:20seq of human marrow showed that the
- 16:24structure and topology is obtained
- 16:26from human bone marrow is actually very
- 16:29similar to that of the mouse and in
- 16:32terms of gene expression for each gene.
- 16:36Steam is aro.
- 16:38We see a very similar pattern
- 16:40in the mouse and in the human,
- 16:42which are represented here as mirror images.
- 16:46And so we can conclude that the
- 16:48mouse is a pretty good model for
- 16:51human hematopoiesis in general.
- 16:53Of course, that we know of some
- 16:56very clear differences as well.
- 16:58So now we were in a position to
- 17:01look at the erythroid trajectory,
- 17:03starting with multipotential progenitors
- 17:05in black and continuing along the
- 17:08array thread branch with the color
- 17:10representing every thread fate probability.
- 17:12We can use this probability to align
- 17:16the cells along a linear axis starting
- 17:19with MPP and ending with the end of ETD.
- 17:23And just to see that things look good.
- 17:29You can look at cells that we know our
- 17:32President Multipotential progenitor's
- 17:33like cities 34 gotta one which is
- 17:37expressed by the entire array thread
- 17:40branch and Alpha globin which is induced.
- 17:43Only with the activation of
- 17:47Arethra terminal differentiation.
- 17:49In order to be able to do experiments
- 17:53with transcriptome information we
- 17:55needed a way of learning how to
- 17:58isolate cells from specific regions
- 18:00of our single cell RNA SEQ graph.
- 18:03And so we've developed a fax approach
- 18:06that gives us five populations and then
- 18:09we sorted each of these five populations,
- 18:13repeated the single cell RNA SEQ work,
- 18:16and then projected them onto our.
- 18:19Original map and what you can see
- 18:22and what's relevant to today's talk
- 18:25is that the P1 and P2 subpopulations
- 18:28project pretty cleanly into this
- 18:30very narrow neck at the beginning
- 18:33of the urethra branch and then into
- 18:37this sort of bulge that follows up.
- 18:41And so we now have a way of isolating
- 18:44cells that correspond to these
- 18:46two regions of the graph.
- 18:48And the P5 population is also pretty
- 18:51good at giving us the multipotential
- 18:54progenitor cells right at the
- 18:57beginning of the erythroid branch.
- 18:59So with these three subpopulations,
- 19:02we can isolate cells from the entire.
- 19:06If we throw brunch and so now we're ready to
- 19:12do some assays and we find that P1 and P2.
- 19:18Almost my stroke of luck.
- 19:20Kivas populations at the highly enriched
- 19:25for CFUE. In fact, P1 contains.
- 19:29Almost all of the CFO is some
- 19:32small number also present in P2.
- 19:36And P2 is the only subpopulation that
- 19:40contains BFUE, so P2 is from the neck.
- 19:44Here represents cells with be
- 19:47a few potential.
- 19:48BF uer colonies that are multifocal either.
- 19:53Small bunches of small foci.
- 19:54Deezer called late be a few E and
- 19:57we see them around day four of.
- 20:00Culture or like might contain much
- 20:03larger column they might have might
- 20:06give rise to much larger colonies
- 20:09after about
- 20:10a week or even 10 days.
- 20:12See if you eat, give rise to 1.
- 20:15Focus of colonies that contain
- 20:17around 30 differentiated red cells,
- 20:20about two to three days after plating.
- 20:24OK, so we now have.
- 20:28Pretty complete set of tools
- 20:31to do our investigation.
- 20:33We can look at multipotential
- 20:36progenitors BFUS&CFUS at the
- 20:38transcriptome levels and we've given
- 20:41them transcriptome related names.
- 20:43You can correlate them with faith
- 20:46assays and isolate them by fax.
- 20:48And so we are in a position to ask,
- 20:52are we looking at a true switch when
- 20:56we transition from S Note 2 S one?
- 20:59Is this a few ITA ET transition
- 21:02at truth Switch?
- 21:03And so here we are looking at
- 21:05genes that are differentially
- 21:06expressed during the linear access.
- 21:09The linear suit I'm going from MPP
- 21:12to terminal differentiation and
- 21:14they arrange arrange story in terms
- 21:16of the peak expression along this.
