Axonal Outgrowth and Synapse Formation 1&2 ppt
Axonal Outgrowth and Synapse Formation in the Developing Nervous System
Introduction: The Challenge of Brain Development
The human brain contains approximately neurons.
Each neuron forms thousands of connections, resulting in about synapses formed during development.
Intriguingly, more than of these synapses are subsequently eliminated.
The central challenge: How does the brain accurately generate the correct number and types of cells, in the precise locations, at the right time, and then assemble them into functional neural circuits?
Challenge 1: Number: The sheer scale of neurons and synapses is immense (Prof. Tom Jessell, HHMI Holiday Lectures).
Challenge 2: Shape: Neurons exhibit incredible morphological diversity, essential for their specific functions. Examples include Pyramidal cells, Purkinje cells, Granule cells, Spindle-Shaped cells, Ovoid cells, and various neurons from different nuclei (e.g., Thalamic, Inferior Olivary, Spinal Trigeminal).
Challenge 3: Spatial Organization: Cells must be positioned and connected in highly specific anatomical regions, such as the distinct fields within the Hippocampus (e.g., CA1).
Aim
To investigate the cellular and molecular mechanisms that underlie the development of neural circuits.
For the brain to be functional, it must be "wired" correctly.
Learning Outcomes
By the end of these lectures, you should be able to:
Explain how inductive signals establish neural identity.
Describe the molecular mechanisms of growth cone guidance.
Describe the molecular basis of selective synapse formation and elimination.
All explanations should include specific examples and experimental evidence.
Recap: Stages of Neural Development (BIOL2051/52)
Neurogenesis: The process where neurons are born.
Differentiation: The determination of specific cell fates (what type of neuron a cell will become).
Migration: Neurons move to find their correct place within the developing brain, for example, to build the layers of the cortex.
Target Innervation: The process by which axons detect and grow towards their specific target regions, involving "address selection."
Synapse Formation: The establishment of functional connections between neurons.
Neural Identity and Patterning
Timing and Location: The "when" and "where" neurons are born are crucial determinants of their eventual fate.
Neural Tube Patterning: The neural tube, the precursor to the central nervous system, is highly patterned along two axes:
Anterior/Posterior (Rostral/Caudal): Along its length.
Dorsal/Ventral: In its cross-section.
Morphogens: These signaling molecules generate this intricate pattern. They are secreted by specific structures, including the notochord, floorplate, and roofplate.
Neural Tube Formation
22 \text{ days}$: Shows anterior neural folds, neural crest, central canal, and the notochord positioned underneath the neural tube.
24 \text{ days}$: Illustrates the formed neural tube, sensory ganglia derived from neural crest, somites (mesodermal structures), rhombencephalon, floorplate, and notochord. The floorplate and roofplate are key signaling centers.
Inductive Development and Cell Fate Determination
Conditional Specification: Cell fate is determined by external "inductive signals" that guide progenitor cells.
Signal Transduction: These signals (e.g., from the notochord) stimulate various intracellular signal transduction pathways.
Gene Expression Alterations: This leads to changes in gene expression (e.g., of HOX genes), resulting in differential gene expression profiles.
Receptivity: A progenitor cell's receptivity to a signal is determined by:
Its distance from the secreting cells (creating concentration gradients).
The availability of the ligand.
The presence and type of receptors on the cell surface.
How Morphogen Gradients Lead to Different Cell Types
Receptor Binding: Morphogens bind to specific receptors on target cells.
Transcription Factor Modulation: This binding activates or represses sets of transcription factors.
Gene Expression Programs: These transcription factors then control distinct programs of gene expression within the cell.
Identity Determination: The resulting gene expression profiles ultimately determine the cell's identity and its differentiation pathway.
Examples of Signaling Pathways for Neural Induction
Sonic hedgehog (Shh):
A peptide signal.
Crucial for the closure of the neural tube and for establishing ventral identity in the spinal cord and hindbrain.
Mechanism: Binds to Patched-1 (PTCH1), which normally inhibits Smoothened. This binding releases PTCH1-mediated inhibition, allowing Smoothened to accumulate on the cell surface. This disinhibits Gli1/2 signaling, leading to gene transcription.
Retinoic acid (RA):
Released by the floorplate and somites.
A derivative of Vitamin A; it's a steroid family, lipophilic molecule that can cross cell membranes.
Mechanism: Binds to the Retinoic Acid Receptor (RAR), which then binds to DNA. Together with co-repressors or co-activators, RAR-RA complexes modulate gene expression profiles.
Fibroblast Growth Factor (FGF):
Released by the mesoderm.
A family of peptides (over different FGFs known).
Act short-range because they bind to the extracellular matrix (ECM).
Mechanism: All FGFs bind to Fibroblast Growth Factor Receptors (FGFRs), which are receptor tyrosine kinases (RTKs). This binding initiates the Ras-MEK-MAPK signaling cascade, leading to gene expression.
Bone Morphogenetic Protein (BMP):
Released by the somites.
Peptides, members of the Transforming Growth Factor $eta$ (TGF$eta$) family.
