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ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 ANAT 416 Lecture 11 – Eye Development Dr. Michel Cayouette – February 14, 2012 NOTE: This NTC is meant to be used as a study aid to supplement your own class notes. Hence, not all of the text contained in the lecture slides will be reproduced here. Please send any comments or questions about NTCs to us through e-mail: [email protected] Announcements: Happy Valentine’s Day! Lecture Outline Part I: Development of the Eye Part II: Development of the Retina Part III: Mechanisms of cell-fate specification in the retina Part I: Development of the Eye The Eye Develops from Embryonic Day 9.5 in Mouse The eye develops from about embryonic day 9.5 in the mouse and in humans it starts from the third week in gestation. It’s an early process in humans and is in mid-embryogenesis in mice. At day 9.5 can see bulge on the head and can recognize as the eye forming. From about E11.5 can see the eye clearly on the head of the mouse. The Eye Develops From a Series of Inductive Events Since the retina develops from the neural tube, it is part of the Central Nervous System (CNS). The neural tube develops from the neural plate. The neural plate is open at the beginning of mouse development and closes to form the neural tube which develops in the CNS. Anteriorly, there are two expansions in the neural tube: o Optic cup – budding of the neural tube o Lens placode - in front of the optic cut and forms the lens. The placode invaginates, pushing the optic cup inside so that it forms a two-layer structure: Anteriorly is the retina, while the retinal pigmented epithelium (RPE – shown in black in the diagram) is more posterior. These two structures come together and form the light-sensing part of the eye, which is the Figure 1: Formation of the Eye function of the retina. 1 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 o RPE – multiple roles: Trophic support: delivers nutrients to the retina. Structural support: removes oxidative damage caused when light hits the retina. Note, the cornea and lens (as well all other parts of the eye) are not derived from the neural tube and thus, are not derived from the CNS. Eye development Using Scanning Electron microscopy (to obtain a 3D perspective of image), you can look at a section through the eye of an embryo. Here, you can see budding of the neural tube and optic cup formation. Note the lens placode forms a thin layer on the neural epithelium, and then thickens. The cells in the periphery will form the developing lens. These cells invaginate, which pushes on the optic cup to form anterior and posterior portions. Therefore, the movement of cells and their distribution in a 3D environment lead to the formation of the optic cup, and other eye structures. Figure 2: Eye Development The adult eye The list on this slide is mostly for your own information. Important to know what the eye looks like. The retina is the only light sensitive area of the eye. Its main role is to transfer light to the CNS. Figure 3: Adult Eye Also have cornea, lens, iris, etc. Part II: Development of the Retina The Retinal Neuroepithelium contains Retinal Progenitor Cells (RPCs) that Divide to Generate Different Retinal Cells The RPCs are also known as the retinal progenitor cells as they give rise to the retina. These cells proliferate and differentiate into different types of neurons and different retinal cell types. The retinal cells in human and mammals do not continue to proliferate throughout life, which is why we can’t regenerate photoreceptors in certain diseases, and thus, should not really be called stem cells. In vivo live imaging of dividing retinal progenitor cells Two videos in zebrafish: you can see these cells divide and expand dramatically in the first few hours of retinal development. Zebrafish are good model organisms for studying eye development because they are transparent and can be immobilized in an agarose gel to observe retinal development in real time. Looked at retinal progenitor cells dividing (labeled with green fluorescent protein, or GFP) Arrows indicate mitotic cells at the apical surface. 2 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 The retinal neuroepithelium contains retinal progenitor cells that divide to generate different retinal cells – part II Usually when look at other epithelia, the apical surface faces the lumen. o Example, in the brain, the apical surface of ventricles faces the lumen. However, in the eye, the apical surface faces the outside. The lumen is the neural tube. After the invagination event, the apical surface will be closely opposed to another apical surface – so two apical surfaces are facing each other. When look at eye, may be tempted to say the apical surface of the retina is facing the lumen so its inside the eye, but it’s not due to folding of the retina. Development of the rodent retina spans a period of two weeks Development of the mouse retina takes about 2 weeks from embryonic day (E) 9.5-10 to postnatal day (P) 7. If focus on retina, and look at E18, see one layer of neuroblast progenitor cells, NBL, (another term for progenitor cells) and a retinal ganglion cell layer. These are neurons. If look at later stages, such as P4, you can see 4 distinct stages: o ONL – outer nuclear layer o Thick layer in middle NBL – neuroblast layer INL – inner nuclear layer o GCL – ganglion cell layer By P7, have final structure of the retina, the ONL, INL, and GCL. Basic organization of the vertebrate retina There are three distinct layers in the retina The ONL contains the cell bodies of the photoreceptors, the rods and the cones. o In this layer, light is transformed into a nervous impulse. o Photoreceptors contain photo pigments: Rods – rhodopsin Cones - opsin o Rods are useful in night vision, as they are very sensitive, but not as good at differentiating details. o Cones are useful in daylight and fine precise vision – looking at colors, reading, etc. The interneuron cells (horizontal, amacrine, bipolar cells) Figure 4: Vertebrate Retina integrate the signals received by the photoreceptors, for example, is it high or low contrast? Is it textured or smooth? o This layer also contains the cell bodies of Muller glia, which are the main cell type of the retina (spanning the entire length). These cells provide structural and trophic support. The retinal ganglion cell (RGC) layer contains RGCs which are the only projection cells of the retina. These neurons send an axon out of the eye to the brain. The axons of multiple RGCs form the optic nerve, which projects to the lateral geniculate nucleus (in humans), and then on to higher visual centers. This happens very quickly. The retina is used as a model system to study nervous system development o ie, when we ask the question: how are different cell types generated from a pool of stem cell or neural progenitor cells? 3 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 o Why is it a good model? It is a simple structure with three distinct layers There is a manageable number of cell types (only 7) Versus cortical progenitors with hundreds of different cell types In addition, we have cell-type specific markers for each of these 7 cell types Immunohistochemistry – technique which uses antibodies to identify proteins expressed in these cells. For example, only rods contain rhodopsin, so we could detect the presence of rhodopsin with an antibody conjugated to a fluorescent molecule. The retina easy to access, as opposed to the brain (would need to drill into the skull, etc). There are two eyes, so one can be used as a control. Example of histological staining: o In the retinal section, see photoreceptor cells stained with protein specific to photoreceptor cells. Also see bipolar and amacrine cells stained with different, cell typespecific marker. Part III: Mechanisms of Cell-Fate Specification How are the different retinal cell types generated? Two different models to do this: o Model one: one progenitor for each cell type o Model two: Population of progenitors that are all similar, but are multipotent and can generate all the different cell types of the retina. How to distinguish between these two different possibilities? o A question still asked for many parts of the developing embryo, too, as we don’t understand how the different cell types of organs are generated. Do they generate from specific Figure 5: Models of retinal cell progenitor cells that make one cell type only, or, from a multipotent progenitor cell? o The retina serves as a model to understand these progenitor cell specifications. o One way we could distinguish between these two models is by labeling the different cell types… Cell lineage studies – Label individual RPCs and analyze their progeny If there are 7 different progenitor cells each generating one cell type, you would expect one cell only yielding progeny. If you label a “grandparent” cell, you can follow whether it differentiates into neural or glial cells. Question: Are the differentiated daughter cells the same type or do they differ and contain combinations of different cell types? Figure 6: Cell lineage model 4 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 Using retroviral vectors for cell lineage tracing Tool to infect only dividing cells, or diving retinal cells. A normal retrovirus would get into the genome and manipulate the host machinery of a cell. You don’t want this virus to be able to spread. People have modified the retrovirus genome to make it replication incompetent. It can still infect the cell, but once it proliferates, it cannot cause cell lysis. The retroviral vectors we use also integrate the host cell’s genome. When a virus enters the cell, it manipulates the host machinery to transcribe their RNA with reverse transcriptase. Using long term repeat sequences, the genome of the retrovirus will integrate into the genome of the host cell. When the host cell divides, it will carry the genome of the retrovirus. If you can manipulate the vector to carry a reporter gene (PLAP, GFP, etc), it will also be replicated. Then, you can use histochemical procedures to indicate which gene has been infected. For example, if you infect the progenitor cell with a retroviral vector and let the retina develop for a couple of weeks (as long as it takes to generate the different cell types) and then get clones. These clones come from a single progenitor cell, that was infected, divided and generated into different cell types. Right away can see heterogeneity in the position of these cones, suggesting the progenitor cell is multipotent and can generate different cell types. If one progenitor generated one cell type, would only see one cell type in each cone (not the case). These retroviral vectors must be diluted so we only