- 21:19Access and without any really
- 21:21fancy analysis you could see that
- 21:24they form kind of three cohorts.
- 21:26There is a cohort of gene expression
- 21:29that happens during a very rapid change.
- 21:32Many streams are being switched on or off.
- 21:36Then we enter a period of relative
- 21:39stability of the ceep progenitors of
- 21:42correspond functionality to see a few E.
- 21:46Jeans and not many genes of
- 21:49turning on or off.
- 21:51Although you can see that there
- 21:53is a progressive slow change
- 21:55and this probably represents an
- 21:57amplification stage where there
- 21:59is little transcriptome change.
- 22:01And then we see.
- 22:05Rapid change or in fact I should say
- 22:07about sharp change from the CFD program
- 22:11to terminal differentiation program.
- 22:13Very few cells expressed genes
- 22:15of both terminal differentiation
- 22:17and sea Fury program.
- 22:18So that tells us that we're looking
- 22:21at a sharp transcriptional switch.
- 22:23So the answer is yes,
- 22:26the transition from this Cepheus
- 22:28Stage 2 terminal differentiation
- 22:29is a sharp transcriptional switch.
- 22:32So what is the context of that switch?
- 22:36And we're in the position to
- 22:38look at that as well.
- 22:40This is a busy slide,
- 22:41but let me take you through it slowly.
- 22:44So if we look at the medial
- 22:46medium panel first in red,
- 22:48we're looking at the expression of CD 71.
- 22:51This is the market we used by
- 22:53fax and we can see that there is
- 22:55a gradual increase in cities of
- 22:5771 throughout the trajectory.
- 22:59But at the time of the switch
- 23:02to terminal differentiation,
- 23:03this becomes a very rapid up regulation,
- 23:05and so the upregulation of CD 71.
- 23:08Which we previously took as a
- 23:10marker of activation of the switch.
- 23:13Acts as a marker of that,
- 23:15also at the single cell RNA level.
- 23:18When we look at either cells of
- 23:21his markers here we're looking at
- 23:24Ipoh receptor expression in blue.
- 23:26EPO receptor is expressed pretty
- 23:29early on in the trajectory in
- 23:31increases gradually subsequently.
- 23:33We can now look at transcription
- 23:36factors and we see in Grey gotta one
- 23:39is high initially and then is downregulated.
- 23:43Gotta sorry got it too in grey.
- 23:47Gotta one is low initially in
- 23:50multipotential parameters and then is
- 23:52expressed induced early in the trajectory.
- 23:55I'm sorry Ann is maintained at pretty
- 23:58high levels throughout the trajectory.
- 24:00Maybe going up a little bit
- 24:03at the time of the switch.
- 24:06Generally speaking,
- 24:07none of the key transcriptional
- 24:10regulators that we know of.
- 24:13Reporters of the timing of
- 24:16the switch from CFUE to ETD.
- 24:19When we look at the cell cycle,
- 24:21however, we see something that
- 24:23does seem to correlate very well
- 24:25with the timing of the switch.
- 24:27So what we're looking at here each
- 24:30each color denotes the average
- 24:32expression of genes in each that are
- 24:36characteristic of each cell cycle phase,
- 24:39either S Phase G,
- 24:412MG1S and so on,
- 24:43and there is very little difference there.
- 24:46Sensually, flat or uninteresting
- 24:48for most of the trajectory.
- 24:50And this really tells us that
- 24:53cells are cycling asynchronously
- 24:55through most of the trajectory.
- 24:58But as we approach the time of the switch,
- 25:01which is this dashed line,
- 25:03you can see the formation of a
- 25:06number of peaks starting with G1,
- 25:08S and then S in red and orange
- 25:11and then G2NG2M.
- 25:13So what we have here in fact
- 25:16is the cell cycle.
- 25:18And the first peak that is formed is S phase.
- 25:23And so it appears that the earliest event
- 25:27at the Switch from Seaview to ET D is
- 25:31marked by cells in S phase of the cycle.
- 25:35So this confirms our earlier functional data.
- 25:38That activation of the TD happens
- 25:42during S phase of the cycle.
- 25:45And So what we asked next is OK.
