Developmental Biology Lesson 29 & 30 Chapter 24 PDF

BIOL 346 Developmental Biology Lesson 29 & 30 Chapter 24: Regeneration: The Development of Rebuilding Fig. 24.1: Representative organisms and their comparative regenerative capabilities Regeneration is the reactivation of development in postembryonic life to restore missing or da...

BIOL 346 Developmental Biology Lesson 29 & 30 Chapter 24: Regeneration: The Development of Rebuilding Fig. 24.1: Representative organisms and their comparative regenerative capabilities Regeneration is the reactivation of development in postembryonic life to restore missing or damaged tissues Mammals unable to rebuild whole appendages, individual tissues and organs do possess variable regenerative capabilities Fig. 24.2: Conceptualized steps of regeneration What Is Required for Regeneration? Pre-Injury: Cells and tissues maintain a "morphological memory map" of their identity and position within the organism. Post Injury: Cells recognize the injury and the need for replacement. Rapid wound closure occurs. Regenerative response: Activation of embryonic-like mechanisms for cell proliferation, tissue growth, and cell patterning to recreate the lost structure. Completion: Regeneration stops once the correct size and shape are achieved, integrated proportionally with the body. Key Requirement: A cell-based system integrates whole-organism awareness, immune response interactions, and developmental morphogenesis. Fig. 24.3: Four different modes of regeneration. Stem Cell-Mediated Regeneration: Stem cells regenerate lost tissues (e.g., hair shafts from follicular stem cells, blood cells from hematopoietic stem cells). Epimorphosis: Dedifferentiation forms a blastema (undifferentiated differentiated cells), which redifferentiates to regenerate structures (e.g., amphibian limb regeneration). Morphallaxis: Re-patterning of existing tissues with little new growth, involving cell death and transdifferentiation (e.g., hydras). Compensatory Regeneration: Differentiated cells divide while retaining their function, without forming undifferentiated tissue (e.g., mammalian liver regeneration).  Is Regeneration a Recapitulation of Embryonic Development? Dual Nature: Regeneration uses mechanisms from embryogenesis while requiring context- specific adaptations due to postembryonic constraints. Key Differences from Embryogenesis: Immune Response: Regeneration begins with injury-induced immune responses, including wound closure and phagocytic cleanup. Induced Reprogramming: Adult cells are reprogrammed into an embryonic-like state to rebuild tissues. System Integration: New cells must integrate with existing tissues (e.g., blood vessels, nerves). Size Recognition and Termination: Regenerating tissues must coordinate growth with neighboring tissues, ensuring proper scaling and stopping once the correct size is reached. Focus of Research: Understanding the interplay between embryonic mechanisms and regeneration- specific responses.  Plants and Animals: Different Regenerative Potentials Plants: Extreme plasticity enables regeneration of tissues and entire organisms (e.g., totipotent cells, green algae, bryophytes, meristems). Full shoot or root regeneration occurs in response to injury. Animals: Regenerative traits are sporadically distributed across taxa. Acoels and planarians exhibit total regeneration via neoblast stem cells, relying on Wnt signaling for anterior-posterior axis formation. Sponges (basal metazoans) demonstrate high plasticity, including: Totipotency to regenerate entire individuals from dissociated cells. Mechanisms like stem cell-mediated regeneration, transdifferentiation (morphallaxis), and blastema formation (epimorphosis). Choanocytes and archeocytes in sponges act as pluripotent stem cells and transdifferentiate to replace lost tissues. Fig. 24.6: Sponge choano­cytes proliferate in response to injury Control State: In uninjured sponges, choanocytes show normal levels of activity and organization. Injury Response: Choanocytes are recruited and exhibit increased proliferation at the wound site. Proliferating cells in the S phase are labeled with EdU (green). Anti-tubulin (blue) marks choanocytes, and red fluorescence identifies all nuclei. Fig. 24.7 Identification and functional characterization of evolutionarily conserved regeneration-responsive enhancers (RREs) in two teleost (bony fish) species Gene Regulation in Regeneration: Regenerative processes are controlled by regeneration-responsive enhancers (RREs), conserved sequences regulating injury and regeneration response genes. Evolutionary pressures may have led some species to retain regenerative abilities while others lost them.  