infect a few cells so they will be sparsely located in the retina (so when there is proliferation and differentiation into a retinal cell, we know whether it is a clone). The chance of finding two cells next to each other that were both infected is pretty rare. Retinal progenitor cells are multipotent Using the same vector as above, if the cell was infected at an early stage (E14), large clones of many different cell types are generated. Using the same vector as above, if the cell was infected at a late stage (P2), small clones of fewer cell types were generated. This suggests that at the late stage, progenitor cells have fewer options and they are unable to generate the early born fates. When it reaches the end of retinal genesis it has lost the potential to generate certain cell types. The reason the clones are smaller at late stage is probably because the retinal progenitor cells will be fully proliferated by P7 and should be ready to differentiate into neuronal and glial cells. Too much proliferation of the retinal progenitor cells could cause problems such as retinoblastoma, a hyperproliferation of retinal ptogenitor cells, which forms into cancer. General conclusions from these studies There are no unipotent (fate-restricted) retinal progenitor cells. RPCs are multipotent (but young progenitors have more potential than later progenitors). Late progenitors have lost the capacity to generate early born cells (ie there were no early born cells in clones generated from progenitors labeled late) Labeling cells undergoing DNA synthesis at various time reveals birth date At P0, noticed some cell types that were missing. For example, don’t get ganglion or horizontal cells. One possibility is that the cells have lost potential to make new cells, as these cells are made at earlier stages where progenitors are more competent to generate these cell types. 5 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 BrdU is a molecule which you can inject into an animal. This will be taken up by the blood circulation rapidly and willl be distributed throughout whole animal. o The “d” stands for deoxy, so it can be incorporated into DNA even though it is a uridine analog. o The bromine acts as the antibody target for localization. Concept: o Inject BrdU. o Cells that are in S-phase will incorporate BrdU at a specific time point in retinal development, say E10, then wait until P7, for example. o Cells uptake the BrdU but after each division, cells become less and less BrdU positive. These cells will soon] o 34 mins Cells uptake the BrdU. Everytime a cell divides it loses BrdU, as it has time to proliferate cells become less and less BrdU positive. Instead of going through mitosis it differentiates into a neuron and it will be labeled in BrdU if it is a terminal cell type. Look at population of neurons that actually had BrdU staining in retina. Count, using BrdU antibodies and cell-type specific markers to determine what type of neuron they are. Different retinal cell types are born at specific times People have injected BrdU into different cell types and looked at BrdU staining in the retina. Using BrdU antibodies and cell-type specific antibodies they determined which neurons were developed when. Hoziontal cells, ganglion cells, and cone cells were induced at early stages. They retain BrdU labeling and were strongly BrdU positively. The other cells had diluted BrdU when injected at early stages. But when injected late, bipolar cells, Muller cells, glial cells, were heavily stained with BrdU. Figure 7: Retinal cell types How do RPCs choose to become a particular cell type? They are multipotent, they generate different cell types at specific times, but how do they generate a specific cell type? This is important because it can be implicated in stem cell therapy. If could regenerate retinal cells from stem cells could use in replacement therapy. In addition, many neurodegenerative diseases would benefit from stem cell therapy. Ultimately, we want to replicate developmental events in stem cell lines. There are two major factors that affect cell specification: o Environmental factors RGCs are one of the first cell types to be generated. It is possible that, as the retina matures, the cellular environment changes such that it is not conducive to RGC production (via secretion of some material) o Intrinsic factors Are retinal progenitor cells intrinsically biased to generate particular cell types at one time? 6 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 Environmental factors There was an experiment done in chicks in which they took early progenitor cells at E4 (in mice at E10) and allow them to dissociate into a little ball of cells and place a membrane around them which allows the fusion of molecules, but which the cells cannot pass. On the other side of the membrane, you put the conditioning cells of increasing agents (start with some at E10 all the way to P0). At E10 there are no RGCs made in retina, but by E13-E14 they are generated. If you increase the age of the conditioning cells, will you decrease the amount of RGCs generated? Use the BrdU thymidine analog to label the cells that are proliferating and then stain with an RGC marker to determine which were actually generated in dish in presence of those conditions cells Experiments I: o Control - if the test cells are E4 in the presence of E4 condition cells, 35% RGCs result. o If do