- 25:49Transcription factors are not good.
- 25:52Taught don't really report
- 25:53the timing of this switch.
- 25:55At least their expression doesn't.
- 25:57It's quite possible that post
- 26:00transcriptional post translational
- 26:01modifications of these do correlate
- 26:03with the timing of this switch,
- 26:05and that's an open question.
- 26:07Um?
- 26:09Can we find something else
- 26:12that might tell us?
- 26:14Something about the timing of the switch.
- 26:17So to do that we went back to the
- 26:20jeans that are expressed during this.
- 26:23See if you E program and we asked
- 26:26whether there are the slow progressive
- 26:29change that happens during this time,
- 26:32which streams are changing during that time?
- 26:35Which teams change their expression
- 26:37in a way that is correlated with
- 26:40progression along suit of time?
- 26:43And when we did that, the top five go.
- 26:48Terms that we got were essentially
- 26:51all to do with DNA replication.
- 26:54The cell cycle, S phase, and so on.
- 26:58And here are some examples.
- 27:01We're looking at cycling
- 27:038 two cycling E1 R&R.
- 27:06Units.
- 27:07Proteins that are associated
- 27:09with the origin of replication.
- 27:11All of these are ramping up their
- 27:15expression throughout the trajectory
- 27:16right through the BFUE&CFU stage
- 27:19and reach a peak at the time of the
- 27:22transcription of switch from CF UE2ET D.
- 27:27Now what does that mean functionally?
- 27:31So to understand the significance of
- 27:34this really quite impressive ramping
- 27:36up in cell cycle X gene expression,
- 27:39we went back to a functional experiment.
- 27:42So here we're looking at the same
- 27:45old experiment where we take a
- 27:48mouse injected with beyond you
- 27:50and then check the cell cycle
- 27:53status and the speed of space.
- 27:56But now we were armed with some more
- 27:59information about early erythropoiesis.
- 28:02We used the slow upregulation of CD 71
- 28:06as a way of measuring sudo time by fax.
- 28:10And we were able to also.
- 28:14Staying for the P1 and P2
- 28:18subpopulations which mark there
- 28:20be a few Ian CF Louise subsets.
- 28:23And so we sort of these cells and
- 28:26looked at the beardi you incorporation
- 28:28in here we're looking at individual
- 28:31cells Bru positive and Bru negative.
- 28:34We arrange them along the CD 71
- 28:37expression suit of time and if I did
- 28:40this sort of time into 14 different
- 28:44Gates 7 seven percentile gates.
- 28:46And then we can look at each of
- 28:50these gates and analyze cell cycle
- 28:52status as well as S phase speed.
- 28:55So the first really quite clear.
- 29:01Finding is that cells in
- 29:03S phase of this cycle.
- 29:05Increased markedly with progression
- 29:06along the earlier we throw trajectory,
- 29:09so maybe 20% of this other INS phase
- 29:11at the early parts of the trajectory,
- 29:14and as we approach this,
- 29:16which essentially all the cells are in
- 29:18space and you could see this right here.
- 29:21So at the time of this which nearly all the
- 29:24cells are in a space where is around here,
- 29:28most of the cells are not.
- 29:31When we look at S phase speed,
- 29:34we see there is there.
- 29:36There is an increment.
- 29:37There is an increase in the
- 29:40speed of West phase early,
- 29:42but then it stays quite stable until
- 29:44the point of the switch to ATD.
- 29:47And so this quite stable speed of essays.
- 29:50In other words,
- 29:52quite stable S faced length
- 29:54can't explain the change in
- 29:56the number of cells in space.
- 29:59And so our interpretation.
- 30:01Is that what's happening is?
- 30:05Shortening in G and in the G1 phase.
- 30:08Of course,
- 30:09this massive increase in the
- 30:11number of S phase cells explains
- 30:14why S phase genes are increased
- 30:16throughout the trajectory.
- 30:21And we suspect that the reason
- 30:23for that is G1 shortening,
- 30:26and so as a proportion the number
- 30:28of cells in S phase increases.
- 30:31The later we are in the Seaview stage
- 30:34as we approach the actual switch.
- 30:38G1 is pretty short.