Why Are So Many Animals Unable to Regenerate? Natural Selection: Rapid scarring often enhances survival by preventing life-threatening blood loss, outweighing the slower benefits of regeneration. Risk of cancer from rapidly dividing cells may also drive the loss of regenerative ability. Environmental Factors: Regenerative capacity is higher in animals living in aqueous environments, suggesting water may play a role in regeneration. Evolutionary Trade-offs: Multiple evolutionary pressures likely reduced regeneration abilities, with no single cause explaining the loss. Future Directions: Understanding mechanisms behind scarring vs. regeneration could lead to pro-regenerative interventions in medicine.  Whole-Body Animal Regeneration Hydra and Planarians: Hydra and planarians can regenerate their entire bodies due to parallels with their asexual reproduction mechanisms. Hydra Anatomy and Regeneration: Hydra's tubular body has a head with tentacles and a basal foot for attachment. Composed of two epithelial layers (ectoderm and endoderm), with multipotent interstitial stem cells (ISCs) enabling regeneration. Routine Cell Replacement: Hydra cells undergo constant mitosis, migrate along the body column, and are shed at the extremities. Regeneration occurs continuously through: Unipotent progenitors for epithelial cells (endoderm and ectoderm). Multipotent ISCs, which form neurons, nematocytes, gland cells, and gametes. Key Cellular Insights: ISCs generate both gland (endodermal) and neural (ectodermal) cells from a shared progenitor. ISCs are primed for rapid proliferation, cycling faster than epithelial stem cells to respond to regenerative needs. Fig. 24.15: Budding in hydra Budding Process: New individuals bud about two-thirds down the adult hydra's body. Cellular Composition: Myoepithelium contains unipotent endodermal and ectodermal cells and multipotent interstitial stem cells (ISCs). Cell Division and Movement: Cell division occurs throughout the body column except at the tentacles and foot. Cells migrate along the column before being shed. ISC Differentiation: ISCs generate nematocytes, nerve cells, and gland cells.  The Head Activator in Hydra Regeneration Ability: Each hydra body segment can regenerate both a head and a foot, but polarity is controlled by morphogenetic gradients. Experimental Evidence: Grafting hypostome tissue (head region) induces a secondary axis with a new hypostome. Grafting basal disc tissue induces a secondary axis with a new basal disc. Grafting both hypostome and basal disc tissue together results in weak or no polarity. Gradients: Head activation gradient: Highest at the hypostome, decreasing toward the basal disc. Foot activation gradient: Highest at the basal disc. Gradient Measurement: Donor tissue from higher levels of the head activator gradient is more likely to induce head formation when transplanted. Fig. 24.16: Grafting experiments demonstrate the different morphogenetic capabilities of regions along the hydra’s apical-basal axis Fig. 24.1: Wnt/β-catenin signaling during hydra budding The Hypostome as Organizer _Organizer Role: The hypostome can induce a second body axis when transplanted. Produces both a head activation signal and a head inhibition signal to regulate axis formation. It is the only "self-differentiating" region of the hydra. _Wnt3 Gradient as head inducer: Wnt proteins (via the β-catenin pathway) act as the major head inducers in the hypostome. Wnt3 is expressed in the apical end of the bud during elongation. Effects of misexpression: Blocking Wnt inhibitors or globally misexpressing β-catenin results in ectopic buds forming throughout the body. Induction of Brachyury: When the hypostome contacts the trunk, it induces Brachyury gene expression in a Wnt-dependent manner, similar to vertebrate organizers. Implication: Wnt3 likely functions as the head organizer during both normal development and regeneration.  