Lecture 11: Regeneration PDF

🥽 Lecture 11: Regeneration LO: Define regeneration, list the different types and give examples of each. Regeneration: the reactivation of developmental processes in post-embryonic tissues to restore missing tissues...

🥽 Lecture 11: Regeneration LO: Define regeneration, list the different types and give examples of each. Regeneration: the reactivation of developmental processes in post-embryonic tissues to restore missing tissues. Types of regeneration: Physiological regeneration Normal process where stem cells regenerate lost cells. Examples Human: Hair shedding and skin renewal, blood regeneration. Deer: Annual regeneration of antlers. Epimorphosis De-differentiation of existing adult cells to form an undifferentiated mass (blastema) that then re-specifies into the lost tissue. Involves significant cell proliferation Examples Lecture 11: Regeneration 1  Salamander/Axolotl: Regenerates limbs. Morphallaxis Regeneration through re-patterning of existing cells with minimal or no cell proliferation. Examples Hydra: When cut in half, it forms two smaller individuals. Compensatory mechanism Differentiated cells divide but maintain their differentiated functions. Examples Mammalian Liver: Capable of regeneration while maintaining liver function. Zebrafish Heart: Regenerates while preserving cardiac function. Physiological Regeneration: Deer Antlers Antlers are replaced annually in deer, elks and moose (starting in spring) A bony pedicle forms from the periosteum of the frontal bones The pedestal supports the growth of the antlers, which develop a core of vascularised bone. The tip of the growing antler comprises a vascular zone of cartilage. Involves Wnt signaling and is stem cell-based (periosteum express Oct4, Sox2, Nanog pluripotency markers) LO: Explain the difference between morphallaxis and epimorphosis. Morphallaxis vs epimorphosis - using the French Flag Model Lecture 11: Regeneration 2  Morphallaxis New boundary regions are established first, and positional values are specified relative to these new boundaries. In morphallaxis, any organism that is cut is regenerated into a complete organism French Flag Model: Imagine cutting the flag halfway through the white part. The white part (new boundary) becomes the highest positional value. Regeneration will restore the flag to its original state with blue, white, and red stripes. Result: A complete flag is regenerated with the same size as the cut portion. Epimorphosis New positional values are linked to the growth from the cut surface. In epimorphosis, the injured/cut part is repaired - limb is regenerated French Flag Model: Cutting the flag halfway through the white part. The cut surface takes on an intermediate positional value. Lecture 11: Regeneration 3  Regeneration grows from this surface to restore the original flag’s blue, white, and red stripes. Result: The flag regenerates from the cut surface, starting with the intermediate value and growing back to its original pattern. LO: Describe the process of limb regeneration in amphibian models. Epimorphic Regeneration of Salamander limbs When an amphibian limb (e.g., salamander) is amputated, the remaining limb cells de-differentiate to form a blastema and then re-differentiate to reconstruct the missing limb Cells retain positional values → ie. cells "know" where they are along the proximal-distal axis and regenerate accordingly Distal Amputation: If the amputation is near the digits, only the distal parts are regenerated. Proximal Amputation: If the amputation is closer to the body axis, all parts relative to that proximal axis are regenerated. Limb reconstruction occurs by epimorphosis via: De-differentiation: Cells lose their specialised functions and become more like stem cells. Proliferation: The de-differentiated cells proliferate to form a blastema. Re-specification: The blastema cells then re-specify into the relevant cell types needed for the regeneration of the missing limb parts. Lecture 11: Regeneration 4  The Process of Limb Regeneration 1. When a limb is amputated - epidermal cells form a wound & form Apical Ectodermal Cap (AEC) on the surface of the wound Pre-Amputation: Includes epidermis, nerves, bone, cartilage, and vasculature. Post-Amputation (Day 7): Macrophage Infiltration - Involved in wound healing. Formation of Apical Ectodermal Cap: On the outer surface of the wound. 2. Cells beneath the cap undergo de-differentiation into lineage-restricted multi-potent progenitors 3. De-differentiated cells proliferate forming the regeneration blastema Lecture 11: Regeneration 5  Blastema: a heterogeneous collection of developmentally restricted progenitor cells derived from the originally differentiated cells at the surface. 