- 30:40The switch itself we think involves
- 30:43S phase shortening. So.
- 30:48To summarize what I've told you so far.
- 30:51We use single cell RNA sequencing
- 30:54to identify the erythroid
- 30:57developmental trajectory in the mouse.
- 31:01We were able to match specific
- 31:04stages that are identified based on
- 31:08transcriptomes to fax populations
- 31:10and to functional subsets.
- 31:13Functional progenitors based on
- 31:16confirmation potential and using
- 31:19these tools we began to analyze.
- 31:22The factors that control
- 31:24the switch from CFUE to ET.
- 31:26We found that this is a
- 31:29true transcriptional switch.
- 31:31And that there is no real clear change
- 31:35in transcription factor levels that
- 31:39reports the timing of this switch.
- 31:42By contrast, we do see really quite
- 31:46marked changes in the cell cycle.
- 31:49Initially we see a gradual shortening in G1.
- 31:54And at the time of the switch we
- 31:57see a further shortening in S phase.
- 32:01So that at the time of this which
- 32:03we have a very short cell cycle,
- 32:06our measurements indicate that this
- 32:07cell cycle is about 6 hours long,
- 32:09with S phase being only four hours.
- 32:13So.
- 32:14What we next asked his first of all,
- 32:19what regulates this really quite
- 32:21dramatic remodeling of the cell cycle?
- 32:24And the second question is,
- 32:26is this cell cycle remodeling
- 32:29relevant to linear development?
- 32:31Does it play a role in these
- 32:34important cell fate decisions?
- 32:36For example, this switch from CFUE to ETD.
- 32:39Is it correlate or does it determine it?
- 32:45And so to begin to look at that.
- 32:49Just before I get to that,
- 32:52I just wanted to show you some of the
- 32:55expression of cell cycle regulators
- 32:58during the original trajectory and
- 33:01what's quite interesting is that.
- 33:03Our different shifting shape of the
- 33:06cell cycle is probably regulated
- 33:08through changing cell cycle regulators.
- 33:11And of course we have no idea
- 33:14how that happens at this point,
- 33:17but we know that for example,
- 33:19the dominant E2F four transcription factor
- 33:22during most of the trajectory is E2F four,
- 33:26but at the time of the switch
- 33:30to ETD it becomes E2F2.
- 33:33Other regulators, for example,
- 33:35when we look at the cycling dies cycling D2,
- 33:40is present early on.
- 33:42But it is gradually
- 33:44downregulated whilst cycling.
- 33:46D3 takes over at the time of this switch
- 33:49so it is quite possible that these.
- 33:53Part of them.
- 33:55So how do I that is able to generate these
- 34:00quite different shapes of the cell cycle?
- 34:04So the first thing that we asked
- 34:07is what are the mechanisms that
- 34:10underlie this very short space?
- 34:13And there are two ways of
- 34:15getting a short space.
- 34:16One is to have more origins of
- 34:19replication and the other is
- 34:22to have folks that move faster.
- 34:24We know from model organisms that
- 34:27have extremely short as phase.
- 34:30Is that the way they manage to
- 34:33replicate the genome very fast is
- 34:36by having all of the origins of
- 34:38replication firing synchronously,
- 34:41and they're all very closely spaced.
- 34:44So having efficient firing of origins
- 34:48of replication would clearly be one
- 34:51mechanism that has a president.
- 34:54At the time that we were doing this work,
- 34:57we started to collaborate with
- 34:59Nick Rind at UMass Medical School.
- 35:02Who is studies replication in yeast?
- 35:04And we through discussion with him for that.
- 35:08That is likely we will find because
- 35:11there was really no precedent for folks
- 35:14speed being regulated physiologically.
- 35:17But so to approach this question,
- 35:19we learned from the rind.
- 35:21Leiber technique called DNA combing here.
- 35:24You take cells,
- 35:25pulse them with finding analogs.
- 35:27We pass them with two distinct
- 35:30finding analogs that we could stay
- 35:32in with two different colors during
- 35:35the pulse with bio deoxy uridine,
- 35:38we were, we are able to follow.
- 35:42Into a incorporation of Iodo.
- 35:46The that by.