this same experiment but in the presence of older aged cells, much less RGCs are produced. In the late environment, there is a signal that prevents the generation of RGCs. What cell type secretes this signal? Is it the RGCs themselves? Experiment II: o Take an E14 chick (E0 in the mouse), kill all the RGCs, do this experiment again, then recover RGC production to level of control. o Suggests the RGCs that are produced and secreting factors to inhibit production of further RGCs. There are active feedback mechanisms in the retina. Intrinsic factors Though RGCs seem under control of environmental factors, these factors don’t tell them what cell type to become. Question: Are RPCs intrinsically biased to generate particular cell types at any one time? Experiment I: can the early environment force late progenitors to make early born cells? o Take a late progenitor cells from P0 cells and put in presence of an excess of older cells. Now look to see if can transform that cell and start making early progenitor cells. Experiment II: Can the late environment force early progenitors to make late-born cells? o Same pattern with opposite conditions. Results: There is a change in cell fate decision. o This tells us that RPCs have a strong intrinsic control whether they were generated at an early or late stage. Seem to be doing so by intrinsic program develops that is modulated by intrinsic feedback decisions in the environment. No matter what environment you put the cell in, it will continue to do what it is supposed to do, suggesting a strong intrinsic program in these cells. A variety of transcription factors (TFs) operate in RPCs to regulate cell fate decisions What could be this intrinsic genetic program? There have been many studies looking at the the production of different cell types, with what TFs influence what cell type. For example, Foxn4 is a transcription factor expressed at early stages. Foxn4 is expressed at the right place and time to specify amacrine and horizontal cell fates Note, the function of a gene is demonstrated only if: o The gene is expressed at certain place and time where cell is o The gene needs to be sufficient for that cell type 7 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 o The gene needs to be required for that cell type Since Foxn4 is expressed at the time amacrine and horizontal cells are produced (and not at later stages), it probably has an effect in early born fate cells. Is Foxn4 required for their expression? Foxn4 inactivation blocks amacrine and horizontal cell production First inactivate Foxn4 by doing a knock out in mouse, then using cell-type specific markers for comparison. In both horizontal and amacrine cells, we observe a strong reduction in the expression of these cells. This suggests Foxn4 function is required for production of amacrine and horizontal cells. Is Foxn4 sufficient for their expression? Foxn4 overexpression increases production of amacrine cells One can use retroviral vectors to force the overexpression of Foxn4 in RPCs to see if can induce more horizontal and amacrine cell production. GFP was used todenote Foxn4 and cell-type specific markers were used to know which cell type we were looking at. Results o There was a strong reduction in rod photoreceptor cells. o Strong increase in production of Syntaxin+ cells (marker for amacrine and horizontal cells). Suggests Foxn4 is sufficient for production of amacrine and horizontal cell… Figure 8: Foxn4 overexpression Asymmetric cell division contributes to generate cell diversity Could there be other intrinsic components controlling diversity? One of the key processes that contributes to cell diversity that is conserved is a process called asymmetric cell divison. You must divide asymmetrically to generate diversity, otherwise, cannot make two daughter cells Two methods of asymmetric cell division: o Extrinsic – stem-cell niche: The mother cell divides which makes two identical daughter cells. One daughter cell exposed to environmental signals sister cell does not see and takes on different cell fate. The division itself was symmetric but influence from environment was asymmetric. Figure 9: Asymmetric cell division 8 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 o Intrinsic Protein or mRNA that is asymmetrically segregated on cortex of mother cell, and is asymmetrically inherited by daughters. Ends up in one of the daughters and not other one – influences fate. It is intrinsic because happening inside cell and division itself was asymmetric. Coordination of cell division orientation with polarized localization of fate determinants generates intrinsic asymmetric cell divisions Using the polarity axis, cell polarizes expression of cell fate proteins such that it is asymmetrically inherited with daughter cell. You can imagine that if the spindle was horizontal, would get symmetric inheritance of protein and thus symmetric division. Figure 10: Asymmetric cell division Mode of cell division: symmetric vs asymmetric Figure 11: Symmetric cell division Figure 12: Asymmetric cell division There is something that influences fate of cell, such as a transcription factor or a protein. When the spindle is horizontal, there is a symmetric division of a factor. Identical cells will be generated, either: o Two progenitor cells – exponential expansion of progenitor cells o Two terminal cells – neurons This type of division allows huge production of neurons in a short time. When the spindle is vertical, there is asymmetric division of a factor. There are three different groups of cells that could be generated: o Two progenitor cells that are different from each other. For example, one could be fated to divide once more and the others to divide three moer times. These are theoretically possible but difficult to study as no good markers for progenitor cells. o “Stem cell mode of division” Division in which a cell divides to make copy of itself, so there is self-renewal but at the same time there is production of neuron. o Terminal asymmetric division Two different types of neurons produced, which increases the diversity of postmitotic cell produced. 