Development and function of spindle neurons

Development and function of spindle neurons

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In his book How to Create a Mind author Ray Kurzweil makes some claims about spindle neurons that he provides no source for.

Concretely he states that spindle cells:

  1. Are Involved in handling emotion and moral judgement;
  2. Are believed to first have occurred in the last common ancestor of chimpanzees and humans;
  3. Are non-existing in newborns, but appear around the age of four months, and increase significantly in number from ages one to three.

Is there a scientific consensus backing these claims?

The spindle-shaped brain cells you refer to are called von Economo neurons -named after the man who first described them (source: Smithsonian).

  1. In humans, von Economo neurons reside only in the anterior cingulate cortex (ACC) and the frontal insula (FI) (Allman et al., 2011). These two regions are particularly active when people experience emotion and they are reportedly important for "self-monitoring," such as noticing bodily sensations of pain and hunger, or recognizing that one has made a mistake. The ACC seems broadly involved in nearly every mental or physical effort (Smithsonian). By contrast, the FI may play a more specific role in generating social emotions such as empathy, social awareness, and self-control (Allman et al., 2011), but also trust, guilt, embarrassment, love, and even the sense of humor Smithsonian).

  2. Humans and the great apes (chimps, bonobos, gorillas and orangutans) have von Economo cells. Lesser primates, such as macaques, lemurs and tarsiers, do not. That means the neurons evolved in a common ancestor of all the great apes about 13 million years ago, after they diverged from other primates but well before the human and chimp lineages diverged about six million years ago (Smithsonian). However, elephants and whales have them too, possibly explaining their complex social behavior (Allman et al., 2011).

  3. I couldn't find literature on the development of these cells in children. Note, however, that, as far as I know, the presence of these cells can be only detected using microscopic techniques, i.e., on coupes of post mortem tissue (e.g., Santos et al., 2010)). This will make research in infants and children difficult.

- Allman et al., Ann N Y Acad Sci (2011); 1225: 59-71
- Santos et al., Brain Res (2011); 1380: 206 - 17

Neuronal Development

The nervous system. Central and peripheral, this system is integral to every bodily function happening in you right now and at every waking moment. From the rhythm of your heartbeat to the tiniest sensation of a gentle itch, the nervous system drives all functions that contribute to your survival.

Without it, well, you would be no more than a bag of bones with the intelligence of a tardigrade (no offense to tardigrades).

So, where does the nervous system begin, and what makes it so important?

Neurons and glial cells are perhaps the most important functioning components of the nervous system. They are vital to the reception and distribution of information from stimuli both inside and outside of your body. These cells emerge before you even become a conscious being: at embryonic development.

Scientists unravel the function of a sight-saving growth factor

NIH study breaks down pigment epithelium-derived factor to understand how it protects and stimulates retinal neurons.

PEDF protein (center) has two domains with different functions. The 34-mer (blue, left) has anti-angiogenic properties. The 44-mer (green and yellow, right) protects and stimulates neurons. The 17-mer (yellow) is a smaller region of the 44-mer with the same function. Lesley Earl, NEI

Researchers at the National Eye Institute (NEI) have determined how certain short protein fragments, called peptides, can protect neuronal cells found in the light-sensing retina layer at the back of the eye. The peptides might someday be used to treat degenerative retinal diseases, such as age-related macular degeneration (AMD). The study published today in the Journal of Neurochemistry. NEI is part of the National Institutes of Health.

A team led by Patricia Becerra, Ph.D., chief of the NEI Section on Protein Structure and Function, had previously derived these peptides from a protein called pigment epithelium-derived factor (PEDF), which is produced by retinal pigment epithelial cells that line the back of the eye.

“In the eye, PEDF protects neurons from dying. It prevents the invasion of blood vessels, it prevents inflammation, it has antioxidant properties—all these are beneficial properties,” said Becerra, the senior author of the study. Her studies suggest that PEDF is part of the eye’s natural mechanism for maintaining eye health. “PEDF may have a role for treating eye disease. If we want to exploit the protein for therapeutics, we need to separate out the regions responsible for its various properties and determine how each of them works.”

The team used a well-known cell culture model system where immature retinal cells are isolated from the eyes of newborn rats and grown in a dish with minimal nutrients. The system includes not only the retina’s light-sensing photoreceptors, but additional types of neurons that help the retina process and transmit visual information to the brain.

“Our model system – using cells isolated from the animal – lets us tease out the individual processes and mechanisms behind PEDF’s protective effects,” said Germán Michelis, graduate student and the study’s first author.

The PEDF protein has functionally distinct domains. The Becerra lab previously found that each domain can work independently. One area, which is called the 34-mer because it is formed by 34 amino acid building blocks, halts blood vessel growth. Aberrant blood vessel growth is central to retinal diseases such as AMD and diabetic retinopathy. The second PEDF domain, called the 44-mer, provides anti-death signals to retinal neurons. The 44-mer can also stimulate neurons to grow neurites, finger-like projections that help the neurons communicate with their neighbors. A shorter version of the 44-mer of only 17 amino acids (17-mer) has identical activities.

Michelis and colleagues tested whether the 44-mer could protect immature retinal cells in a dish. Without the presence of proteins and other cells in their usual retinal environment, immature photoreceptors quickly die but can be preserved with PEDF.

They found that both the 44-mer and 17-mer were as capable of saving these photoreceptors as full-length PEDF.

The researchers also found that PEDF activity appears to be most needed at a specific point in photoreceptor cell development. Light detection takes place in a part of the photoreceptor known as the outer segment, where light-sensing opsin proteins are concentrated. The scientists found that when a photoreceptor cell is just beginning to create its outer segments, PEDF triggers the movement of opsin into the budding outer segment, where it belongs.

Along with photoreceptors, the retina is packed with several other types of neurons, which work together to process visual signals. Via neurites, amacrine neurons form connections, called synapses, to the cells that forward these visual signals to the brain. Becerra and colleagues found that PEDF stimulates amacrine cells to develop neurites in their cell culture model and that the 44-mer and 17-mer were at least as effective - or better - at stimulating these connections than the native protein.

Further, the 44-mer and 17-mer peptides work by binding to a protein receptor (PEDF-R) on the surface of neurons. PEDF activates PEDF-R, which processes molecules like docosahexaenoic acid (DHA), an omega-3 fatty acid critical for babies’ development and for eye health. PEDF-R was discovered previously by the Becerra lab.

“We’ve known for a long time that DHA is important for retinal health. We think PEDF signaling might be a key component of regulating omega-3 fatty acids like DHA, both during eye development and in maintaining the eye’s health over time,” said Becerra. “We’re hoping that we can harness some of these protective effects in a peptide-based therapeutic approach in the near future.”

This study was funded by the National Eye Institute Intramural Program and the National Research Council of Argentina.

Spindle neurons of the human anterior cingul. Ate cortex

The human anterior cingulate cortex is distinguished by the presence of an unusual cell type, a large spindle neuron in layer Vb. This cell has been noted numerous times in the historical literature but has not been studied with modern neuroanatomic techniques. For instance, details regarding the neuronal class to which these cells belong and regarding their precise distribution along both ventrodorsal and anteroposterior axes of the cingulate gyrus are still lacking. In the present study, morphological features and the anatomic distribution of this cell type were studied using computer-assisted mapping and immunocytochemical techniques. Spindle neurons are restricted to the subfields of the anterior cingulate cortex (Brodmann's area 24), exhibiting a greater density in anterior portions of this area than in posterior portions, and tapering off in the transition zone between anterior and posterior cingulate cortex. Furthermore, a majority of the spindle cells at any level is located in subarea 24b on the gyral surface. Immunocytochemical analysis revealed that the neurofilament protein triplet was present in a large percentage of these neurons and that they did not contain calcium-binding proteins. Injections of the carbocyanine dye DiI into the cingulum bundle revealed that these cells are projection neurons. Finally, spindle cells were consistently affected in Alzheimer's disease cases, with an overall loss of about 60%. Taken together, these observations indicate that the spindle cells of the human cingulate cortex represent a morphological subpopulation of pyramidal neurons whose restricted distribution may be associated with functionally distinct areas.