Stem Cell-Mediated Regeneration in Flatworms Reproductive Regeneration: Planarians reproduce asexually via binary fission, splitting into two parts, with each half regenerating missing structures using pluripotent stem cells. Whole-Body Regeneration: Cutting a planarian in half results in the head regenerating a tail and the tail regenerating a head, forming smaller versions of the original due to limited mass. Mechanisms: Regeneration combines morphallaxis (tissue remodeling and cell death) with stem cell proliferation for regrowth. Polarity and Gradients: Polarity determines regeneration direction: Middle pieces regenerate a head at the anterior and a tail at the posterior. Extremely thin middle segments fail to regenerate properly due to lack of discernible morphogen gradients. Fig. 24.18: Flatworm regeneration and its limits Head Regeneration (A): Planarians regenerate a new head after amputation. Bisection (B): Anterior of the lower half regenerates a head, and posterior of the upper half regenerates a tail. Regenerated individuals are smaller versions of the parent. Trisection (C): The middle piece regenerates a head from its anterior end and a tail from its posterior end. Thin Middle Slices (D): Thin segments fail to regenerate properly due to lack of a discernible morphogen gradient. Fig. 24.19: Cell production during planarian regeneration is accomplished by a pluripotent stem cell population of neoblasts Blastema Formation: Regeneration blastema A regeneration blastema is a mass of forms from clonogenic neoblasts (cNeoblasts), undifferentiated cells at a wound site that not dedifferentiated old cells. forms new tissues and structures. Pluripotent Neoblasts (A): Neoblasts, labeled with soxP-2, generate colonies of dividing clonogenic neoblasts (cNeoblasts) that produce differentiating cells for regeneration. Neoblasts are distributed throughout the body except in the pharynx. Regeneration Evidence: 1750 rad irradiation kills most neoblasts, but a single surviving cNeoblast can restore all differentiated cells. 6000 rad eliminates all dividing cells: Transplanting one donor cNeoblast restores all cell types and regenerative capacity. Fig. 24.20: Wnts specify tail and repress head cell fates during planarian regeneration Head-to-Tail Polarity Signaling Pathways: Planarians maintain active signaling pathways for tissue turnover, using molecules like BMPs (dorsal-ventral patterning), Wnts and FGFs (anterior-posterior patterning), and Slit proteins (medial- lateral patterning). Wnt Expression Gradient: Wnt transcripts form a gradient from posterior to anterior, highest in the tail (A, B). Function of Wnt Signaling: Wnts promote tail cell fates and repress head cell fates during regeneration. RNA Interference Effect: Blocking Wnt signaling via RNA interference (wnt1 mRNA) causes the posterior blastema to regenerate a head, creating a two-headed worm Fig. 24.21: Restoration of head regeneration in planarian (D. lacteum) Wnt/β-Catenin Signaling and Head Regeneration Inhibitory Role of Wnt: Wnt signaling represses head specification, with β-catenin activated in the posterior-facing blastema to form tails. Repression of Wnt Signaling: Blocking β-catenin via RNA interference (RNAi) allows head formation in the posterior blastema. Restoring Regeneration: In regeneration-deficient species (e.g., D. lacteum), RNAi-mediated β-catenin knockdown enables functional head regeneration over 21 days. Excessive Inhibition: Complete elimination of β-catenin in planarians causes multiple heads with functional eyes to form around the periphery. Fig. 24.23: Positional control gene expression is distributed by the spatial orientation of muscle fibers in planarians Morphological Memory Map in Planarian Regeneration