Intestinal Villi Assume the Fetal Position in Response to Injury

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00:00:12.14 My name is Takahiro Ohara,
00:00:14.19 and I’m an MD-PhD student at Washington University in St. Louis.
00:00:19.24 When I was a medical student a few years ago
00:00:22.04 — this is me as a newly minted student
00:00:24.16 and me with my classmates at a local health fair —
00:00:27.25 I had the opportunity to talk to and learn
00:00:31.02 from many different patients.
00:00:32.16 And one patient in particular made a big impact on me.
00:00:37.00 This patient was a 64-year-old male
00:00:39.10 who came into the hospital with
00:00:42.10 stomach pain, diarrhea, weight loss, and fatigue.
00:00:44.24 And when I talked to this patient,
00:00:47.01 I remember he was visibly distressed.
00:00:49.08 He told me that he was tired all the time
00:00:52.07 and that he had trouble getting out of bed every morning,
00:00:55.14 and no matter how much he ate
00:00:57.23 he felt like he was literally starving to death.
00:01:00.15 We suspected that there might be something going on
00:01:03.11 in his intestinal tract,
00:01:05.16 and so we decided to do a biopsy
00:01:07.23 where we took a small piece of tissue from his gut
00:01:10.26 and looked at it under the microscope.
00:01:13.06 Normally, the intestine is lined by millions
00:01:16.20 of these finger-like structures called villi.
00:01:18.29 These villi are extremely important for nutrient absorption.
00:01:22.27 But in this patient, his villi were completely destroyed.
00:01:27.11 And so we decided to give this patient
00:01:28.29 the current standard of care,
00:01:31.08 which is a dietary intervention,
00:01:33.17 and so, removing any food that may be causing the damage.
00:01:37.14 However, many months later, this patient came into the hospital again
00:01:42.17 and he looked even worse.
00:01:44.14 He appeared even more lethargic and emaciated,
00:01:48.07 and his villi were still damaged.
00:01:50.19 And I remember just feeling helpless
00:01:53.01 because there weren’t any medications,
00:01:55.06 any lifestyle modifications,
00:01:57.00 that we could provide to help this patient.
00:02:00.28 After witnessing firsthand the importance of having healthy villi
00:02:05.06 for our well-being,
00:02:07.09 I was really motivated to find new ways
00:02:09.24 to treat these patients.
00:02:11.28 This patient had what’s called persistent villus atrophy,
00:02:14.27 whereas his villi appeared continuously damaged
00:02:17.12 even with treatment.
00:02:19.23 Unfortunately, this is not uncommon.
00:02:22.13 Over one-third of patients with damaged villi
00:02:24.21 can experience this.
00:02:27.00 Currently, many treatment efforts are focused on
00:02:30.05 removing the agent that’s causing the damage.
00:02:32.28 However, this doesn’t always work.
00:02:35.07 In fact, there’s not a lot known about
00:02:38.03 how villi restore normally after injury.
00:02:41.18 And so, I wondered,
00:02:43.28 by understanding how villi normally heal,
00:02:46.18 can we develop new therapeutics
00:02:49.20 around promoting this process?
00:02:52.07 And so, during graduate school, I decided to study this question.
00:02:56.02 That is, what molecular and cellular processes
00:03:00.00 drive villus recovery after injury?
00:03:03.00 So, here’s what the villus looks like at the cellular level
00:03:05.23 using a standard dye.
00:03:08.02 Notice the finger-like structures
00:03:10.19 that project into the lumen of the intestine.
00:03:13.13 In the image on the right,
00:03:15.25 each villus is predominantly lined
00:03:18.21 by a cell type known as enterocytes.
00:03:21.13 Enterocytes are specialized epithelial cells
00:03:24.10 that mediate nutrient uptake and metabolism.
00:03:28.24 Now, studying villus injury and repair in humans
00:03:32.09 is quite challenging.
00:03:34.10 I can’t really ask healthy people
00:03:36.19 to let me damage their villi
00:03:38.26 and perform multiple biopsies on them.
00:03:41.06 So, instead, I turned to an animal model
00:03:43.18 that is commonly used in the laboratory: the mouse.
00:03:46.24 Mice are great models because,
00:03:48.12 one, their intestine looks just like humans,
00:03:51.11 with villi,
00:03:52.24 and, two, it is quite easy to change their genetics.
00:03:56.12 Now, going back to our patient,
00:03:58.19 we ultimately didn’t know what exactly was causing
00:04:01.00 the damage in his gut lining.