Emeriti and Professors of the Graduate School

John Ngai
Professor Emeritus of Neurobiology
Molecular and cellular mechanisms of olfaction

W. Geoffrey Owen
Professor Emeritus of Neurobiology
Membrane biophysics retinal neurophysiology

Mu-ming Poo
Professor Emeritus of Neurobiology

Frank Werblin
Professor Emeritus of Neurobiology
Neurophysiology of vision

Gerald Westheimer
Professor of the Graduate School Division of Neurobiology
Neurobiology psychophysics

Bob Zucker
Professor of the Graduate School Division of Neurobiology
Cellular neurophysiology synaptic biophysics


I would like to thank my many coworkers for their various excellent contributions and dedication to our research mission over the years, my two career mentors Pico Caroni and Thomas Jessell for their strong support and valuable advice, and I would like to apologize to other researchers in the motor control field for not being able to describe and cite their work in this short article due to space limitations. Research described here was supported by an ERC Advanced Grant, the Swiss National Science Foundation, the Kanton Basel-Stadt and the Novartis Research Foundation.

Part 2: Asymmetric Cell Division From Drosophila to Humans

00:00:13.04 Hello.
00:00:14.04 My name is Juergen Knoblich.
00:00:15.04 I am a scientist at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences
00:00:21.00 in Vienna.
00:00:22.00 And, in this second part of my lecture, I would like to tell you about how we can recapitulate
00:00:28.06 human brain development, in the lab, starting from pluripotent stem cells, using three-dimensional
00:00:35.05 culture methods.
00:00:36.05 Now, I'm sure every one of you would agree with me that the human brain is the most complex
00:00:43.06 but also the most fascinating structure that nature has generated.
00:00:47.08 It contains about 87 billion neurons that have to be born at the right time, migrate
00:00:52.21 to the right position, and wire up in the right way in order to allow us to perform
00:00:56.24 the cognitive processes that we're able to do.
00:01:00.06 But despite its complexity, the brain develops from a limited number of stem and progenitor cells
00:01:07.19 in a set of predefined rules.
00:01:10.14 And essentially everything that we know about those rules comes from experiments that were
00:01:14.13 done in rodent model systems, particularly in the mouse.
00:01:18.16 So, this shows you a very simplified view of mouse cortical development.
00:01:24.24 The mouse neocortex develops from a neural epithelium that lines a liquid-filled cavity,
00:01:31.08 which is called the lateral ventricle.
00:01:33.21 It's a polarized epithelium with basal to the outside, apical to the inside, and all
00:01:39.06 the different cell types in this epithelium are nicely arranged in a layer-type fashion
00:01:43.19 along the apical-basal axis of the epithelium.
00:01:46.17 So, here, on the more basal side, is the so-called cortical plate, and this is where the neurons
00:01:51.17 are and where they send out their axons.
00:01:53.16 And, here, on the more [apical] side, is the so called ventricular zone, and this is where
00:01:58.16 the stem and progenitor cells reside.
00:02:01.05 And these progenitor cells undergo either one of three different types of division.
00:02:05.16 Early during development, they divide symmetrically and this leads to an initial amplification
00:02:10.23 of the progenitor pool.
00:02:12.11 Later, they divide asymmetrically, where one progenitor generates one progenitor cell and
00:02:17.24 one cell that migrates out to become a neuron.
00:02:21.00 Or, the other cell forms a so-called intermediate progenitor, which divides once more into two
00:02:27.03 terminally differentiated neurons.
00:02:29.01 And this is called direct or indirect neurogenesis.
00:02:33.15 Now, when we move from rodents to humans, there is two characteristic differences.
00:02:40.01 First of all, the initial set of symmetric divisions lasts a lot longer
00:02:47.02 in humans and in primates.
00:02:49.16 This is a rodent cortex in. a rodent brain and a primate brain at about. at a comparable
00:02:57.17 stage of development, magnified to a comparable size.
00:03:02.01 And what you can see is that, while the more posterior brain parts are actually very similar,
00:03:07.17 the cortex is vastly expanded in the primate brain.
00:03:11.16 And this is because these cells had a lot more time to divide symmetrically and to amplify.
00:03:18.14 The second big difference is that, in addition to the ventricular zone and the cortical plate,
00:03:26.15 the primate brain contains an additional layer that is called the outer subventricular zone.
00:03:33.16 The outer subventricular zone is characteristic for primate brains and it contains a cell
00:03:38.24 type that is called outer radial glia cells or basal radial glia cells.
00:03:44.05 These cells are not, or in only very small numbers, present in rodent brains.
00:03:49.19 But, in primates, they act as a transit amplifying population during neurogenesis.
00:03:55.21 So, while in the rodents, per progenitor division, either one neuron or two neurons are generated,
00:04:04.02 in the primates, the progenitors generate these outer radial glia cells, which continue
00:04:09.12 to divide asymmetrically, generating hundreds of intermediate progenitors
00:04:14.03 and hundreds of neurons.
00:04:16.05 So, this is good news because it explains why we have so many more neurons than a mouse,
00:04:21.14 but it's also bad news because it means that there are certain brain developmental processes
00:04:25.19 that cannot be modeled in a rodent model system.
00:04:30.02 And this is particularly emphasized. empha. emphasized on this slide.
00:04:35.17 This shows you an MRI scan of a patient who suffers from a very severe neurodevelopmental
00:04:41.03 disorder that is called microcephaly.
00:04:43.19 What you can see is that the patient's brain is much smaller than that of a healthy patient.
00:04:49.11 This patient carries a mutation in a gene that is called Nde1.
00:04:53.03 Nde1 is conserved all the way through evolution, from yeast to humans, but when you make a
00:05:00.06 mutation in a mouse in Nde1 you see no, or only a very weak, phenotype.
00:05:06.15 So, certain neurodevelopmental disorders cannot properly be modeled in rodent models.
00:05:13.23 And, for this reason, there have been many attempts to actually model brain development
00:05:19.12 in a human setting.
00:05:20.15 So, how can we actually do this?
00:05:24.11 The easiest way, of course, is to use human tissue.
00:05:28.12 Human cortical tissue can be obtained from aborted fetuses, it can be fixed,
00:05:33.14 it can be stained.
00:05:35.07 There. one can even do live imaging and cell tracing on this.
00:05:41.20 But, of course, such experiments place a huge experimental burden and ethical burden, and
00:05:48.08 are very complicated and usually suffer from low N numbers.
00:05:51.14 And, for this reason, people have been trying to model brain development starting from
00:05:56.12 pluripotent stem cells.
00:05:58.03 The easiest way of doing this is to turn the pluripotent stem cells into neural stem cells,
00:06:03.17 which can then be forced to undergo neuronal differentiation, and one can study the phenotype
00:06:10.01 in those patient-derived neurons.
00:06:12.07 But, of course, neurons like to be in a three-dimensional environment and so there's many limitations
00:06:19.18 to those two-dimensional experiments.
00:06:21.03 And so, there have been many attempts to actually model the development of human tissues in
00:06:27.05 three-dimensional culture.
00:06:28.16 And, really, the pioneer of this has been a Japanese scientist named Yoshiki Sasai.
00:06:36.19 Yoshiki Sasai, around 2012, was able to recapitulate the development of a human eye in culture.
00:06:49.16 And this was clearly a pioneering experiment in the field of
00:06:55.12 generating human tissues in culture.
00:06:58.12 Now, I think Yoshiki Sasai clearly was one of the leading developmental biologists of
00:07:04.19 our times and it's very sad that he's no longer with us.
00:07:08.15 And so, based on his work, but also adding other experimental approaches to it,
00:07:13.24 Madeline Lancaster, a couple of years ago, in my lab, has decided to develop an in vitro three-dimensional
00:07:22.18 culture system that we can use to model the development of a human cortex in culture.
00:07:27.24 And here's the method that she came up with.
00:07:30.14 We start with pluripotent human stem cells, which we dissociate and then rapidly reaggregate
00:07:36.23 on the bottom of a 96-well plate.
00:07:39.09 We then allow those cells to develop into embryoid bodies, giving them just enough time
00:07:45.18 to form the three germ layers.
00:07:47.15 We then replace the medium with a neural induction medium, so that only the neural ectoderm survives.
00:07:53.18 And these balls of neural ectoderm are then placed into droplets of Matrigel gel, which
00:07:58.19 is a collagenous 3-dimensional support matrix that supports the development of
00:08:06.00 stem cell-derived tissue.
00:08:08.06 We then culture these Matrigel droplets, first in floating culture and later either in a
00:08:13.10 spinning bioreactor or, more recently, in an orbital shaker.
00:08:17.18 And, over time, Madeline saw the development of fairly complex tissues.
00:08:22.19 So, these are two examples of what we call cerebral organoids.
00:08:27.07 Here is one example and you can see a developing human cortex, here.
00:08:32.07 Here is a lateral ventricle.
00:08:34.17 Here is another piece of cortical tissue.
00:08:37.06 And here is another one.
00:08:39.01 This is another example.
00:08:40.21 There is cortical tissue, here, and over here.
00:08:45.06 And down here you see the development of a human eye.
00:08:50.20 This is a cross-section through a cerebral organoid.
00:08:54.03 There's cortical tissue, here.
00:08:55.20 You see a ventricular zone, here.
00:08:58.04 Here is the lateral ventricle.
00:09:00.14 And here are the differentiating neurons.
00:09:03.24 Down here is what we call the choroid plexus, which is the area that generates the
00:09:09.02 cerebral spinal fluid.
00:09:10.19 And here are other brain areas -- in the absence of markers, I do not know what they are, but