- 35:47Later on stretching DNA fibers
- 35:50along a cover sleep,
- 35:52the green regions are the regions that were
- 35:57replicating at the time of the iota pals.
- 36:00Then we followed that 10 minutes
- 36:02later with a pass of chloro,
- 36:04uh.
- 36:04Deoxy uridine and here we see the
- 36:07red regions are being labeled that
- 36:10this is where Clara gets incorporated
- 36:13and by doing that we get both the
- 36:16speed of the fork and its direction.
- 36:18Ality because we know that green
- 36:21replication proceeds read replication
- 36:23and with that we can place the origins.
- 36:26So here we have a fork moving
- 36:28in One Direction,
- 36:29another fork in another service
- 36:32must be a replication bubble
- 36:34with origin in the center.
- 36:36And with this approach.
- 36:38We discovered it was actually no difference.
- 36:42No significant difference in the
- 36:44distance between origins of replication,
- 36:46but there was a marked difference
- 36:49in the speed of replication Forks.
- 36:52So on average we're looking
- 36:55globally in the genome here.
- 36:59The replication folks in S1
- 37:01move at 50% faster speed than
- 37:05replication folks in S note,
- 37:08and that really entirely accounts for
- 37:12this speed of four S8 shortening.
- 37:17And So what might be regulating that?
- 37:19And is it really relevant to self it?
- 37:23Well, the answer is still not fully clear,
- 37:26but we know of examples where
- 37:28esfe shortening is at least
- 37:30associated with cell fate decisions.
- 37:32Here we're looking at gastrulation,
- 37:34in mammals where we know that
- 37:37there is dramatic space shortening
- 37:39from 7 hours to 2 1/2 hours.
- 37:42We know more recent examples
- 37:45running Yale from Shank Xingguo,
- 37:48where ultrafast cycles.
- 37:52Mark the cells that are most likely
- 37:55two week program into I PS cells,
- 37:58so this is clearly worth following up.
- 38:01So what might be regulating
- 38:04as they shortening?
- 38:06We looked at one key regulator P 57.
- 38:10Keep two. This is a CD K inhibitor.
- 38:16An IT regulates all it inhibits all
- 38:18city case, except I think City K 6.
- 38:22Um? What we found is it that
- 38:25it is present in S, not cells,
- 38:29but is rapidly downregulated in S1.
- 38:32And here you can see that,
- 38:35unusually, it's expressing S phase
- 38:37of those cells rather than in G1,
- 38:40suggesting it play some kind
- 38:42of role in S phase here.
- 38:45Looking at our Western and at Q.
- 38:48PCR, which both indicated the same thing,
- 38:50and so we looked at the P57.
- 38:53Keep two deficient embryos
- 38:55is in the imprinted gene,
- 38:57so we can look at the heterozygous and
- 39:01have essentially a knockout phenotype.
- 39:04And we see that these mice are anemic.
- 39:07They die in the prenatal stage.
- 39:10I have multiple developmental abnormalities.
- 39:12We found that they were anemic.
- 39:14They had fewer cells in the fetal
- 39:17liver and when we looked at the
- 39:20differentiation they had trouble
- 39:22generating differentiated and we
- 39:24throw blasts from the early CF you.
- 39:26So what's going on here Weekly looked
- 39:30thought to look at the S phase.
- 39:33So here we're looking at S
- 39:35phase again in CFUE.
- 39:37It is maintained as along as
- 39:39phase and we in wild type.
- 39:42Projectors see shortening at the
- 39:44time of the switch from C FE2ET D.
- 39:48And here we're looking at RC71
- 39:50so that I'm in the wild type
- 39:53and in knockout littermate and
- 39:55what you can see is that S phase
- 39:59speed is consistently faster.
- 40:01Prematurely fast in the CF,
- 40:03UE of the P57 knockout embryos,
- 40:06so we have a premature shortening of space.
- 40:13When we did DNA combing,
- 40:14we found that the folks were moving
- 40:17faster in their knockout in S not
- 40:20fork speed was already almost as
- 40:22fast as it would be in the wild
- 40:25types of in the water plasma cells.