00:04:02.23 But the most likely culprits were some sort of inflammation
00:04:05.23 — so, an overactive immune system —
00:04:08.21 or an infection
00:04:10.18 — for instance, due to a virus.
00:04:12.14 We can model inflammation and a viral infection in mice
00:04:16.07 using a double-stranded RNA analog called poly(I:C).
00:04:20.00 So, think of poly(I:C) as a viral mimic.
00:04:23.11 When poly(I:C) is given to mice,
00:04:26.03 there is rapid induction of cell death
00:04:28.01 within just a few hours of injection in the villus compartment,
00:04:31.12 as shown by the brown staining.
00:04:34.10 This causes the villi to collapse,
00:04:36.26 similar to what we observed in our patient.
00:04:40.27 The power of this model is that
00:04:43.13 once the villi get damaged,
00:04:45.12 these structures can repair and regenerate
00:04:48.09 in a reproducible fashion.
00:04:50.11 And so, this offered me the unique opportunity
00:04:53.12 to study how villi repair after injury,
00:04:56.21 and potentially understand how this process can go awry in disease.
00:05:02.05 Using this model, I first asked the question,
00:05:04.18 what happens to the enterocytes?
00:05:07.08 These are the main cells that line the villi,
00:05:11.14 and they play a crucial role in nutrient absorption.
00:05:14.00 What I found was that, during villus atrophy,
00:05:16.05 at the 24-hour time point,
00:05:18.05 the enterocytes were essentially lost.
00:05:21.28 As you can see, there’s no more red cells
00:05:24.04 covering the damaged villi.
00:05:26.23 But during regeneration, the enterocytes came back,
00:05:29.24 and one week after injury the villi returned to normal.
00:05:34.01 And so, there was something really interesting going on,
00:05:36.22 transiently,
00:05:38.17 during the atrophy phase,
00:05:40.14 and so I decided to focus on this time point.
00:05:44.01 Now, if you look very closely,
00:05:45.24 there are still cells lining the rudimentary villi,
00:05:48.06 as pointed out by the black triangles.
00:05:51.28 But these cells are really small.
00:05:54.28 And so, because these cells look nothing like any cell
00:05:57.28 that you see in the normal gut,
00:06:00.14 I decided to call these cells atrophy-induced villus epithelial cells
00:06:03.24 or aVECs.
00:06:05.28 Here’s a high-power view of these cells using electron microscopy.
00:06:10.22 On the top are normal enterocytes,
00:06:13.10 and here we’re only seeing part of the cell
00:06:15.15 because enterocytes are quite tall.
00:06:18.14 On the surface of enterocytes are these hair-like extensions
00:06:21.01 called microvilli,
00:06:23.03 which give enterocytes their absorptive function.
00:06:26.20 On the bottom are the aVECs,
00:06:28.21 and here we can see the entirety of the cell because,
00:06:32.13 again, these cells are quite small
00:06:35.00 and also they have poorly developed microvilli
00:06:38.08 and they contain a lot of lipid droplets,
00:06:40.26 which are the circles that you see within the cell.
00:06:43.16 This is typically a sign of malabsorption.
00:06:47.06 Recall from our patient that he had trouble taking up nutrients
00:06:50.22 and that he kept on losing weight.
00:06:53.14 And part of the reason is that his villi were not functioning well.
00:06:57.10 By microscopy, we can see that these aVECs,
00:07:00.04 which line damaged villi, look unhealthy;
00:07:04.14 they look like immature versions of enterocytes.
00:07:08.19 And so, to further characterize these cells
00:07:11.01 and understand how they’re different from normal enterocytes,
00:07:14.09 I performed a microarray analysis.
00:07:17.11 A microarray is essentially a small chip
00:07:19.07 that allows you to compare the gene expression profile
00:07:21.19 between two different samples or cell types.
00:07:25.19 Each circle in the chip represents a single gene.
00:07:29.00 And if there’s more of the gene in one sample,
00:07:31.02 based on RNA levels,
00:07:32.27 then the circle gives a color:
00:07:35.24 red if there’s more in the red sample,
00:07:38.01 blue if there’s more in the blue sample.
00:07:41.01 And so, using this method,
00:07:43.19 I can determine the expression of practically all of the genes
00:07:46.03 within the cell type or sample of my interest.
00:07:50.05 Now, the intestine is composed of multiple different cell types,
00:07:53.06 including epithelial cells,
00:07:56.13 immune cells,
00:07:58.17 and stromal cells.
00:08:00.02 And to specifically compare the gene expression between enterocytes versus aVECs,