00:09:15.24 I want you to note that they have a different histology, indicating that they are different
00:09:23.01 areas of the developing human brain.
00:09:25.14 So, cerebral organoids can be used to model the development of
00:09:30.09 various parts of the human brain.
00:09:33.08 But, for our analysis, we focused on the developing cortex, which is the most complex but also
00:09:39.05 the most fascinating part of our brain, and also the one that is most different between
00:09:43.16 us and rodents.
00:09:45.15 This is a cross-section through a cerebral organoid.
00:09:48.18 This is the cortical part.
00:09:51.02 In red are progenitors, in green are the differentiating neurons, and what you can see is that, at
00:09:54.23 this stage, the organoid histology is essentially indistinguishable from that of a developing
00:10:01.09 mouse cortex.
00:10:02.09 So, cerebral organoids can recapitulate the three-dimensional organization of a developing
00:10:08.07 human cortex.
00:10:10.03 We also analyzed neuronal differentiation.
00:10:14.04 The neurons in the cerebral organoids send out long axons that contain growth cones,
00:10:21.01 they often branch out, they can bundle together, and the neurons also send out
00:10:27.12 large dendritic trees.
00:10:29.06 But, most importantly, the neurons in our organoids are electrically active.
00:10:34.06 This is a calcium imaging experiment where you can see that the neurons spike action
00:10:40.01 potentials, and they communicate with each other, and the pattern of these electrical firings
00:10:46.08 is far from random.
00:10:48.01 And. and. and there are certain correlated neurons and anti-correlated neurons.
00:10:54.08 So, cerebral organoids recapitulate both the three-dimensional organization of the developing
00:11:01.02 human cortex, and proper neuronal differentiation and function.
00:11:06.14 Now, the neurons in the developing cortex actually come in different flavors.
00:11:11.15 They are arranged in a layer-type fashion and these layers are formed in an inside-out
00:11:17.17 manner, where the deep layers are formed first and then newly formed neurons have to migrate
00:11:23.23 through these deep layers, adding additional layers to the outside.
00:11:27.09 There are various markers for these layers -- SatB2 is a layer for the out. is a marker
00:11:31.09 for the outside neurons, Ctip2 for the inside.
00:11:35.03 When we stain early organoids, essentially all of the neurons are positive for the deep
00:11:40.05 layer marker, Ctip2.
00:11:42.06 A bit later, SatB2-positive neurons appear, initially they are intermingled, and then
00:11:46.23 they show some kind of a sorting out, although we do not really see the formation of proper
00:11:53.11 neuronal layers.
00:11:54.11 But, the temporal specification of various different neuronal subtypes can be recapitulated
00:12:00.21 in the cerebral organoids.
00:12:03.10 So, we've generated a three-dimensional cultural system that we can use to recapitulate the
00:12:09.06 development of the human cortex in culture.
00:12:11.14 So, what can we do with it?
00:12:13.13 In my view, we are currently experiencing a complete revolution in the way of how we
00:12:18.24 do biomedical research.
00:12:20.13 And this is because there are currently four technological developments that are
00:12:25.02 coming together.
00:12:26.11 The first one is the availability of more and more complete human genome sequences,
00:12:32.10 many of them associated with complete patient records.
00:12:36.00 The second one is our ability to generate pluripotent stem cells
00:12:40.07 from each of these patients.
00:12:42.21 And the third one is our ability to generate, in the lab, more and more different tissues
00:12:48.06 as organoids, that recapitulate particular organs in those patients.
00:12:52.23 And, finally, we can edit the genome and introduce or remove mutations from any of these
00:12:59.14 pluripotent stem cells.
00:13:00.21 And how we can combine these technologies to analyze human neurodevelopmental disorders
00:13:05.23 will be shown on the next couple of slides.
00:13:08.18 For this, we teamed up with Andrew Jackson, a pediatric neurologist at the University
00:13:13.12 of Edinburgh, who works with a patient who suffers from a severe form of microcephaly.
00:13:18.02 This is a MRI scan of the patient you can see the brain is much smaller than that of
00:13:22.06 an age-matched healthy patient.
00:13:25.10 We obtained a biopsy from the patient, reprogrammed the cells into iPS cells, and then generated
00:13:31.05 organoids from them.
00:13:32.17 These are healthy, control organoids and patient-derived organoids.
00:13:37.06 And what you can see is that the control organoids contain a nice differentiated ventricular
00:13:44.15 zone, large ventricles, and many neurons, but the patient-derived organoids are much
00:13:49.17 smaller, they contain a lower number of neurons, and only a very tiny ventricular zone.
00:13:54.21 So, using our organoid system and patient-derived iPS cells, we can recapitulate the small-brain
00:14:01.07 phenotype of a microcephaly patient.
00:14:03.17 And we can now go back in history and ask, why are there so fewer neurons in these
00:14:10.02 microcephaly-derived organoids?
00:14:13.10 This is a control organoid and a patient-derived organoid at a much earlier stage of development.
00:14:19.18 At this early stage, in the control organoid, all the progenitor cells are still undergoing
00:14:25.16 the symmetric divisions, and neurogenesis has not started.
00:14:29.19 This is very different in the patient-derived organoids, where we can already see the appearance
00:14:34.02 of individual neurons.
00:14:35.14 And we conclude from this experiment, and many others that I don't have the time to
00:14:38.24 show you, that the cells have switched to an asymmetric division pattern in a premature
00:14:45.01 state, this leads initially to the formation of too many neurons at a too early stage,
00:14:51.02 but at the same time the progenitors are not sufficiently amplified.
00:14:54.15 And we believe that this is the reason for why those organoids are so much smaller.
00:14:59.13 So, premature neurogenesis and incomplete progenitor amplification are correlated with
00:15:05.18 the appearance of a microcephaly phenotype.
00:15:07.18 So, how can we explain this?
00:15:10.14 For this, we performed whole exome sequencing on the patient and we found that the patient
00:15:16.01 carries a compound heterozygous mutation in a gene that is called CDK5Rap2.
00:15:23.23 Both of these mutations introduce premature stop codons and, consistent with this, we
00:15:28.13 do not detect the CDK5Rap2 protein in the patient-derived cells.
00:15:33.19 So, what is CDK5Rap2?
00:15:36.19 CDK5Rap2 is a protein that originally has been identified in the fruit fly Drosophila,
00:15:41.09 where it was called centrosomin.
00:15:43.01 It's a centrosomal protein that is absolutely required for the correct orientation of the
00:15:48.15 mitotic spindle.
00:15:49.15 And, consistent with this, while in the control organoids, at an early stage, all the mitotic
00:15:56.01 spindles are precisely aligned with the ventricular surface, in the patient-derived organoids
00:16:01.01 they assume a more or less random orientation.
00:16:04.07 And so we believe that, during the early stages of cortical development, the orientation of
00:16:09.04 the mitotic spindle is very important to ensure the equal distribution of the apical and basal
00:16:15.23 lateral plasma membrane domains, and to make sure that both daughter cells can maintain
00:16:20.24 the progenitor fate.
00:16:23.09 In the patient-derived organoids, however, a tilt of the mitotic spindle no longer allows
00:16:28.12 the symmetric inheritance, so that some cells start undergoing neuronal differentiation
00:16:33.01 and only a few maintain the progenitor status.
00:16:37.04 So, a defect in spindle orientation, leading to a lineage defect, might be responsible
00:16:44.00 for the microcephaly phenotype.
00:16:46.11 So, this is where a normal human genetics analysis would stop we've recapitulated the
00:16:51.21 disease, we've found a plausible gene, and a plausible mechanism.
00:16:56.15 But, now, genome editing tools allow us to unambiguously ask, was it really this mutation
00:17:04.04 that was responsible for the disease phenotype?
00:17:06.13 And for this, we used genome editing to repair one of the two premature stop codons.
00:17:12.13 This is a control organoid, a patient-derived organoid, and an organoid that's derived from
00:17:18.17 cells where we have repaired one of the two mutations.
00:17:22.15 And what you can see is that both the size defect, as well as the premature neuronal
00:17:27.08 differentiation, can be rescued.
00:17:30.00 So, cerebral organoids can be used to recapitulate neurodevelopmental disorders.
00:17:35.20 And they can also be used to unambiguously associate
00:17:39.20 particular mutations with those disorders.
00:17:43.18 Now, microcephaly is a very severe disorder and, ultimately, we would like to recapitulate
00:17:51.19 more subtle disorders like epilepsy or autism.
00:17:56.16 And the holy grail of neurodevelopmental disease modeling is a type of neurons that have a
00:18:03.05 very complicated developmental origin, and these are the GABAergic interneurons.
00:18:11.05 This is a schematic view of the developing cortex.
00:18:15.03 The cortex develops from the dorsal neuroepithelium and all the neurons that arise in the dorsal
00:18:22.21 neuroepithelium express an excitatory neurotransmitter, glutamate, and become excitatory neurons.
00:18:30.06 In addition to those neurons, there are inhibitory GABAergic interneurons, which arise from the
00:18:36.07 ventral part of the developing cortex.
00:18:38.21 They then migrate tangentially to go into the dorsal areas and integrate into the circuits
00:18:44.21 of the dorsal cortex.
00:18:46.08 And it is thought that migrating interneurons is what actually is absolutely essential for
00:18:55.20 creating functional neuronal circuits.
00:18:58.22 And the types of mutations that are associated with diseases like epilepsy or autism suggests
00:19:06.07 that defects in interneuron formation and migration
00:19:10.06 may be associated with many of these diseases.
00:19:13.13 And so we asked, can we actually model this very complex developmental event in our