Mechanism: Activate receptor serine kinases. These kinases phosphorylate receptor-regulated SMADs (R-Smads), which then associate with co-SMADs (e.g., Smad4). The SMAD complex translocates to the nucleus and acts as transcription factors.
In the ectoderm, BMPs typically induce skin formation. However, in the context of the neural tube, their activity is tightly regulated.
Sources of Key Morphogens
Notochord: Essential for early neural induction and the formation of the floorplate.
Floorplate: A major source of Shh, Retinoic acid (RA), and Chordin.
Roofplate: A major source of BMPs, Retinoic acid (RA), and Noggin.
Somites (mesodermal structures): Give rise to the skeleton and muscle, and are sources of BMPs and RA.
Regulation of Bone Morphogenetic Proteins (BMPs)
Extracellular Regulation: BMP activity can be regulated extracellularly by antagonist molecules.
Noggin, Chordin: These molecules bind directly to BMPs.
Effect: This binding prevents BMPs from interacting with and activating their receptors, thus inhibiting BMP signaling.
Significance: This regulation is crucial to prevent neuroectoderm from turning into epidermis, ensuring proper neural development.
Later Roles: After initial neural identity is established, BMPs can still signal to influence the fate of differentiating neurons.
Mechanism of BMP Signaling: BMPs bind to Receptor Type II (BMPRII) and Receptor Type I (BMPRI). This leads to phosphorylation of R-Smads. Phosphorylated R-Smads associate with Smad4 (a co-Smad) and sometimes cofactors. This complex translocates into the nucleus, binding to DNA, often with co-activators/repressors, to regulate gene transcription.
How a Gradient Establishes Different Identities: Transcription Factor Interactions
Initial Response: The amount of transcription factor (TF) available and the sensitivity of the cell (determined by receptor expression) dictate the initial cellular response.
Hierarchical Control: The first set of TFs activated by morphogens often determines the expression of a second set of TFs.
TF Interactions: TFs can interact in various ways:
Activation/Repression: They can activate or repress the expression of other genes, including other TFs.
Mutual Repression: Activation of one TF can initiate a program that actively represses another TF within the same cell or in neighboring cells (e.g., via Delta-Notch signaling).
Boundary Formation: Mutual repression is a key mechanism for establishing sharp boundaries between different cell populations.
Examples of Transcription Factor Interactions
Patterning of the Early Cortex (Anterior-Posterior Axis):
Pax6: Expressed at high levels in the anterior part of the developing cortex; it instructs the development of the frontal and motor cortex.
Emx2: Expressed at high levels in the posterior part; it instructs the development of the visual cortex.
Interactions: These TFs, along with signaling molecules like FGFs, BMPs, and Wnt, establish a gradient that patterns the cortical areas. Disruption of this balance (e.g., altered Emx2 or Pax6 levels) leads to mispatterning of cortical regions.
Spinal Cord Patterning (Dorso-Ventral Axis):
Shh Gradient: A ventral concentration gradient of Shh from the floorplate is critical.
TF Domains: This gradient establishes distinct domains of transcription factor expression (e.g., Dbx2, Pax6, Nkx6.1, Nkx2.2, Pax7, Olig2).
Class I/Class II Transcription Factors: These TFs are often categorized by their response to Shh levels, leading to the specification of different neuronal fates along the ventral spinal cord.
Consequences of Morphogen Signaling Errors
Lack of Shh Signaling: A severe disruption in Shh signaling can lead to significant developmental defects:
Loss of ventral identity in the neural tube.
Enlargement of dorsal areas.
Lack of segregation between distinct neural areas.
Synophthalmia ("cyclopia"): A dramatic malformation characterized by the partial or complete fusion of the eyes, often due to severe defects in midline brain structures that rely on Shh signaling.
Reminder: Neuronal Migration
Neurons must migrate to their correct positions after birth.
Cortex Example: The cerebral cortex develops in an "inside-out" manner, with later-born neurons migrating past earlier-born neurons to form superficial layers.
Axonal Outgrowth and Growth Cone Guidance
To be functional, the brain needs to be "wired" correctly, which means axons must navigate precise paths to their targets.
Cell Polarity: A crucial early step is the establishment of neuronal polarity, where one process becomes the axon and others become dendrites. This process involves specific "Partitioning genes."
Overview of Growth Cone Guidance
Guidance Cues: Growing axons use a variety of cues or signals to navigate from "stepping stone" to "stepping stone" through the complex environment.
Fasciculation: Axons often grow in bundles, adhering to each other along the way.
Types of Cues: Guidance cues can be:
Attractive or Repulsive (e.g., Netrins, Semaphorins, Ephrins, Slits).
Short-range (cell-to-cell contact) or Long-range (diffusible).
Gradients: These cues often act via concentration gradients.
Interpretation: The growth cone, located at the tip of the growing axon, interprets these signals and responds accordingly.
The Growth Cone
Pioneer Description: Ramón y Cajal (in ) first described the growth cone as the motile structure leading axonal extension.
Transient Structure: Growth cones are dynamic, temporary structures.
Sensing and Guidance: They are primarily responsible for sensing the extracellular environment and guiding the axon across considerable distances.