00:08:03.29 I first used a laser to isolate
00:08:08.03 or capture these cells under the microscope
00:08:10.28 to get a purified population.
00:08:12.19 This is called laser-capture microdissection.
00:08:15.23 Once I got my purified sample of enterocytes
00:08:18.07 and purified sample of aVECs,
00:08:20.27 I submitted them for microarray analysis.
00:08:24.16 This is a pretty standard volcano plot that you get
00:08:28.26 from analyzing microarray data,
00:08:30.10 where each dot represents a single gene.
00:08:34.17 The x-axis here is the fold change,
00:08:37.17 which is how big the difference is,
00:08:39.14 and the y-axis is the p-value,
00:08:41.19 which is how meaningful that difference is.
00:08:45.11 And the genes that are significantly different are colored here,
00:08:48.17 and as you can see there’s a lot of genes there.
00:08:51.15 And so, I decided to focus on the top-most
00:08:56.11 differentially expressed genes in aVECs.
00:08:58.03 So, these are the genes that are on the top right corner of the plot,
00:09:00.13 within the dashed box.
00:09:03.18 So, here’s a heat map showing you
00:09:06.05 some of those top-most differentially expressed genes.
00:09:08.28 Each column here represents a sample,
00:09:11.11 and each row represents a specific gene,
00:09:13.04 as indicated.
00:09:15.18 And what I found was that genes that are expressed
00:09:17.16 in the fetal intestine
00:09:20.06 — so, that includes mesothelin, clusterin, IL1RN, etc —
00:09:24.03 were all highly upregulated in aVECs
00:09:27.00 compared to enterocytes.
00:09:29.09 Furthermore, when I took all of the genes
00:09:32.15 that are expressed in the fetal intestine
00:09:34.23 and then asked whether that fetal signature
00:09:39.17 is more enriched in aVECs or enterocytes…
00:09:44.01 this is called a gene set enrichment analysis.
00:09:47.17 In the graph on the right,
00:09:49.21 each black line represents a single fetal gene
00:09:51.22 and the green curve represents the degree of enrichment.
00:09:56.08 And as you can see, the majority of the fetal genes
00:09:59.02 were more expressed in aVECs compared to enterocytes,
00:10:02.27 and thereby the enrichment score
00:10:05.22 was much higher in aVECs,
00:10:07.09 and the green curve skewed towards that direction.
00:10:10.24 This demonstrated that a fetal or developmental program
00:10:13.06 gets turned on after injury
00:10:16.01 within these cells.
00:10:18.15 And so, this is really interesting…
00:10:21.24 but whenever you do a gene expression analysis
00:10:24.09 such as a microarray,
00:10:26.08 you want to try to validate your results
00:10:28.04 using another technique.
00:10:29.16 Here, I performed a technique called in-situ hybridization,
00:10:33.20 which allows me to take a gene of interest
00:10:36.04 — in this case clusterin, which is one of the fetal markers —
00:10:38.28 and look at the expression of this RNA within the tissue.
00:10:42.14 As you can see, in normal villi,
00:10:47.06 clusterin was not expressed.
00:10:49.05 But in… during villus atrophy,
00:10:51.14 clusterin was highly expressed in aVECs,
00:10:54.11 as you can see by the red staining.
00:10:58.09 And so, I saw a similar pattern of expression
00:11:01.22 with the other fetal markers as well using this technique.
00:11:07.00 And so, what is driving this fetal program
00:11:09.05 that appears to be a defining feature of aVECs?
00:11:13.20 Could it be some kind of developmental pathway that’s involved?
00:11:18.27 One pathway that caught my attention
00:11:22.16 was the Hippo signaling pathway.
00:11:24.16 It’s called the Hippo pathway because, back in 2003,
00:11:27.29 scientists discovered a mutant fly
00:11:30.29 that had abnormally large organs.
00:11:34.10 They named the gene that was mutated in these flies Hippo
00:11:37.07 because the flies resemble a hippopotamus.
00:11:40.09 It turns out the Hippo pathway is very important
00:11:43.12 in controlling organ size during development,
00:11:47.09 but recent research has also suggested that
00:11:51.29 this pathway is crucial for tissue regeneration,
00:11:54.11 and in disease processes, in adult animals.
00:11:58.21 And so, this Hippo pathway
00:12:01.26 was an interesting candidate for potentially controlling
00:12:04.17 the fetal program in my injury model.
00:12:07.08 So, briefly, how does the Hippo pathway work?
00:12:09.18 Well, the main effector of the Hippo pathway
00:12:12.05 is called YAP, or yes-associated protein.