00:19:20.04 cerebral organoids?
00:19:21.15 The first thing we wanted to know is, do we actually have ventral and dorsal cortex in
00:19:27.00 the organoids?
00:19:28.05 This is shown here.
00:19:30.07 The dorsal cortex can be identified, because the neurons and the intermediate progenitors
00:19:35.19 express a gene that is called Tbr2, whereas the ventral cortex, the lateral ganglionic eminence,
00:19:41.15 in this case, can be identified by the expression of Gsx2.
00:19:45.03 When we section an organoid, we find, typically, that the organoids contain both Tbr2-positive
00:19:52.11 dorsal regions and Gsx2-positive ventral regions.
00:19:56.20 So, both ventral and dorsal areas are actually present in our cerebral organoids.
00:20:01.08 So, can we use them to model interneuron migration?
00:20:05.05 This slide shows you a dorsal cortical area where the intermediate progenitors express
00:20:10.13 the dorsal marker Tbr2, and when we stain the adjacent section with a vGad, an interneuron
00:20:18.06 marker, and somatostatin, a marker of particular interneuron subtypes, we find that interneurons
00:20:24.05 are actually present in the dorsal cortical areas of the organoid.
00:20:30.05 So, interneuron migration from the ventral into the dorsal part can actually be modeled
00:20:35.21 in cerebral organoids.
00:20:37.13 But there's one key problem with this.
00:20:39.20 And this is illustrated here.
00:20:41.20 In the current protocol, organoids are a little bit like a car where the wheels are up, the
00:20:46.22 engine is up, the seat is in the back, the windshield is in the front. so, all the
00:20:51.05 individual parts are present, but they are in a random arrangement.
00:20:54.17 And so, if we make a section through such an organoid, we have to be very lucky to hit
00:21:00.00 both a dorsal and a ventral part, and to be able to model interneuron migration.
00:21:07.05 And so, we asked, can we actually introduce a polarity axis in organoids, for example,
00:21:14.03 a dorsal-ventral axis?
00:21:16.01 And so, in order to do this, Josh Bagley, a postdoc in my lab, set out to develop a
00:21:21.08 protocol where he separately generates dorsal organoids and ventral organoids.
00:21:28.06 And then he places these two organoids together into one droplet of Matrigel.
00:21:36.03 One organoid is labeled with RFP expression another organoid is labeled with GXP.
00:21:41.00 GFP expression.
00:21:42.00 Initially, when the two organoids are co-embedded, they lie next to each other, but over time
00:21:48.10 they fuse with each other and, if I would not show you the red and green color, you
00:21:52.19 would not be able to distinguish the boundary between the two organoids.
00:21:57.05 So, we have developed a co-culture protocol that we can use to generate organoids where
00:22:03.02 one part is dorsal and the other part is ventral.
00:22:06.19 So, can we use this to model interneuron migration?
00:22:12.04 This slide shows you a typical dorsal-ventral fusion organoid, and what you can see is that,
00:22:17.16 within the dorsal area, you see these green spots.
00:22:22.05 When we focus in on this, we find that these green areas are actually full of cells that
00:22:28.10 have the migratory appearance of interneurons.
00:22:31.21 When we fuse dorsal with dorsal organoids, we no longer see this.
00:22:36.15 And so, from this experiment and many others that I don't have the time to show you, we
00:22:40.19 conclude that, when we fuse ventral with dorsal organoids, there are cells that are migrating
00:22:47.04 from the ventral into the dorsal part of the organoid.
00:22:50.23 So, what are those cells?
00:22:53.07 This slide actually summarizes a very large number of experiments and I only show you
00:22:57.20 the key experiments.
00:22:59.12 Here, I show you that the migratory cells express GFP and all of them also express GAD1,
00:23:08.02 which is a marker for developing interneurons.
00:23:11.24 This is a high magnification view and you can actually see the elaborate cell shapes
00:23:17.01 that these develop. these migrating interneurons actually have.
00:23:20.15 We also use many other markers of migrating interneurons and various interneuron subtypes,
00:23:27.11 like parvalbumin, somatostatin, neuropeptide Y and also calbindin and calretinin, and we
00:23:34.05 find all of these subtypes in our migrating fused organoids.
00:23:39.12 So, taken together, this tells us that we can use fused organoids, where we have regenerated
00:23:46.12 a dorsal-ventral axis, to model the long-range interactions of various parts of the human
00:23:52.22 brain, in particular the migration of cells from one part of the brain into the other.
00:23:59.19 Now, of course, ideally, we would like to image this process of interneuron migration
00:24:05.07 in real time.
00:24:06.11 And, in order to do so, Josh developed a method that we can use to do long-term imaging of
00:24:13.01 migrating interneurons and this is shown here.
00:24:15.20 For this, Josh makes very thick slices from fused organoids and those slice. slices
00:24:23.17 are then imaged on a spinning disk time-lapse microscope for about three days.
00:24:29.07 This is a still of one of these migratory movies and what you can see is that we can
00:24:34.24 actually see migrating cells that go from one part of the organoid into the other.
00:24:41.17 This slide shows you a movie of migrating interneuron.
00:24:46.19 The interneuron can be seen here here is the cell body and here are two processes.
00:24:52.15 I would like you to focus on this one cell and you will see that it migrates across the
00:24:57.09 entire field of the movie.
00:25:00.10 And interneuron migration is one of the most complex but, in my view, also both beautiful
00:25:06.00 processes that occur during development.
00:25:09.03 You can see the cell extends many processes, then some of them are retracted, and it migrates
00:25:14.11 along other processes.
00:25:17.10 The second movie shows you a more realistic view.
00:25:20.23 It shows you how crowded the field is.
00:25:23.06 There are many different migrating interneurons.
00:25:27.11 I want you to focus on this one.
00:25:31.00 And you will be able to see that this cell actually migrates across the entire field
00:25:35.20 of the image field.
00:25:38.01 So, here, you can see that the cell extends different processes, some of the processes
00:25:44.05 are then retracted.
00:25:45.15 It makes decisions to actually follow processes which initially are weaker.
00:25:52.12 And so, with this, we can now use organoids to model long-range interneuron migration,
00:26:00.22 and we're in the process of deriving iPS cells from patients that actually suffer from neurodevelopmental
00:26:07.06 disorders to see whether interneuron migration is affected.
00:26:10.22 Now, it is known that the migration of interneurons is in response to a chemokine that is called
00:26:21.01 CXCL12.
00:26:23.18 And the receptor for this chemo. for this chemokine is CXCR4.
00:26:29.03 And there is a drug called AMD3100, or Plerixafor, which actually is an inhibitor of
00:26:36.20 this chemokine receptor.
00:26:38.06 And so we asked, is interneuron migration that we actually see in our cultures dependent
00:26:44.13 on this receptor?
00:26:46.00 This is a normal organoid, where you can see a lot of interneurons migrating from one into
00:26:50.23 the other part, and this is an organoid that has been cultured in the presence of this
00:26:55.12 CXCR4 inhibitor.
00:26:57.18 And what you can see is that the migration of the interneurons is
00:27:01.19 actually almost completely suppressed.
00:27:04.08 I want you to note that the growth of axons from one part of the organoid into the other
00:27:09.08 is actually not affected, indicating that the other part of the organoid is actually
00:27:14.00 still alive.
00:27:15.04 So, we can use fused organoids to model interneuron migration and also to test chemical compounds
00:27:22.17 for their effect on interneuron migration.
00:27:25.14 And we are currently generating various models for neurodevelopmental disorders that we can
00:27:33.16 use to then test whether interneuron migration is affected.
00:27:38.24 So, in the end, I would like to summarize what I've told you.
00:27:42.24 I've told you that we've generated a three-dimensional culture system that we can use to recapitulate
00:27:49.20 early human brain development in culture.
00:27:52.19 Our cerebral organoids generate various parts of the human brain that can actually functionally
00:28:00.07 interact with each other.
00:28:01.12 We can use the organoids to model neurodevelopmental disorders and we can generate various parts
00:28:08.10 of the human brain separately in culture, then fuse them together and the interactions
00:28:14.05 between these various parts are maintained.
00:28:16.23 And, particularly, the last part of this of course has huge potential.
00:28:22.02 We will be able to generate any pairs of brain regions and watch the migration of cells from
00:28:30.01 one into the other, or a migration of axons, and hopefully eventually even be able to visualize
00:28:36.08 the formation of some of the major axon tracts.
00:28:39.15 In the end, I would like to acknowledge people who actually contributed to this work.
00:28:43.22 The hero of the organoids is Madeline Lancaster.
00:28:47.12 She was a postdoc in my lab, now has her own lab at the LMB in Cambridge.
00:28:53.01 For looking at dorsal-ventral patterning, she worked together with a grad student,
00:28:56.04 Magdalena Renner.
00:28:57.23 The fusion organoid system was generated by Josh Bagley, another postdoc in my lab.
00:29:03.07 These are the names of other people who work together in these various projects.
00:29:08.09 The reprogramming of the cells was a collaboration with the lab of Joseph Penninger.
00:29:12.07 I would particularly like to thank the patient and their family for allow. for allowing
00:29:16.11 us to use the cells in our experiments.
00:29:21.21 And our collaborator at the University of Edinburgh is Andrew Jackson.
00:29:25.21 I would like to thank the Austrian Academy of Sciences for funding our work.
00:29:30.17 In addition, we obtained funding from the European Research Council, the Austrian Science
00:29:35.16 Fund, and from EMBO.
00:29:36.24 And I would like to thank my entire lab for being such a fantastic group.