Role in Force Generation: Growth cones generate the force required to extend and retract, pushing the axon through surrounding tissue.
Inside the Growth Cone: Structure and Function
Key Features: Hand-like structures with fine extensions.
Receptor-Mediated Sensing: Receptors on the growth cone's surface enable it to detect environmental signals.
Cytoskeletal Changes: Activation of these receptors drives rapid and dynamic changes in the organization of the cytoskeleton.
Adhesion: Adhesion molecules facilitate anchoring to the substrate, providing traction.
Directionality and Force: These combined actions provide the necessary directionality and mechanical force for axonal outgrowth.
Cytoskeletal Dynamics Driving the Growth Cone
The growth cone's motility is driven by the dynamic interplay of two main cytoskeletal components:
Actin Cytoskeleton: Drives rapid changes in growth cone shape.
Actin Treadmilling: A continuous process of actin polymerization at the leading edge and depolymerization at the trailing edge, generating protrusive force.
Rho Family GTPases: Critical regulators of actin dynamics (Rac, Cdc42, RhoA).
Myosins: Motor proteins that interact with actin filaments to generate contractile forces.
Microtubule Cytoskeleton: Provides stability and directionality to the axon.
Dynamic vs. Stable Microtubules: Microtubules in the growth cone are highly dynamic, whereas those in the axon shaft become more stable.
Bundling: Microtubules are bundled together to form the core of the axon.
Coupling: A crucial aspect is the precise coupling and coordination between the actin and microtubule cytoskeletons.
Actin Dynamics in the Growth Cone
Sensing Structures: Lamellipodia (broad, flat protrusions) and filopodia (thin, finger-like projections) are highly dynamic, actin-based structures that rapidly sense the environment and change shape.
Actin Treadmilling Proteins: Actin dynamics are driven by proteins that polymerize, depolymerize, or sever actin filaments:
Arp2/3 complex: Creates branched actin networks, driving the formation of lamellipodia.
Formins: Nucleate straight, unbranched actin filaments, promoting the formation and extension of filopodia.
Rho Family GTPases in Growth Cone Guidance
Signal Transduction: These small GTPases (Rac, Cdc42, RhoA) translate extracellular guidance signals into specific changes in cytoskeletal organization.
Regulation of Protrusion/Retraction: They regulate protrusion (extension) and retraction (collapse) of the growth cone by controlling actin-binding proteins.
Rho GEFs/GAPs: Guanine nucleotide Exchange Factors (GEFs) activate Rho GTPases, while GTPase Activating Proteins (GAPs) inactivate them.
Specific Roles:
Rac, Cdc42: Generally associated with attraction and growth, promoting protrusions. They activate proteins like Arp2/3, PAKs (which link to adhesion molecules), and cofilin (which severs actin to create new barbed ends).
RhoA: Generally associated with repulsion and collapse of the growth cone. It activates myosin II, leading to contractility and retraction.
Microtubules in the Growing Axon
Organization: Microtubules are regulated by Microtubule-Associated Proteins (MAPs).
Proteins (e.g., EB3, CLASP): Bind to the growing of microtubules.
Function: They protect microtubules from "catastrophe" (rapid depolymerization) and are implicated in directing growth cone guidance.
Stabilizing Proteins (e.g., Tau): Promote microtubule assembly and stability.
Destabilizing Proteins (e.g., Stathmins): Promote microtubule disassembly.
Severing Proteins (e.g., Spastin, Katanin): Cut microtubules into shorter filaments, influencing their dynamics and organization.
Integration of Actin and Microtubule Dynamics for Guidance
Guidance Cue Gradient: A guidance cue gradient is sensed by receptors on the growth cone.
Protrusion (Attraction):
Actin Bundle Activation: Tip complex activation leads to bundled actin polymerization and filopodial extension.
Actin Capping: Creation of a dendritic network for lamellipodial protrusion.
Actin Severing: Generates new barbed ends for continued growth.
Microtubule (MT) Growth and Stabilization: Directional growth along actin bundles, polarized delivery of membrane and receptors.
Retraction (Repulsion):
Actin Bundle/Network Loss: Inactivation of barbed end protector proteins and bundling proteins.
Severing without Polymerization: Retrograde actin flow is maintained or increased, but new polymerization is suppressed.
Microtubule (MT) Shrinkage/Destabilization: Increased catastrophe (due to active destabilizing proteins) and decreased rescue (due to inactive stabilizing proteins).
Actin and Microtubule Linkage: Crucial for guidance, with microtubules often growing along actin filament bundles to provide structural support and direct growth.
Summary
Neural Identity: Morphogens establish neural identity by driving specific transcriptional programs.
Migration and Polarization: Neurons migrate to their designated locations, polarize to distinguish axons and dendrites, and then extend their axons.
Growth Cone Motility: Cytoskeletal dynamics, particularly the coordinated actions of actin and microtubules within the growth cone, drive the axon's forward movement and navigation.
Next Topics
Cues that guide the growth cone to its final destination.
Developmental cell death in the nervous system.
Principles of selective synapse formation and elimination.