00:12:15.20 YAP is typically inhibited by the Hippo proteins,
00:12:18.14 as represented by the hippopotamus.
00:12:22.04 And so, generally YAP is inhibited.
00:12:25.17 However, when YAP is relieved from the inhibition
00:12:28.27 of the Hippo proteins,
00:12:30.10 it moves into the nucleus and activates gene expression.
00:12:34.16 And so, the key point here is that when YAP is cytoplasmic
00:12:37.04 it’s inactive,
00:12:38.20 and when YAP is nuclear it’s active.
00:12:41.26 And so, if YAP is involved,
00:12:43.22 looking at the RNA level or looking at my microarray data
00:12:46.28 wouldn’t necessarily be helpful.
00:12:49.24 Instead, by looking at the localization of the YAP protein
00:12:54.11 within the cell,
00:12:55.29 I can tell whether or not it’s activated.
00:12:58.25 Here’s a bar graph showing you
00:13:01.15 the localization of YAP in normal villi versus atrophic villi.
00:13:06.02 Cytoplasmic YAP is represented by a beige color,
00:13:08.29 and nuclear YAP is represented by a dark brown color.
00:13:12.29 And if the YAP is both nuclear and cytoplasmic,
00:13:16.04 that’s represented by a light brown color.
00:13:18.27 And what I found was that YAP was largely cytoplasmic in normal villi,
00:13:23.23 but during villus atrophy
00:13:26.01 YAP becomes predominantly nuclear.
00:13:29.00 These are representative images showing you that
00:13:32.12 YAP is in fact nuclear within the aVECs,
00:13:36.01 suggesting that it’s activated in these cells.
00:13:39.17 And so, to functionally test whether YAP
00:13:42.27 is in fact controlling the fetal program,
00:13:46.25 I looked at mice that were deficient in the YAP gene,
00:13:48.29 which I refer to as YAP knockout mice.
00:13:52.05 As you can see, compared to control mice,
00:13:55.03 the expression of YAP was completely lost in the knockout mice,
00:13:58.24 as you’d expect.
00:14:00.26 Notice that this is without injury,
00:14:03.11 and these mice do pretty fine.
00:14:05.13 The villi are normal,
00:14:07.28 and that’s likely because YAP is generally inhibited,
00:14:11.24 and so taking away something that’s inactive
00:14:13.26 doesn’t necessarily do anything.
00:14:16.07 And so, the interesting question is,
00:14:17.26 what happens after the injury,
00:14:19.23 when the activity of YAP gets turned on?
00:14:23.26 So, now we’re looking at post injury,
00:14:25.25 during villus atrophy.
00:14:27.27 On the left is control mice, and on the right is YAP knockout mice.
00:14:31.18 And like I’ve shown you before,
00:14:34.14 in control mice the damaged villi
00:14:36.26 are lined with aVECs that express fetal markers.
00:14:40.03 In this case, I’m showing you clusterin expression again.
00:14:43.22 In the YAP knockout mice,
00:14:45.13 the expression of clusterin, and many of the other fetal markers
00:14:47.26 that I’m not showing you here,
00:14:49.26 were gone.
00:14:52.09 And so, now we have a scenario
00:14:55.19 where we have normal aVECs in control mice
00:14:57.20 with the fetal program
00:14:59.26 and abnormal aVECs in YAP knockout mice
00:15:02.14 stripped of the fetal program.
00:15:05.21 And so, what is the consequence of having abnormal aVECs?
00:15:09.26 Because aVECs are at the surface of the gut
00:15:12.12 and they directly interface with the external environment,
00:15:15.06 I hypothesize that these cells play and… serve as a shield, or as a barrier,
00:15:22.14 against the harmful substances that may be present in the gut lumen
00:15:26.23 from reaching the host.
00:15:29.15 I can assess the intestinal barrier function in mice
00:15:32.25 by orally delivering a green dye
00:15:35.12 and then measuring the serum levels of the dye.
00:15:38.26 How this works is that when the barrier or shield
00:15:42.07 is intact, the dye cannot go through,
00:15:45.14 and so it gets flushed out.
00:15:48.10 But when there’s a breach in the barrier,
00:15:50.07 the dye can seep through and enter the bloodstream.
00:15:53.13 And so, by measuring the level of the dye in the blood,
00:15:57.03 I can assess the quality of the intestinal barrier.
00:16:01.20 And so, when I performed this experiment,
00:16:03.25 I found that in YAP knockout mice
00:16:07.26 the level of the dye in the serum
00:16:11.06 was much higher compared to control mice.
00:16:13.01 This suggested that aVECs play a critical role
00:16:16.01 in maintaining barrier integrity,
00:16:18.15 and that this function was dependent on YAP.
00:16:22.15 And so, finally, what happens when your barriers compromise