  • Part 1: Asymmetric Cell Division From Drosophila to Humans

Mechanisms and functions of endocytosis

A recent EMBO-FEBS workshop entitled Endocytic Systems: Mechanism and Function, organized by Howard Riezman in Villars-sur-Ollon (Switzerland), showcased the multifaceted approaches and model systems used to study endocytosis. The meeting revealed how endocytosis controls multiple aspects of biology, ranging from development to immunity and neurotransmission.

Mechanisms of clathrin-dependent endocytosis

Although clathrin-mediated internalization has been investigated for many years, new technologies keep providing us with novel insights into its underlying mechanisms, with an unprecedented scale down to molecular details regarding the structure and dynamics of the proteins involved. The plasticity of clathrin-mediated internalization was well illustrated by the fact that this pathway is used not only to traffic extracellular molecules or cellular proteins, but also to mediate entry of certain toxins, viruses, and bacteria.

The issue of lifetimes of clathrin-coated pits (CCPs) and vesicles (CCVs) has remained controversial, as values reported in the literature range from seconds to minutes. Sandra Schmid (The Scripps Research Institute) described quantitative computational analyses to track the dynamics of CCP/CCV formation on the plasma membrane. In this way, three kinetically distinct populations of CCPs could be distinguished, two short-lived (early- and late-abortive with lifetimes in the range of seconds) and one long-lived productive population stable for over one minute. Interestingly, cargo appears to increase a number of productive, long-lived CCPs/CCVs without affecting their lifetimes, which can in turn be regulated by the activity of dynamin.

Morphological heterogeneity of CCVs was emphasized by Tomas Kirchhausen (Harvard Medical School). Cryo-electron tomography of individual CCVs revealed a broad range of patterns used to organize a clathrin lattice, with asymmetrically located membrane vesicles buried inside the shell (Cheng et al., 2007). Moreover, high-resolution imaging of live cells based on total internal reflection fluorescence technology indicates that AP-2 adaptor proteins are also localized nonsymmetrically within an individual CCV. This may result from an initially restricted localization of adaptors, as they are captured during the nucleation and early phases of coated pit assembly, while retaining the adaptors concentrated at the place of their original recruitment at the time of vesicle pinching and CCV formation.

Clathrin-mediated endocytosis serves some specialized functions in various tissues, including the nervous system. Knockout (KO) studies in mice, reported by Pietro De Camilli (Yale University School of Medicine), demonstrated that dynamin-1 appeared not to be essential for the biogenesis and endocytic recycling of synaptic vesicles (Ferguson et al., 2007), although studies of dynamin mutants in cultured cells would have predicted a crucial role for this protein in vivo. The role of dynamin-1 in synaptic vesicle endocytosis is activity dependent and becomes evident during strong stimulation of neurons. The morphology of KO nerve terminals was visualized by EM tomography followed by tridimensional reconstruction. Such synapses are filled with clusters of clathrin-coat components, forming tubular networks capped by clathrin-coated pits that open to the plasma membrane.

Role of actin in clathrin-dependent endocytosis

Because of the ease of genetic manipulations, the yeast Saccharomyces cerevisiae has been very useful for dissecting the molecular machineries of endocytosis. Genetic studies have revealed an essential role for actin in endocytosis in yeast, and a key question concerns how actin functions together with clathrin in endocytosis. Using real-time image analysis of yeast cells expressing fluorescently tagged versions of more than 40 endocytic proteins, David Drubin (University of California, Berkeley) has analyzed the dynamic appearance, movement, and disappearance of these proteins at endocytic sites. Drubin presented data indicating that these proteins can be grouped into four functional modules that mediate coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. Maria-Isabel Geli (Instituto de Biología Molecular de Barcelona) described an in vitro assay to reconstitute the complex actin structures that participate in the formation of endocytic profiles and the use of immuno-electron microscopy to define the primary endocytic profiles in yeast and the localization of the actin machinery.

Given the importance of actin and clathrin in endocytosis, proteins that link actin and clathrin functions are of special interest. Genetic studies in yeast have indicated that clathrin light chain may regulate the ability of Sla2 to control actin dynamics in endocytosis (Newpher et al., 2006). Frances Brodsky (University of California, San Francisco) described a study of Hip1 and Hip1R, the mammalian homologues of Sla2, which have overlapping but not identical functions in endocytosis. Brodsky presented evidence that Hip proteins interact sequentially with clathrin and actin rather than functioning as bridges between the two.

Clathrin-dependent endocytosis and pathogen entry

Certain toxins and pathogens harness clathrin-mediated internalization to enter cells. Endocytosis of anthrax toxin, described by Gisou van der Goot (Federal Polytechnic School of Lausanne), is clathrin- and dynamin-mediated but requires also the presence of lipid rafts, a classical hallmark of clathrin-independent entry routes. The protective antigen (PA) subunit of the toxin binds to cell surface receptors (TEM8 and CMG2) and induces their multiple post-translational modifications, such as palmitoylation, phosphorylation, and ubiquitination, which differentially regulate toxin internalization (Abrami et al., 2006). Yet another new player implicated in this process appears to be the Wnt coreceptor LRP6, which interacts with TEM8 and CMG2 and its depletion results in reduced toxin uptake. Anthrax toxin thus provides an interesting example of using complex intracellular endocytic and signaling mechanisms for precise regulation of its internalization in time and space.

Semliki forest virus (SFV) was one of the first viral pathogens identified to exploit clathrin-dependent internalization mode, as recalled by Ari Helenius (ETH Zurich) in his plenary lecture, along with a number of viruses using other pathways (see Fig. 1). Further viruses, such as species C adenovirus type 2 (Ad2) and Ad5 reported by Urs Greber (UZH Zurich), enter cells via clathrin- and dynamin-dependent mechanisms but escape the classical Rab5-Rab7-EEA1-Hrs pathway from early to late endosomes and are redirected to trans-Golgi compartments in an Arf1-dependent process.