4. Cells in the blastema re-differentiate to form the missing parts of the limb, including bone, dermis and cartilage precursors Proliferation of cells in the blastema is dependent on the presence of nerves Cell Fate and Differentiation in Epimorphic Regeneration Cell Fate and Redifferentiation Cell memory - cells in the blastema retain their identity and knowledge of their original tissue type (e.g., cartilage, nerve, muscle). Study: GFP axolotls: Cells from Different Tissue Origins Labelled with GFP → “Track” the Fate of Cells Lecture 11: Regeneration 6  Each tissue produces progenitor cells with restricted potential → THUS, the blastema is a heterogeneous collection of restricted progenitor cells (not pluripotent) GFP-labeled cartilage cells will form cartilage in the regenerating limb but will not differentiate into other tissue types like bone or muscle. Cells maintain a memory of their tissue of origin and predominantly regenerate into the same type of cells they were before amputation. Factors Affecting Epimorphic Limb Regeneration Apical Ectodermal Cap secretes FGF8 - crucial for blastema growth FGF8 was important in the apical ectodermal ridge during early embryogenesis. Limb regeneration depends on its nerve supply Lecture 11: Regeneration 7  Denervated Limbs: If a limb is amputated and the nerve is removed, it does not regenerate properly. NAG (Nerve Anterior Gradient Protein): A growth factor secreted by the nerve and promotes regeneration (essential for adult newt limb regeneration) Removing the nerve and then adding NAG can allow for some regeneration. Both anterior and posterior cells must be present in the blastema for proper limb regeneration. Diverting the nerve to only the anterior or posterior part of the limb leads to incomplete regeneration. Proper regeneration requires nerve supply along the entire limb length, providing signals to both anterior and posterior regions. FGF8: Secreted by anterior cells in the blastema. Sonic Hedgehog: Secreted by posterior cells in the blastema. Lecture 11: Regeneration 8  Cross-Induction: Anterior FGF8 stimulates posterior Sonic Hedgehog, and posterior Sonic Hedgehog stimulates anterior FGF8. Regeneration in salamanders always occurs in a distal direction. Eg) if the hand is amputated, regeneration will proceed from the proximal to the distal part of the limb. Experiments Involving Limb Inversion and Amputation When a limb is amputated and inverted, with vascular connections reestablished, regeneration still occurs in a distal direction. Even if both proximal and distal surfaces are involved, the regenerating structures will always develop distal elements. Cells in regenerating limbs have positional values—they "know" where they are in relation to the cut surface. The outcome of regeneration depends on the location of the cut rather than more proximal tissues - ie. the limb reads the positional values of the cut site and regenerates all structures distal to it. What is the Nature of the Positional Value? Lecture 11: Regeneration 9  Positional values in regenerating limbs are thought to involve a graded signal related to cell adhesiveness: Proximal tissue is more motile. Distal tissue is more adhesive. In limb regeneration, most structures form from the proximal stump Even when a distal blastema is grafted onto a proximal stump, the proximal stump takes precedence in regenerating structures. Proximal tissue plays a dominant role in regeneration due to a factor called Prod1. Lecture 11: Regeneration 10  Role of Prod 1 Prod1 is a small glycolipid-anchored protein that helps confer positional information It interacts with NAG (nerve anterior gradient protein), which is secreted by the nervous tissue. Prod1 is expressed in a graded manner along the proximal-distal axis, with higher levels proximally. Lecture 11: Regeneration 11  This graded expression allows cells to regenerate the appropriate distal structures based on their positional information. Proximal cells (high Prod1 levels) are less adhesive and more motile, while distal cells are more adhesive Experiment show: The proximal blastema (red) wraps around the distal blastema (yellow), demonstrating that proximal cells are more motile and less adhesive. When Prod1 is blocked using an antibody, the movement of the proximal blastema is inhibited. When a distal blastema is transplanted into the dorsal surface of a proximal blastema, the distal blastema cells retain their positional memory and know they are from the distal region of the limb. These cells will, therefore, regenerate distal structures (e.g., hand, fingers) rather than proximal ones (e.g., humerus or radius). However, the proximal blastema has its own positional identity and will regenerate the missing proximal structures based on its location and memory. Both the proximal