00:16:27.21 as a result of aVECs becoming dysfunctional?
00:16:30.29 Well, it turns out to have a big impact.
00:16:34.16 In this graph, we’re looking at villus length
00:16:37.03 at various time points during injury repair.
00:16:39.28 Recall that the taller the villi,
00:16:41.23 the more surface area for nutrient absorption,
00:16:43.28 the better it is for the animal.
00:16:46.16 And the 24-hour time point in both control and YAP knockout mice,
00:16:50.25 the villi get damaged and undergo atrophy
00:16:53.22 to a similar degree.
00:16:55.12 But then we start seeing a striking difference
00:16:57.24 during the regenerative phase,
00:17:00.15 where in control mice, represented by the blue line,
00:17:04.10 the villi regenerate robustly,
00:17:07.23 but in YAP knockout mice,
00:17:09.20 represented by the dashed red line,
00:17:11.27 villus regeneration is greatly impaired.
00:17:15.03 Here’s what the regenerating villi look like
00:17:17.13 at the 48-hour time point.
00:17:19.06 On the top are control mice,
00:17:21.18 and here the villi are robustly regrowing
00:17:24.00 back to their original length.
00:17:26.04 On the bottom are YAP knockout mice.
00:17:28.12 Notice that the villi here are shorter,
00:17:30.16 they look stunted,
00:17:32.07 and then they’re also aggregated.
00:17:35.03 This reminded me of the persistent villus atrophy pathology
00:17:38.18 that I saw in our patient.
00:17:41.05 And so, together these results
00:17:44.08 demonstrated that YAP plays an essential role
00:17:47.17 in villus regeneration after injury.
00:17:51.15 As humans, when we sense danger or when we’re hurt,
00:17:55.09 we instinctively assume a fetal position to sort of protect ourselves.
00:18:01.14 When the villi in our gut gets damaged,
00:18:03.20 a similar phenomenon occurs
00:18:06.00 at the molecular and cellular level,
00:18:07.14 where after a villus injury
00:18:09.16 a unique cell type called aVECs
00:18:12.09 emerges and covers the damaged villi.
00:18:15.16 A fetal or developmental program
00:18:18.12 gets re-initiated in these cells,
00:18:20.10 and this program is crucial for repair
00:18:22.29 as well as for protecting the host.
00:18:26.03 As an aspiring physician-scientist,
00:18:29.03 my goal is to make discoveries in the lab
00:18:31.19 and translate those findings into the development of better therapeutics.
00:18:37.02 Going back to my patient,
00:18:39.04 where he had a long-standing problem with his gut health,
00:18:42.17 I hope that my finding could open new possibilities
00:18:46.29 for better treatment options for patients like him.
00:18:50.13 As the Nobel Prize-winning physicist
00:18:52.25 William Lawrence Bragg once said,
00:18:55.14 “The important thing in science is to [not only] obtain new facts
00:18:59.17 [but also] discover new ways of thinking about them.”
00:19:05.09 I would like to thank my advisor, Dr. Thaddeus Stappenbeck,
00:19:09.13 and my co-mentor, Dr. Marco Colonna,
00:19:12.13 for their mentorship.
00:19:15.06 I would also like to thank the cores at Washington University,
00:19:18.06 my thesis committee,
00:19:19.24 my funding sources,
00:19:21.24 and iBiology for giving me this opportunity to share my research.

This Talk

Speaker: Takahiro Ohara

Audience:

Student
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Researcher
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Recorded: June 2021

Talk Overview

Intestinal villi are finger-like projections that line the small intestine, increasing surface area to help with absorption of nutrients. Injuries to the intestinal villi can cause a range of health problems, and can even be life-threatening. In this Share Your Research Talk, physician-scientist trainee Takahiro Ohara discusses his research on the molecular and cellular processes that drive recovery of intestinal villi. He describes the fetal-like state that cells on damaged villi take on, and how adaptive differentiation of these cells leads to proper villus recovery.

Speaker Bio

Takahiro Ohara

Takahiro Ohara is an MD-PhD student at Washington University in St. Louis. Interested in regenerative medicine, his research focuses on how intestinal villi recover from injury. He received a B.S. from UCLA in Molecular, Cell, and Developmental Biology with a Minor in Biomedical Research. Continue Reading

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Chris George (iBiology): Graphics
Maggie Hubbard (iBiology): Graphics

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