However, the clathrin-mediated pathway appears to be exploited not only by viruses, but also by much larger bacterial pathogens such as Listeria monocytogenes, as reported by Pascale Cossart (Pasteur Institute of Paris). In a process induced by the bacterial surface protein InlB, clathrin and auxilin are recruited around the entering bacteria, followed by actin polymerization. Nevertheless, despite certain similarities and common players involved (dynamin, Eps15, and the E3 ubiquitin ligase Cbl), this process appears mechanistically and kinetically different from the canonical clathrin-mediated internalization of macromolecules. This in turn argues that the networks of protein–protein and protein–lipid interactions involved in clathrin-mediated internalization can assemble in various combinations. This plasticity may be exploited not only by pathogens, but also under physiological conditions by different types of cells or tissues with particular needs for specialized forms of internalization.

Clathrin-independent endocytosis pathways

Once obscure, clathrin-independent internalization routes are a focus of intense research revealing the molecular players involved and cargos entering via these mechanisms. This allows us now to redefine these pathways more precisely and in positive terms, in contrast to their initial collective description as “non-clathrin endocytosis”. Some classification schemes have been already proposed (Mayor and Pagano, 2007), based primarily on the dependence on dynamin and various small GTPases however, the exact number of clathrin-independent pathways and their mutual relations remain unclear.

Satyajit Mayor (National Centre for Biological Sciences, Bangalore) reported progress toward further characterization of a clathrin-, dynamin-, and caveolae-independent internalization route that transports GPI-anchored proteins (GPI-APs) and involves GEECs (GPI-AP–enriched early endosomal compartments) as intermediates. This constitutive pinocytic pathway is initiated by a cholesterol-dependent recruitment and stabilization of active Cdc42 on the plasma membrane, which leads to localized actin polymerization (Chadda et al., 2007). Interestingly, the activity of Cdc42 is controlled by an Arf1-dependent recruitment of RhoGAP, such as ARHGAP10. This mechanism represents an interesting example of a cross-talk between different small GTPases of the Ras superfamily in regulation of endocytosis.

Flotillins appear to be essential structural components of the internalization pathway independent of clathrin and caveolae, as reported by Ben Nichols (MRC Laboratory of Molecular Biology). Flotillin-1 and -2 coassemble on the plasma membrane into microdomains, which are laterally mobile, distinct from CCPs or caveolae but bearing certain features of lipid rafts (Frick et al., 2007). Electron microscopy and live-cell imaging demonstrated that these microdomains can induce membrane invaginations and eventually bud off from the plasma membrane in a process stimulated by overexpression of flotillins. The resulting primary endocytic structures contain cholera toxin B subunit, but not transferrin. They appear morphologically different to GEECs, although patched GPI-APs partly colocalize with flotillins.

Shiga toxin B-subunit (STxB) uses clathrin-dependent and -independent modes of internalization. Ludger Johannes (Institut Curie) reported that STxB can induce tubular invaginations, possibly acting as endocytic internalization intermediates. Their formation does not require clathrin, actin, dynamin, or caveolins, and is also observed on energy-depleted cells. Interestingly, the STxB-induced tubulation could be reproduced in vitro on giant unilamellar vesicles in the absence of cytosolic protein machinery. While the exact mechanisms underlying this phenomenon await further characterization, the findings indicate that some endocytic internalization events can be mediated by protein-induced rearrangements of a lipid bilayer as a driving force of membrane deformation.

Macropinocytosis represents clathrin-independent internalization of vast areas of plasma membrane and large volumes of extracellular fluid that are enclosed in macropinosomes, big (>1 μm) vacuolar structures. Such voluminous membrane rearrangements involve actin ruffles and are driven by Rac1 and Src kinases. Fission of nascent macropinosomes does not involve dynamin, but no alternative mechanisms have been proposed. As reported by Prisca Liberali (Consorzio Mario Negri Sud), CtBP1/BARS, a protein controlling membrane fission in other transport steps, assumes this role also in macropinocytosis. CtBP1/BARS is locally recruited to the closure site of a macropinocytic cup where it is phosphorylated by p21-activated kinase-1 (Pak1). This is a key event in the fission process in which CtBP1/BARS acts as a scaffold in a larger complex coupling membrane rearrangements with cytoskeletal machinery.

The morphological features and the molecular players involved in the entry of vaccinia virus make it a new addition to the list of cargo internalized via macropinocytosis, as presented by Ari Helenius (ETH Zurich). Besides exploiting features of regular macropinocytosis (dependence on actin, Rho GTPases, cholesterol, and Pak1), the virus uses additional specialized mechanisms, involving formation of transient blebs on the plasma membrane induced upon virus binding and preceding its internalization. This surprising effect was tracked down to the presence of phosphatidylserine (PS) on the viral particle, as a key factor for successful infection. PS externalization is a known hallmark of apoptosis, resulting in extensive plasma membrane blebbing. Thus, this “apoptotic mimicry” used by the virus causes a transient stimulation of membrane rearrangements to ensure an efficient entry.

Even within one family of viruses, different species can use various internalization routes. In contrast to its relatives entering via clathrin-dependent mechanisms (see above), the species B adenovirus type 3 (Ad3) described by Urs Greber (UZH Zurich) induces macropinocytosis to mediate its own entry. This process requires cell surface receptor CD46 and αv integrins, in addition to other proteins regulating macropinocytosis, including CtBP1/BARS.

Although phagocytosis represents a very distinct and specialized class of endocytic internalization, it shares certain mechanisms and machinery with other pathways. Sergio Grinstein (Hospital for Sick Children) reported that lipid remodeling and the resulting changes in membrane surface charge can regulate protein recruitment in phagocytosis, a mechanism that likely could be extended to endocytosis in general. Novel genetic probes revealed a decrease of surface potential upon sealing of the phagocytic cup, due mainly to hydrolysis of phosphoinositides (Yeung et al., 2006). This affects binding of several signaling and regulatory molecules (e.g., K-Ras, Rac1, c-Src) recruited to the plasma membrane via electrostatic interactions. Internal endocytic compartments have lower negative surface charge than the plasma membrane, so depending on the strength of the cationic targeting sequences, proteins can be differentially recruited to the plasma membrane or endocytic compartments, whereas proteins lacking such motifs localize increasingly to the endoplasmic reticulum.

Systems biology approaches to study endocytosis

Current technologies enable global analyses of physiological pathways, and a few systems biology approaches to study endocytosis in various organisms were presented. Genome-wide analysis of endocytic recycling in S. cerevisiae was reported by Liz Conibear (University of British Columbia). The screen involved measuring an increased or decreased presence of the v-SNARE Snc1p on the plasma membrane, thus identifying both exocytic and endocytic defects among the mutant collection encompassing deletions of all nonessential genes. Genetic interaction analysis of the top hits established a network of gene clusters involved in various intracellular processes contributing to Snc1p trafficking, containing both known and novel components.

Endocytic internalization of transferrin (destined for recycling) and epidermal growth factor (EGF directed for degradation) in mammalian cells has been a target process for a multiparameter, genome-wide RNAi screen undertaken by Marino Zerial (Max Planck Institute of Molecular Cell Biology and Genetics) and colleagues. Given the high frequency of off-target effects by commercially available siRNA libraries, the screen has been performed using at least seven independent siRNA oligonucleotides and a mixture of endoribonuclease-prepared siRNAs (esiRNA) per gene, making this an effort of unprecedented scale. The initial analysis of the screen results included over 60 highly quantitative parameters describing the morphology, intracellular distribution, and cargo content of transferrin- and EGF-bearing endosomes. In addition to the identification of novel genes regulating endocytosis, the study revealed interesting correlations and general principles pertaining to the organization of the endocytic pathway in mammalian cells.

Another effort based also on siRNA high-throughput screening technology was described by Lucas Pelkmans (ETH Zurich) to determine infectomes, and reveal detailed infection pathways for 15 mammalian viruses. This approach allows for identification of critical host genes and grouping together viruses using similar intracellular components for establishing infection. However, the efficiency of viral infection depends strongly on several parameters such as local cell density, cell and colony size, or number of cells. These factors, describing cell population properties of infection, are also quantitatively assessed as a part of a tri-dimensional dataset along with the corresponding viruses and siRNA phenotypes. Secondary screens for endocytosis of various cargo types will complement the infection screens in order to pinpoint the common machinery involved.