- 40:28And in the X one of the P57 deficient
- 40:32embryo S1 cells will break,
- 40:34virtually breaking the speed
- 40:35limit on fork speed,
- 40:37and so we have very fast folks.
- 40:40What's the significance of that functionally?
- 40:44Anemia is 1,
- 40:45but why exactly do we get anemia?
- 40:48We didn't find that out until we
- 40:51looked at CF self renewal in the dish.
- 40:55So it turns out that CF UE can
- 41:00undergo self renewal in the dish
- 41:04for quite prolonged periods of time.
- 41:08Up to a month for adult.
- 41:12See a few ehad is documented in the
- 41:16literature in our hands up to about 2 weeks.
- 41:19And this contrasts with CF you,
- 41:21even they differentiate their form rec
- 41:24cells within a matter of two to three days.
- 41:27And what stops a fuse from differentiating
- 41:30it keeps them in itself for in
- 41:33your state are glucocorticoids.
- 41:34And so when we placed glucocorticoid
- 41:3857 knock hard to safely.
- 41:41In this self renewal cocktail.
- 41:44We found that they failed to
- 41:47undergo efficient self renewal.
- 41:49And so that is very likely
- 41:52the reason for the anemia.
- 41:54So for some reason they fail
- 41:57to sell from you,
- 41:58and the reason we can clear when we
- 42:01looked at the expression of P57 in cells
- 42:04in the presence of glucocorticoids.
- 42:07Here we're looking at using Dex,
- 42:09which is a synthetic glucocorticoid,
- 42:12so cells express P57 and then P 57
- 42:14isn't used further by DEX and so it
- 42:17seems that dex and glucocorticoids
- 42:19in general probably work at least
- 42:22in part by inducing P.
- 42:2557.
- 42:25Inhibiting Assface CK activity and
- 42:28that in turn allows a stabilizes
- 42:31and long essays and promotes
- 42:34the safe UE self renewal state.
- 42:37So here we see a connection
- 42:39between along S phase and self
- 42:42renewal or persistence of a
- 42:45particular transcriptional state.
- 42:47Suggesting that maybe a fast S
- 42:51phase is somehow destabilizing
- 42:53to transcriptional program.
- 42:56Now we were able to rescue.
- 42:59The situation by adding
- 43:01to the knockout cells P.
- 43:0457 knockout cells acidic A2 inhibiting
- 43:07drugs so this will reduce CD K
- 43:11activity will inhibit CK activity
- 43:13in S phase cells and you can see
- 43:17that compared to the city 57.
- 43:19Knock ourselves the knockout cells
- 43:22treated with the drug almost completely
- 43:25resume normal self renewal activity.
- 43:30And so, um. You can we have a
- 43:34bit more data here, so yeah,
- 43:37so here we're looking at wild type cells.
- 43:40We can take this paradigm,
- 43:42feather and say, well,
- 43:43OK we have CF uees long essays.
- 43:46Stable self renewal can
- 43:47make it even more stable.
- 43:49Can we enhance the sea of yourself
- 43:52on your potential by adding city
- 43:54K inhibitors to the medium and
- 43:56prolonging S phase even more?
- 43:58And that indeed happens so you
- 44:00can see that compared to control
- 44:02cells undergoing self renewal
- 44:04in just dexamethasone.
- 44:05When we add the city K2 inhibitor with,
- 44:08these cells are completely blocked.
- 44:10They don't up regulator 119.
- 44:13When the S phase is slower and
- 44:16we can amplify them much further,
- 44:19so in red at the cells that have
- 44:23sydicate 2 inhibitors added on so we
- 44:26can amplify the CFL stage even further.
- 44:30And clearly this may have some
- 44:33translational applications.
- 44:34Maybe Syndicate two inhibitors will assist,
- 44:37or perhaps replace glucocorticoids in
- 44:40therapeutic approaches that target the CFUE.
- 44:43But it also for fundamental
- 44:45biology point of view.
- 44:47It tells us that stabilizing and
- 44:50prolonging S phase delays the switch.
- 44:56So I will. I'm running a little
- 44:58bit out of time so I will quickly
- 45:02summarize what we what I've shown
- 45:04you in the second part of the talk.
- 45:07See if you self renewal.
- 45:09Depends on a long space.