Two Regeneration Methods: Blastema generates new cells at the wound site. Existing tissues change cell identities to create smaller, proportionate regenerates. Positional Control Genes (PGCs): PCGs guide cell fate based on positional information. Encode signaling ligands, receptors, regulators, transcription factors. Muscle Fiber Coordinate System: PCG expression aligns with circular, diagonal, and longitudinal muscle fibers. Muscle fibers provide a spatial framework for regeneration. Fig. 24.24: Overall model for planarian regeneration A Model for How Injury Changes Us Morphological Memory Map: Muscle-mediated PCG expression guides positional information for regeneration. Polarity Establishment: Wnt signaling promotes tail development while repressing head formation. Notum, expressed in the head, inhibits Wnt signaling, allowing head regeneration. Gradient Restablishment: After amputation, Notum is upregulated in anterior-facing wounds; Wnt is expressed posteriorly. Gene Activation: Injury induces ~200 genes; notum and wnt1 reset PCG patterns. Wnt1 promotes posterior neoblast specialization; Notum supports anterior specialization. Regeneration Process: Opposing gradients reassign PCG expression in muscle cells. Neoblasts proliferate in the blastema, enabling morphallaxis and forming a proportionate flatworm. Fig. 24.25: Bioelectric regulation of planarian regeneration An Alternative Morphological Memory Map: "It’s Bioelectric" Bioelectric Pattern: Proposed as a second positional map based on endogenous electrical properties of cells. Bioelectric Signaling: Communication between cells via changes in transmembrane voltage potential (Vmem) caused by ion concentration differences. _Injury Induced Changes: Injuries disrupt cell membranes, causing ion movement and altering Vmem (hyperpolarization or depolarization). Planarian Regeneration: Vmem Gradient: Depolarized head cells; hyperpolarized tail cells. Ion Manipulation Effects: Depolarization induces two heads in the midbody. Hyperpolarization prevents head formation. Fig. 24.26: Bioelectric interactions with Wnt/β-catenin signaling Membrane Voltage and Tissue Reconstruction Integration of Bioelectricity and Genetics: Membrane voltage influences positional control gene (PCG) expression and neoblast activity. Bioelectric states interact with Wnt/β- catenin signaling, crucial for anterior-posterior regeneration. _Experimental Evidence: Double Heads: Loss of Wnt/β-catenin signaling in a midbody slice causes double heads. Rescue with Hyperpolarization: A hyperpolarizing drug restores normal regeneration in similarly treated slices. Dynamic Morphogenetic Map: Bioelectricity dynamically translates physiological states into genetic programs, a form of “physiological epigenetics.” Fig. 24.27: Regeneration of a salamander forelimb Tissue-Restricted Animal Regeneration Whole-Body Regeneration: Absent in vertebrates, but some species regenerate specific tissues or organs. Salamanders: Epimorphic Limb Regeneration: Limb amputation triggers reconstruction of missing structures only. Regeneration respects the proximal-distal axis, forming parts only from the severed point onward. Example: A wrist-level cut regenerates a wrist and foot, not an elbow. Positional Information Restored: Normal limb structure is regenerated within 72 days. Fig. 24.28: Anatomy of the limb blastema Limb Regeneration in Salamanders Regeneration Blastema: Forms at the distal end of the amputated limb. Aggregation of undifferentiated progenitor cells drives regeneration. Anatomy of the Limb Blastema _Apical Epidemeral Cap (AEC): Thickened epidermal layer covering the wound. Preexisting Tissues: Muscle (M), skeleton (S), nerves, and connective tissue (C) lie proximal to the cut plane. Dedifferentiation: Cells at the distal tip of existing tissues dedifferentiate to form lineage-restricted progenitors (muscle, skeleton, fibroblast). Blastema Formation: Proliferative mass of progenitor cells beneath the AEC. Includes muscle progenitors (MP), skeleton progenitors (SP), fibroblast progenitors (F), and regenerating axons. Regenerating Axons: Graded from