blastema and the distal blastema form tissues according to their respective positional cues, which results in the formation of two hands: 1. The proximal blastema regenerates the entire limb including proximal structures like the humerus, radius, and ulna. 2. The distal blastema will regenerate distal structures, including another set of hand-like structures at the transplanted location. Summary: Salamander Limb Regeneration Epimorphosis requires AEC & Blastema (heterogeneous mass of restricted progenitor cells) Blastema must contain cells from anterior and posterior of limb (FGF8 and Shh signalling) Lecture 11: Regeneration 12  Requires growth factor (nAG) from nerves Regeneration proceeds in proximal to distal direction with the positional value defined at the cut site Positional values defined by graded signals, including Prod1 expression LO: Explain the head inhibitory model in the patterning of hydra. Overview of Hydra Hydra is a simple invertebrate organism with two germ layers: ectoderm and endoderm (no mesoderm). It has a hypostome (head structure with tentacles for feeding) and a basal disc (attaches to substrate). Continuous cell growth occurs, with cells constantly moving along the body. Asexual Reproduction via Budding Hydra reproduces asexually through budding, where a new hydra forms and detaches to become independent. Lecture 11: Regeneration 13  Cells proliferate and move over time, leading to re-specification as new tentacles form and eventually detach. Morphallactic Regeneration in Hydra Morphallaxis is the process of regeneration without significant new growth. When a hydra is cut: The remaining cells repattern to regenerate the lost structures (head or foot). This involves reorganisation of existing cells rather than new cell division. Regeneration is polarised If the head is cut off, the foot area regenerates a new head. Lecture 11: Regeneration 14  If the foot is cut off, the remaining tissue regenerates a new foot. Regeneration does not lead to immediate growth; the new hydra is smaller and will grow over time to match the size of the parent. Characteristics of Hydra Regeneration No significant cell division is required for initial regeneration as it can occurred in irradiated individuals The regeneration process involves re-differentiation of cells to replace lost parts, with little or no extra growth occurring initially. Newly regenerated hydra individuals are small and take time to reach the size of the original hydra. The Head and Basal Disc Act as Organising Centres Lecture 11: Regeneration 15  The head (hypostome) and basal disc in hydra act as organising centers Organising centers induce the formation of new structures in the host tissue Transplantation experiments have shown that new structures form from host tissue but not from the donor graft - ie. the transplanted hypostome acts as an organizing center, signaling the host’s cells to regenerate the structure, but it is the host own tissue that generate the new structure, not the donor graft If the hypostome (head) is transplanted into the mid-region of a host hydra, it induces the formation of a new head structure on the side of the animal. If the basal disc is transplanted into a recipient, it induces the formation of a new basal disc. Lecture 11: Regeneration 16  Role of Organising Centers There are gradients associated with the head and foot: Head activation gradient: Strong near the head, declining towards the body. Foot activation gradient: Strong near the basal disc, declining towards the body. Head Inhibition Gradient There is a head inhibition gradient that prevents the formation of additional heads along the body. This gradient is strongest near the head and declines toward the basal disc. Tissue from below the head does not form a new head when transplanted into a recipient unless the recipient’s head is removed. The head inhibitory factor is present along the length of the hydra, and so tissue from below the head can still induce a new head structure if: The head of the recipient is removed, eliminating the head inhibition gradient → new head structures can form, allowing for the creation of a secondary axis. The tissue is transplanted into a region far from the head inhibition gradient, such as the bottom of the animal. Lecture 11: Regeneration 17  Wnt Signals are Produced by the Hypostome The major inducer of the head (hypostome) is a set of Wnt proteins. Wnt signaling works through the canonical beta-catenin signaling pathway to establish the hypostome, which is the key head-signaling region. Wnt3a is one of the important molecules involved in this process. Wnt Staining in Budding: During the budding process, where a new head is forming, in situ hybridization shows strong expression of Wnt in the