Presenting a genome-wide RNAi screen for endocytic regulators in Caenorhabditis elegans, Barth Grant (Rutgers University) identified 168 candidate genes. Surprisingly, a group of proteins known for their role in embryonic and epithelial polarity, PAR-3, PAR-6, PKC-3, and CDC-42, were identified in this screen. A closer analysis revealed that ablation of these proteins disrupted the morphology and function of recycling endosomes. Consistent with these data, CDC-42 and its mammalian homologue Cdc42 were found on recycling endosomes in C. elegans and mammalian cells. One possible function of the polarity proteins may be, together with actin, to mediate scission of vesicles or tubular elements from the recycling endosome. These findings are consistent with the view that membrane trafficking, and in particular endocytic recycling, may contribute to epithelial polarity (Balklava et al., 2007).

Trafficking to lysosomes

Many endocytosed membrane proteins, including receptors for growth factors, cytokines and hormones, are transported to lysosomes for degradation. Conjugation with ubiquitin serves as a signal for this pathway, and endosomal ubiquitin-binding proteins have consequently been sought as components of the sorting machinery. Through genetic and biochemical studies of yeast mutants defective in vacuolar protein sorting, Scott Emr (Cornell Institute for Cell and Molecular Biology) has identified three endosomal sorting complexes required for transport, ESCRTs, that mediate degradative sorting of ubiquitinated membrane proteins (Saksena et al., 2007). Emr showed evidence that ESCRT-II may nucleate the formation of ESCRT-III multimers and suggested that some ESCRT-III subunits may function as capping proteins that prevent chain elongation. This controlled multimerization, reversed by Vps4, may drive the membrane rearrangements that underlie MVB biogenesis. In support of this hypothesis, Phyllis Hanson (Washington University, St. Louis) presented a poster showing that overexpressed ESCRT-III subunits assemble on membranes to form curved filaments that associate into circular arrays. The ESCRTs are evolutionarily conserved, and consistent with their role in lysosomal receptor down-regulation they function as tumor suppressors in Drosophila (Hariharan and Bilder, 2006). Harald Stenmark (University of Oslo) presented evidence that these complexes may also protect against neurotoxicity by facilitating autophagic degradation of toxic protein aggregates (Rusten et al., 2007).

Jean Gruenberg (University of Geneva) showed that back-fusion of intralumenal vesicles (ILVs) with the limiting membrane of the MVB is exploited by vesicular stomatitis virus (VSV), which enters MVBs through endocytosis and subsequent trafficking and releases its nucleocapsid to the cytosol when VSV-containing ILVs fuse with the limiting MVB membrane. This appears to happen in late MVBs and depends on the late endosome–specific lipid LBPA and its effector protein Alix. Gruenberg presented an in vitro assay to study back-fusion of ILVs and showed evidence that ESCRT-I is required for back-fusion, and Alix depletion stimulates this process. He proposed that Alix stabilizes a post-fusion, pre-fission intermediate between ILVs and the limiting MVB membrane, whereas ESCRT-I controls ILV biogenesis directly.

Even though the canonical function of lysosomes is in degradation of endocytosed and autophagocytosed material, these organelles do have additional functions. Norma Andrews (Yale University School of Medicine) explained that lysosomes are the major vesicle type responsible for calcium-dependent exocytosis in nonsecretory cells to re-seal the plasma membrane after injury. Other cell types contain specialized lysosomes that fuse with the plasma membrane independently of cell damage. Such secretory lysosomes are exemplified by granules in cytotoxic T lymphocytes (CTLs), which contain enzymes that kill target cells, as presented by Gillian Griffiths (University of Cambridge).

Endocytosis in signaling and development

In addition to its well-described roles in nutrient uptake and receptor down-regulation, endocytosis plays a direct role in the modulation of cell signaling responses (Miaczynska et al., 2004). Marcos Gonzalez-Gaitán (University of Geneva) presented studies on Smad anchor for receptor activation, SARA, which links Dpp (TGFβ) receptors with Smad signaling adaptor proteins and is a central component of the Dpp signaling pathway in Drosophila. SARA is located to a subset of endosomes that mediate cellular memory of Dpp signaling in wing imaginal discs by distributing evenly between the two daughter cells during cell division (Bokel et al., 2006). Using development of the sensory organ precursor (SOP) as a model for asymmetrical cell division, Gonzalez-Gaitán showed that SARA-containing endosomes accumulate at the central spindle during cell division and then partition asymmetrically into the signal-receiving daughter cell. Even though SARA is a mediator of Dpp signaling, the SARA-containing endosomes could well function as vehicles for other signaling pathways, and Gonzalez-Gaitán is currently investigating the possibility that asymmetrical Notch-Delta signaling, strongly implicated in binary fate decisions, could be mediated via SARA-containing endosomes. Another evidence that endocytosis regulates SOP development was provided by Roland Le Borgne (Université de Rennes), who proposed that transcytosis of Delta may be required for Notch-Delta signaling in the SOP.

Recent studies have shown that recycling endosomes contribute membrane to the advancing cleavage furrow during the cytokinesis phase of cell division (Strickland and Burgess, 2004). How endosomes are targeted specifically to the cleavage furrow and the midbody is still an open question. Philippe Chavrier (Institut Curie) presented evidence that a novel family of effectors of the endocytic small GTPase Arf6 are involved in the completion of cytokinesis via control of membrane delivery at the midbody through interactions with microtubule motors.

One interesting function of endocytosis in developing tissues is its involvement in cell migration, and transparent zebrafish embryos have been one of the favorite models for such studies. Carl-Philipp Heisenberg (Max Planck Institute of Molecular Cell Biology and Genetics) showed that mesoderm polarization and directed migration strongly depend on Rab5-dependent trafficking and cellubrevin-mediated recycling, indicating that the endocytic cycle is required for proper mesoderm cell migration. Endocytosis and recycling may be important for membrane rearrangements during cell migration, but could also play a role in signal sensing during migration. An interesting example of the latter was discussed by Erez Raz (University of Münster), who has been studying the involvement of the chemokine SDF-1a and its receptor CXCR4b in germ cell migration in zebrafish. The receptor is expressed on the surface of the primordial germ cells, which receive directional cues from somatic tissues that secrete SDF-1a. While wild-type germ cells put on the brakes as they approach their target tissue where the gonad develops, cells expressing a non-internalizable CXCR4b mutant fail to down-regulate signaling, which causes longer runs and less precise targeting (Minina et al., 2007).

Among clathrin-independent endocytic mechanisms, an internalization route involving caveolae has been investigated in much detail. Unlike constitutive clathrin-dependent internalization, caveolar endocytosis is inducible upon signaling triggers. As presented by Miguel Del Pozo (Universidad Complutense de Madrid), cell adhesion and integrin signaling play a crucial role in this process, with global implications for cell polarity, motility, and invasiveness.


In conclusion, significant progress has now been made regarding the mechanisms of endocytosis and post-endocytic trafficking, and their roles in cell signaling, development, and host–pathogen interactions. The recent introduction of systems biology approaches promises to provide a deeper understanding of how endocytosis works, and how it serves to orchestrate biological processes.


Motor neuron organization and the assembly of circuit for locomotion

The precise spatial organization of neurons in the nervous system is thought to be an important determinant of identity, connectivity and ultimately function. In the central nervous system (CNS), neurons are broadly arranged following two anatomical plans that involve laminar and nuclear organization, where distinct neuronal subtypes are often found in stereotyped positions that are predictive of their input-output connectivity patterns. A striking example of nuclear organization is apparent in the positioning of limb innervating motor neurons, which are grouped into discrete structures, termed motor pools, occupying conserved and stereotyped positions in the ventral spinal cord. Motor neurons grouped together not only share positional coordinates but also send projections to the same muscle targets in the periphery. Thus, precise spatial organization appears to represent a major strategy to simplify the problem of wiring the motor system. However, the molecular mechanisms controlling motor pool organization and its contribution to the assembly of spinal circuits are not fully understood. We address these problems using mouse genetics to generate models with scrambled motor neuron organization to study the mechanisms controlling precise positioning and then investigate the effects on circuit assembly and motor behavior.

The functional organization of spinal somato-sensory circuits

The ability to monitor changes in the environment and generate appropriate behavioral responses is critical for survival. A fundamental question in neuroscience regards the circuit logic that is used to detect a virtually unlimited amount of stimuli and encode them to create a coherent perception of the world. In particular, the somatosensory system is given the complicated task of encoding a wide variety of different information including pain, touch, itch, proprioception, interoception, and temperature sensation. Much progress has been made in the anatomical, physiological and genetic characterization of primary somatosensory neurons, leading to a quite extensive description of sensory neurons specialized in the detection of different stimuli. In comparison, little is know about how sensory modalities are encoded by spinal circuits to influence, locally, motor function and, in higher centers, our perception of somatic sensation. In particular, despite recent efforts in untangling the identity and function of interneurons participating to spinal somatosensory networks, circuit organization of sensory afferents encoding different modalities and their spinal targets are not clear. We seek to define, at anatomical and functional level, how distinct primary somatosensory neurons subtypes wire in the central nervous system to control processing of somatic information. In order to achieve this goal we combine mouse genetic, anatomical tracing and behavioral approaches to study spinal circuits controlling perception of somatic sensation and the generation of appropriate motor responses.