proximal to distal within the blastema. Fig. 24.29: Stages of limb regeneration in the salamander Blood and Immune Response: Flooding of cells, formation of blood clot. Cell Activation: Wounding triggers stem/progenitor cell proliferation. Wound Epidermis Formation: Epidermal cells migrate over the wound. Apical Epidermal Cap (AEC): Wound epidermis thickens into the AEC. AEC Signals: Stimulate blastema development beneath it. Blastema Growth: Proliferation and differentiation fuel limb outgrowth. Fig. 24.30: Cells of the connective tissues invade the blastema and adopt an embryonic limb-budlike identity in axolotl but not in regeneration-incompetent, postmetamorphic frogs Axolotl Regeneration: Connective Tissue Invasion: Extensive infiltration of connective tissue cells into the limb bud and blastema, marked by PRRX1 (A). Embryonic Gene Expression: Connective tissue cells adopt an embryonic limb-budlike gene expression identity, critical for regeneration. Regeneration-Incompetent Frogs: _Lack of Reprogramming _______________________: Post- metamorphic frogs do not exhibit similar connective tissue cell reprogramming (B). Gene Expression Profiles: Violin plots show divergence in transcriptional profiles compared to axolotls, emphasizing regenerative differences. Fig. 24.31: Blastema cells retain their specification even when they dedifferentiate Cell Memory in Regeneration Blastema Key Question: Do blastema cells retain "memory" of their original tissue type? Findings (Kragl et al., 2009): _Tissue-Specific Regeneration: Muscle cells regenerate only from dedifferentiated muscle cells. Dermal cells regenerate only from old dermal cells. Cartilage regenerates from old cartilage or dermal cells. Blastema Composition: Heterogeneous mix of tissue-specific progenitor cells. Not a collection of unspecified multipotent progenitor cells. Evidence (FIGURE 24.31): GFP-labeled cartilage cells contributed only to regenerated cartilage, not other tissues. Conclusion: Blastema cells retain tissue-specific "memory," maintaining their lineage during regeneration. Fig. 24.32: The requirement for nerves and sufficiency of neuregulin for axolotl limb regeneration Nerve Requirement for Regeneration: Denervation significantly reduces cell proliferation in the blastema, halting regeneration. Role of Neuregulin (Nrg1): Neuregulin-1-coated beads implanted in denervated limbs rescue regeneration up to digit formation. Control beads soaked in PBS fail to restore regeneration. Blastema Observations (FIGURE 24.32): Normal blastema: Highly innervated and densely populated with proliferating cells (BrdU-labeled, green). Denervated blastema: Lacks neural input and cell proliferation. Conclusion: Nerves are essential for regeneration, and neuregulin-1 is sufficient to partially restore regenerative capacity. Fig. 24.33: Induction of ectopic limbs in salamanders Dependence on Nerves and Apical Epidermal Cap (AEC): Both nerves and the AEC are essential for blastema formation and limb outgrowth. Induction of Ectopic Limbs: Diverting a nerve to a wound site induces an ectopic blastemalike bud, but not a complete limb. Complete ectopic limb formation requires both nerve signals and an epidermal graft from a different positional location. Conclusion: Nerve signals are critical for regeneration but must work alongside positional cues from the AEC (secret Fgf) and epidermis for successful limb patterning and growth. Alternative Induction: Accessory limbs can also be induced using beads coated with BMP2/BMP7 and Fgf2/8 applied to the wound site (E). Fig. 24.34: Lens regeneration in newts is depicted in cross sections through the eye, from intact eye to 80 days post-lentectomy _Lens Regeneration: Regeneration is unaffected by age or repeated injuries (e.g., lentectomies). Transdifferentiation: Only dorsal pigmented epithelial cells (PECs) undergo transdifferentiation into lens fiber cells. Ventral iris PECs do not contribute to regeneration. Regeneration Process: Dorsal PECs lose pigment, re-enter the cell cycle, and proliferate to form lens fiber cells. Regenerating lens is eventually fully crystalline. Significance: Regeneration does not follow the typical embryological pathway. Provides insights into transdifferentiation mechanisms.  