hypostome. Lecture 11: Regeneration 18  This head structure also acts as an organizing center, inducing the formation of tentacles nearby. Inhibition of GSK3 and Ectopic Structures: GSK3 is part of the destruction complex for beta-catenin. If GSK3 is inhibited, causing beta-catenin accumulation and thus activation of the Wnt pathway, ectopic tentacles can form anywhere on the body. This activation can also stimulate the outgrowth of new buds. Summary: Hydra Regeneration Hydra grow continuously, losing cells from ends and “budding”, the constant cell movement means constant re-patterning Regeneration not dependent on growth, it involves re-patterning of existing tissues (transdifferentiation) The head (hypostome) and the foot (basal disk), both act as organising centres There is a head activation gradient and a foot activation gradient There is a head inhibitory gradient emanating from the head region Wnt signalling is required to establish the head/hypostome LO: Describe the process of heart regeneration in zebrafish and the steps and tissues involved. How to mend a broken heart? ⇒ Possible approaches: Lecture 11: Regeneration 19  Induce cardiomyocytes within the heart to proliferate Produce new cardiomyocytes and transplant into the heart. Enhance stem cell or progenitor cells within the heart to produce more cardiomyocytes. But do stem cells exist? Heart Regeneration in Zebrafish Adult zebrafish can regenerate damaged heart Following a cut: The epicardium covers the damaged area through the formation of a fibrin clot (shown in red). Cell migration occurs, with migrating cells shown in green. New cardiac tissue is produced, replacing the fibrin clot. Cell proliferation (depicted in green) leads to complete wound healing. The process results in a fully regenerated heart. Role of FGFs and Epicardial Cells: The new heart tissue produces fibroblast growth factors (FGFs). FGFs signal epicardial cells to: Migrate into the damaged area. Generate new blood vessels / vascularise the area / new muscle Over time, wounded zebrafish heart returns to nearly its original shape, size, and pumping ability. What is the Cellular Origin of Heart (Cardiomyocyte) Regeneration in Zebrafish? Cardiomyocytes responsible for heart regeneration in zebrafish are derived from differentiated cardiomyocytes. This is an example of compensatory regeneration, where existing cells (not stem cells) regenerate damaged tissue. Fibroblast Growth Factor (FGF) signaling plays a role in this regeneration process. Lecture 11: Regeneration 20  Research Methodology - Reporter fish: Researchers use transgenic zebrafish with inducible reporter constructs Cmlc2-alpha promoter drives expression in cardiomyocyte-specific tissues. LoxP sites flank a stop codon, preventing GFP expression. A Cre-inducible system is activated upon the addition of tamoxifen, allowing GFP to be expressed. Once tamoxifen is added, Cre enters the nucleus, excising the LoxP-flanked stop, enabling GFP expression. After tamoxifen induction, GFP-labeled cardiomyocytes are seen in green. When the heart is cut, these green-labeled cardiomyocytes migrate into the damaged area, confirming their origin from differentiated cardiomyocytes, not stem cells or blastema. Lecture 11: Regeneration 21  Cardiomyocytes undergo a limited de-differentiation process Sarcomeres, the muscle structures, are disassembled and reassembled prior to cell proliferation. Stem or progenitor cells are not significantly involved. Heart Regeneration in Various Model Organisms Zebrafish and salamanders can regenerate their hearts, while rodents have limited regeneration capacity: Only embryonic or very young mice show some heart regeneration. In humans, heart injuries like myocardial infarctions result in fibrotic scars, with no regeneration. Future Directions - Transgenic Approaches: Lecture 11: Regeneration 22  Scientists are exploring whether these regenerative genes can be used in mammals, potentially in humans, to induce heart regeneration. Pros of Transgenic Heart Regeneration: Potential to reverse heart damage: Using regenerative genes could offer a way to heal damaged human hearts after myocardial infarctions. Inspiration from natural processes: Zebrafish and amphibians provide a natural model for heart repair, which may be leveraged in medical applications. Cons and Challenges: Complexity of gene regulation: Introducing or activating the right genes in a human heart requires precise gene editing and control, which is technologically challenging. Unpredictable outcomes: Human hearts may respond differently, leading to unwanted side effects or improper tissue formation. Ethical concerns: Genetic modifications raise ethical questions about the extent to which we should alter human biology. Lecture 11: Regeneration 23