9 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 PCs divide along different planes Is there oriented cell division in the retina? You can see horizontally and vertically dividing cells through their chromosome orientation. Perhaps there is asymmetrical inheritance in vertical divisions How could we know if vertical vs horizontal division affects fate? Orientation of RPC division correlates with cell fate decisions Using time lapse electron microscopy, you can take the retina out of the animal, then take a slice of tissue and put it on a filter (which are used to grow organs, example for skin grafts). You can take a retina at E13 and put on this filter and it will follow normal development, generating all the different layers for study ex vivo. Using a retroviral vector to induce GFP expression in RPCs. These cells will divide on the apical surface, and every 10 minutes you take a picture then reconstruct a movie. See whether it is horizontal or vertical division. In 90% of the time, when cells divide horizontally, two daughter cells of the same type result. In 80% of the time, when cells divide vertically, the daughter cells become different types. Suggests that cell division orientation is a random process; there is a strong correlation between orientation of division and fate of cell. Are there any asymmetric fate determinants that would regulate this? Asymmetric inheritance of cell-fate determinants contribute to generation of cell diversity Numb “numbs the effect of Notch” ie is an antagonist of Notch. Notch is important in cellular differentiation, so a protein that would inhibit Notch would affect cell fate. Numb is asymmetrically localized to the cortex of cells, which would divide asymmetrically if the spindle was vertical. What would asyemmetric inheritance of Numb do? Must inactivate it to study it’s function. Clonal genetic inactivation of Numb Study cell autonomous function Allow temporal-specific inactivation Lineage analysis To inactive Numb, must do it clonally, in a single cell. This cell would divide and make two more progenitors, or two neurons of same type, or two neurons of different type. Figure 13: Clonal genetic inactivation of Numb Take advantage of the Cre-Lox system, in which LoxP are sequences that are integrated into the genome of the mouse, flanking an important axon in the Numb gene. Cre Recombinase expression is induced and removes the region between the LoxP sites. In this experiment, they took the retina out of an animal, put on filters, then infected them with Cre-expressing viral vector that is also expressing GFP. Cre inactivates Numb and GFP tells us which cell was infected. Then, you allow the retina to develop and differentiate for 17 days, fix the tissue, and use celltype markers to determine whether division was symmetric or asymmetric. 10 ANAT 416 Dr. Cayouette February 14, 2012 Lecture 11 When do this, don’t know if inactivation of this gene could lead to some non cell-autonomous effect. Only want to inactivate Numb in a single cell, otherwise could disrupt signaling of some secreted factors, for example, if affected many cells. Can do this early, late, or in middle of retinal development and can identify lineage of entire infected cells. Inactivation of Numb increases symmetric divisions and decreases asymmetric divisions If you inactivate Numb, you find a dramatic increase in the proportion of symmetric divisions in versus asymmetric divisions. There is an increase in photoreceptor/photoreceptor daughter cells (for example) and a decrease in production of terminal asymmetric products (many types). Suggests Numb is required for those terminal divisions. But is Numb sufficient? Figure 14: inactivation of numb Numb gain-of-function increases symmetric terminal divisions at the expense of asymmetric terminal divisions Now overexpress Notch, and would expect opposite results but actually observed the same results. In both cases, you broke the asymmetry by inactivating Numb or by overexpressing it and thus, lose assymetric Numb presence in the daughter cells. A model of Numb function in terminal asymmetric cell divisions In a normal animal, Numb is asymmetrically inherited and that goes on to activate Notch in only ONE of the daughter cells (while the other cell has Numb and thus inactivates Notch). Now, when there is over-expression of Numb, you force symmetrical inheritance in both cells so they both inhibit Notch and so both take on same fate. The asymmetric presence of those proteins during division instructs asymmetric division. It doesn’t instruct a specific cell type but instructs asymmetry or symmetry. Figure 15: Numb function in terminal asymmetric cell division 11