Luring the Mechanisms of Regeneration from Zebrafish Organs Zebrafish as a Model for Regeneration: Regenerates diverse organs: fin, heart, CNS, eye, liver, kidney, pancreas, sensory hair cells. Offers genetic and technical advantages for studying molecular mechanisms. Upon Amputation: Regeneration involves wound closure by epidermal cells, dedifferentiation, proliferation, and formation of a blastema. Wnt/Beta-Catenin Signaling in Fin Regeneration: Role: Essential for blastema proliferation and regenerative growth. Effects of Misexpression: Misexpression of Axin1 (β-catenin inhibitor) reduces fin regeneration and impairs fin ray ossification. Interactions: Modulates other pathways like Hedgehog, Fgf8, retinoic acid, and IGF. Experimental Results: Loss of Wnt signaling results in reduced blastema proliferation and slower regeneration rates. Fig. 24.35: Testing the spatiotemporal requirements of Wnt/β-catenin signaling during fin regeneration Ubiquitin (global expression). Her4.3 (osteoblast progenitors). DOX: Induces expression of Axin1- YFP (β-catenin inhibitor). Global Inhibition (Ubiquitin Promoter): Reduced fin regeneration when Axin1 is misexpressed throughout the fin. Targeted Inhibition (Her4.3 Promoter): Severely impaired ossification of fin ray bones. Reduced red staining indicates disrupted bone formation.  Regeneration in Mammals1 _Limited Mammalian Regeneration: Liver compensatory regeneration, neonatal mouse heart regeneration, and spiny mouse skin regeneration. Liver Regeneration Compensatory Regeneration: Remaining liver lobes enlarge to restore function after partial hepatectomy. Does not remake missing lobes; maintains liver-to-body weight ratio (“hepatostat”). Mechanism: Mature hepatocytes re-enter cell cycle and proliferate. Hepatic progenitor cells activate during severe injury. All liver cell types (hepatocytes, cholangiocytes, Kupffer cells, etc.) divide while retaining identity.  Regeneration in Mammals2 Liver Regeneration continued Bloodstream Signaling: Shear forces on endothelial cells activate Wnt/β-catenin signaling in hepatocytes. Bile acids regulate liver size via Fxr transcription factor activation. Pro-Regenerative Factors: Hepatocyte growth factor (HGF), transforming growth factor α (TGFα), fibroblast growth factors (FGF1/FGF2), and VEGF promote cell proliferation. Termination of Regeneration: ECM restoration, dormancy of HGF, and IlK protein regulate regeneration cessation. Loss of IlK or WNT5a prolongs regeneration, resulting in enlarged livers. Research Significance: Understanding liver regeneration mechanisms could advance treatments for damaged livers. Fig. 24.36: Changes in gene expression correlate with increases in liver mass following partial hepatectomy DNA Synthesis Peaks: Hepatocytes (H DNA) synthesize DNA first, followed by nonparenchymal cells (NP DNA). Peaks correspond to liver mass recovery after partial hepatectomy. Gene Expression Dynamics: Growth-Regulated Genes: Upregulated during initial liver regrowth phase; taper as liver mass normalizes. Cell Cycle-Regulated Genes: Follow a similar pattern, supporting cell division during regeneration. Post Regeneration Activity: Overall gene expression remains high, reflecting functional liver activity in the regenerated tissue. Fig. 24.37: The spiny mouse regenerates instead of scarring Skin Regeneration: Spiny mouse sheds fragile skin down to muscle, regenerating it fully, unlike other mice species. Ear Pinna Regeneration Model: 4-mm ear hole regenerates without scarring, completing regeneration within 40 days. Regeneration is concentrated along the proximal half of the injury, forming a significant blastema by day 30. Role of Macrophages: Macrophage depletion (using clodronate liposomes) inhibits regeneration. Regeneration resumes as macrophages return after stopping depletion treatment. Key Findings: Regeneration involves full restoration of all tissue types, highlighting the critical role of macrophages in regenerative processes.