- 45:12And when that is.
- 45:15Impaired, for example,
- 45:17in the P57 knockout mouse.
- 45:21There are insufficient cycles of
- 45:22self renewal, and that causes anemia.
- 45:25And we can rescue that and in fact enhance.
- 45:29See if you see few suffering you even
- 45:32in wild type progenitors by inhibiting
- 45:35SDK activity and prolonging essays.
- 45:39So it appears that exercise shortening
- 45:41may be causally related to the switch,
- 45:44although the underlying mechanism
- 45:47of course isn't clear yet.
- 45:50And then in the last few minutes of my talk,
- 45:53I'll take just five minutes to tell you
- 45:57knew story that isn't yet published.
- 46:00Again, that seems to highlight the
- 46:03importance of the cycle in this
- 46:07time in terminal differentiation.
- 46:09So we know that E praeceptor becomes
- 46:11essential during terminal differentiation.
- 46:13Although it begins to be expressed much
- 46:17earlier in earlier with the Preseas.
- 46:20And with that,
- 46:21the power sector I've shown you earlier,
- 46:23we have no cells in terminal differentiation.
- 46:26And so we asked.
- 46:27And this is work by Daniel Hidalgo in my lab.
- 46:31We asked whether.
- 46:35The emperor sector has added
- 46:36functions other than survival
- 46:38during terminal differentiation,
- 46:40and two answer that we developed a
- 46:42genetic model in which we take the
- 46:45perceptive nokut fetal livers and
- 46:48introduce back either the EPO receptor
- 46:50obiesie ELEX to stop them from dying.
- 46:53And with this model system we
- 46:55were able to see that in fact
- 46:57you can get full differentiation
- 46:59without any equal receptor at all,
- 47:01as long as you keep the cells from dying by.
- 47:06D. Transgenic expression of BCLX.
- 47:10However, there were various abnormalities,
- 47:11and the biggest one was that the
- 47:14Sea Fury colonies that were formed
- 47:16were much smaller than the area,
- 47:19which is much smaller.
- 47:22And the second finding was that
- 47:24S phase wasn't as fast,
- 47:26so it was still pretty fast,
- 47:29but not as fast as in cells that
- 47:32were expressing the E prospectors.
- 47:35So clearly E.
- 47:36Praeceptor can further accelerate
- 47:38space speed.
- 47:39When we look at cell growth,
- 47:41this is in vitro.
- 47:44And see that cells expressing the ape
- 47:47receptor grow much faster than cells
- 47:50expressing BCLX and the doubling time.
- 47:53Let's look at control cells.
- 47:55That doubling time is only six hours in
- 47:58our cells that express the equal sector,
- 48:00but it climbs to a towers in cells that
- 48:04don't have a protective signaling.
- 48:06We were fortunate to be able to
- 48:09collaborate with Sean James Goose Lab,
- 48:11who developed a beautiful Reporter mice.
- 48:14This mouse reports the length of the
- 48:17cell cycle using a Fusion protein.
- 48:20H2B fluorescence timer Fusion.
- 48:23These Fusion is fluorescent blue
- 48:25when it is first synthesized and
- 48:28then becomes a fluoresces red
- 48:29about an hour or two later.
- 48:32And so cells that have a short cycle IBB
- 48:36lower than cells that have a longer cycle.
- 48:40And so with her lab,
- 48:42we injected mice with either Ipoh or Saline.
- 48:45And found that two mice injected
- 48:48with Saline here in blue.
- 48:50Um?
- 48:52Here we're looking at the ratio
- 48:54of blue to red fluorescence.
- 48:56We see a shift in that ratio when we look
- 48:59at two mice that are injected with people.
- 49:03And here we're looking at a specific
- 49:05erythroblast earlier Race for Life stage,
- 49:07and we can look at a number of our
- 49:10way through glass stages and at
- 49:12each stage we see a clear difference
- 49:14in cell cycle length.
- 49:16So it seems that if receptor
- 49:18can shorten space even further,
- 49:20and in fact shorten the cycle.
- 49:23I'm going to skip this next part,
- 49:26which very briefly,
- 49:27you would think that if a preceptor
- 49:30drives as shortest cycle and more
- 49:33numerous cycles in terminal differentiation,
- 49:35the red cells that would result
- 49:38would be smaller.