Development and function of spindle neurons - Biology

Our lab investigates central nervous system development in Drosophila. His lab is currently interested in (1) temporal identity programs used to generate an ordered series of neural progeny from a single progenitor, (2) how spatial patterning and temporal identity are integrated to generate heritable neuronal identity, (3) how neuronal progenitors change competence to respond to intrinsic and extrinsic cues over time, and (4) the developmental mechanisms driving neural circuit assembly, with a focus on larval locomotor circuits and adult central complex circuits.

The role of “temporal transcription factors” in generating an ordered series of neural subtypes

Neuronal diversity is generated in a stepwise fashion. In the first step, neuroectodermal spatial patterning cues (homeodomain proteins expressed in columns and rows) assign a unique identity to every neural progenitor (called neuroblasts in Drosophila). This results in 100 unique neuroblasts in each brain lobe, and 30 unique neuroblasts in each hemisegment of the ventral nerve cord (VNC). The second step of generating neuronal diversity is temporal patterning (the ordered production of different neural subtypes from a single progenitor). Although much is known about spatial patterning mechanisms, relatively little is known about temporal patterning mechanisms.

Embryonic neuroblasts sequentially express transcription factors (Hunchback, Krüppel, Pdm, Castor) that specify temporal identity of their neuronal progeny. Isshiki et al., Cell (2001).

There are five “temporal transcription factors” (TTFs) sequentially expressed in embryonic VNC neuroblasts: Hunchback (Ikaros class) > Krüppel (zinc finger class) > Pdm (Pou/homeodomain class) > Castor (Casz1 class) > Grainy head (CP2 class). Each of these factors is sequentially expressed by neuroblasts, and maintained in the neural progeny born during each expression window. Each temporal transcription factor is necessary to specify the neuronal identity produced during its expression window, and early TTFs can suppress the expression of later TTFs to generate ectopic early-born neurons (see Kohwi and Doe, 2014, Nature Reviews Neuroscience).

It is unknown if larval neuroblasts have a similar temporal transcription factor cascade to increase the diversity of neurons in the adult CNS. There are two types of larval neuroblasts: canonical type I neuroblasts bud off progeny called GMCs that differentiate into a pair of neurons, whereas type II neuroblasts produce transit-amplifying cells called intermediate neural progenitors (INPs) that themselves bud off

12 neural progeny. Thus, type II neuroblasts generate much larger, and possibly more complex, cell lineages than type I neuroblasts. Interestingly, type II neuroblasts produce most of the intrinsic neurons of the adult central complex (a brain region with beautiful laminar/columnar organization that is used for multimodal sensorimotor processing).

We are interested in identifying temporal transcription factors in larval type II lineages (both in neuroblasts and in the INPs). We have recently shown that INPs sequentially express Dichaete (Sox family) > Grainyhead > Eyeless (Pax6) transcription factors, and that these temporal transcription factors are required for the production of distinct neural subtypes. Moreover, young type II neuroblasts also transiently express at least three transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. Thus, neuroblast and INP temporal patterning axes act combinatorially to generate increased neural diversity within the adult brain (Bayraktar and Doe, 2013, Nature). How these two “axes of information” are integrated to generate the specific neurons of the adult central complex remains an open question.

Currently there are many experiments ongoing to identify and functionally characterize both neuroblast and INP transcriptional cascades. For example, we are using TU-tagging (Miller et al., 2009, Nature Methods) to identify novel TTFs during larval type II neuroblast lineages, and to determine their role in assembling the adult central complex. We are also investigating the embryonic origin of type II neuroblasts, to determine (a) if they form as type II neuroblasts “de novo” or by transition from a simpler type I neuroblast (b) if they use the Hunchback>Kruppel>Pdm>Cas>Grh cascade of TTFs during their embryonic lineages, and if these factors are maintained in INP lineages and (c) to identify the neurons produced by embryonic type II neuroblasts and determine if they are the pioneers of the adult central complex neuropil.

The integration of spatial and temporal patterning to generate heritable neuronal identity

Spatial patterning to specify neuroblast identity occurs in the neuroectoderm, just prior to neuroblast delamination and initiation of its cell lineage. Yet these transient spatial patterning cues somehow generate a heritable cell fate that is maintained by neuroblasts cultured in isolation or transplanted into a new spatial location within the embryo. It remains unknown how transient spatial cues lead to heritable neuroblast identity. Even more interesting, the spatial cues that specify unique neuroblast identity must be combined with the sequential expression of temporal transcription factors to generate lineage-specific cell fates. For example, NB7-1 is specified by the combination of Engrailed (row 6/7) and Ventral nervous system defective (Vnd row 1) the first temporal transcription factor Hunchback induces a motor neuron identity (U1) in this lineage. In contrast, NB7-2 has a different spatial code: Engrailed (row 6/7) and Intermediate nervous system defective (Ind row 2) and the first temporal transcription factor Hunchback induces an interneuron identity in this lineage. How do the spatial patterning genes shift the output of Hunchback from making a motor neuron in one lineage (NB7-1) and an interneuron in the adjacent lineage (NB7-2)? Or a serotonergic neuron in the next adjacent lineage (NB7-3)?

The question of how transient spatial patterning cues impart a heritable neuroblast identity, and how this neuroblast identity “flavors” the output of subsequent temporal transcription factors, are two of the most important open questions for understanding the generation of neuronal identity.

Larval type II neuroblasts undergo changes in gene expression that indicate temporal patterning, while their intermediate neural progenitors (INPs) sequentially express three transcription factors (Dichaete, Grainyhead, Eyeless). The combination of neuroblast and INP temporal identity axes increases the neural diversity generated by a single progenitor.

The development and function of locomotor circuits

Over the past 30 years we have learned a great deal about the specification and connectivity of motor neurons to their target body wall muscles. To determine how these motor neurons are used to generate larval locomotion it is essential to identify the interneurons in the locomotor circuits. Yet almost nothing is known about interneuron specification (just a few exemplar interneurons have been characterized, out of the

270 interneurons per hemisegment of the VNC), and even less is known about interneuron function in locomotor behavior.

Confocal image of an entire Drosophila larva stained using the Multicolor Flip Out (MCFO) method of Nern et al. (2015) PNAS 112:E2967. Note that staining the intact larva required development of a novel fixation method (Manning and Doe, unpublished) Image: Laurina Manning.

To initiate a comprehensive analysis of interneuron diversity, including their role in locomotion, we have identified several hundred Gal4 lines expressed in 1–5 interneurons (Manning et al., 2012, Cell Reports). We have used these lines in two ways. First, we have mapped them into a three-dimensional atlas that allows us to uniquely identify more than 50 percent of all interneurons in the ventral CNS (Heckscher et al., 2014, Development), and are currently linking each neuron to its developmental origin by adding lineage and TTF markers to the atlas. Second, we are using these lines to screen the interneurons for a role in larval behavior. We have expressed the warmth-activated TrpA1 channel in each “sparse interneuron Gal4 line” and found lines where neuronal activation leads to behavioral defects such as reverse locomotion, turning only, feeding only, left-right uncoordinated locomotion, pausing, and rigid paralysis (Matt Clark, submitted). We have investigated one phenotype (left-right uncoordination), showing that the affected interneurons are Even-skipped (Eve)+ contralateral ascending interneurons (that are conserved in mouse Evx1/2). We used thermogenetics and optogenetics to determine the function of these interneurons in larval locomotion and used a TEM (transmission electron microscopy) serial reconstruction of the entire larval CNS to identify pre- and postsynaptic partners to define a proprioceptive sensorimotor circuit (Heckscher et al., 2015, Neuron).

The characterization of interneurons in other phenotypic categories remains to be studied. In addition, we are using the circuits we identify as an entry point for testing hypotheses for how interneurons and motor neurons assemble locomotor circuits: common transcriptional programs, common birth order, or common lineage. Future directions include studying plasticity and compensation within these circuits, as well as their remodeling and participation in adult locomotor circuits.

Watch the video: What is a neuron? (June 2022).


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