- 49:39But in fact we find exactly the opposite.
- 49:42We find that the rattles are formed at
- 49:46larger and I won't go through the data,
- 49:49but we see a larger diameter in cells that.
- 49:53Experienced high ipho and so
- 49:56that's a sort of paradox.
- 49:58This is interesting in its own right.
- 50:01I'm.
- 50:03And so I will summarize
- 50:04what I've told you so far.
- 50:07And summarize my talk.
- 50:08What we find is that during
- 50:10the arethra developmental trajectory,
- 50:13the cell cycle takes up a
- 50:16number of different shapes.
- 50:17If you like, it is long,
- 50:20early in the trajectory with
- 50:23along for a space and along G1.
- 50:26During the gradual progression
- 50:28through to see if you re staged
- 50:31towards the switch to ETD we have
- 50:35initially gradual shortening of G1.
- 50:37And sorry, gradual shortening of D1.
- 50:40And then at the time of this which we have.
- 50:44An abrupt shortening of essays,
- 50:46the shortening West phase is the result
- 50:49of downregulation of P57 Kip too,
- 50:51which is acidic Lee,
- 50:53two inhibitor.
- 50:53It inhibits S phase city K activity
- 50:56and that leads to an increase in
- 51:00the speed of replication Forks.
- 51:02The long ass face in the CFG stage
- 51:06is critical for the stability of that
- 51:09stage in it for its self renewal,
- 51:12and it appears that glucocorticoids
- 51:15utilize that by enhancing expression
- 51:18of of the silicate to inhibit P 57 and
- 51:22prolonging the CFO is suffering you'll stage.
- 51:25And we may be able to use that
- 51:27therapeutically with silicate
- 51:29two inhibitors as well.
- 51:30Once we cross this this late
- 51:33into terminal differentiation,
- 51:34the initial cycles are very short.
- 51:36In fact I forgot to mention this cycles
- 51:40of the earlier throw blasts as some
- 51:43of this shortest in the bone marrow.
- 51:47And these can be even further
- 51:51shorter when we.
- 51:53Use that when we simulate these cells.
- 51:56With people so at high levels of people
- 51:59which we would find in stress situations,
- 52:03high altitude or disease or bleeding.
- 52:06Cycles become even shorter.
- 52:09And that in itself is raises
- 52:12the question of why is that?
- 52:15And I did not have time to discuss that,
- 52:20but all the work shows that the
- 52:23fast cycles of earlier reefer blasts
- 52:26contribute to rapid DNA demethylation,
- 52:29and that interns assist the speed
- 52:32of the ATD transcriptional program.
- 52:35So it's possible that that might
- 52:38be the reason why.
- 52:40This takes place here,
- 52:41but of course we don't know that for sure.
- 52:45So I'd like to thank the people that
- 52:49did the work and these people in my lab.
- 52:55Including include US Warrior, Sami, Nathan.
- 52:57Better bear to see who did a lot
- 53:00of the single cell RNA SEQ work.
- 53:03Melinda Futron who worked in the DNA
- 53:07combing together with young Kwang
- 53:09who did all the work on P57 and
- 53:11Daniel Hidalgo who did the work on
- 53:14the ape receptor signaling and our
- 53:17collaborators were very fortunate
- 53:19in our collaborators and old clients
- 53:21lab with a single cell RNA SEQ.
- 53:23Necrime at UMass Medical School.
- 53:26Who helped us with the DNA combing
- 53:29and shanting Google in Yale who
- 53:32had been a great colleague,
- 53:34was also interested in the cell cycle and
- 53:38we've collaborated with her transgenic mouse.
- 53:41So thank you.
- 53:46Thank you very much for a great talk.
- 53:48We have two questions for
- 53:50you in the Q&A session.
- 53:51I don't know if you can see them wrong,
- 53:54but I can read them to the group.
- 53:57The first is from Doctor Liu says great talk.
- 54:00Any speculation in how CD K2 and
- 54:02P57 regulate fork speed in S phase.
- 54:05We would like to know that yeah,
- 54:07so we don't know there are many.