19 - Neurogenesis - Part 1 Study Notes

Neurogenesis - Overview

1. Interlude: Two Key Invertebrate Model Systems

   • Neurogenesis in the Roundworm, Caenorhabditis elegans

   • Neurogenesis in the Fruitfly, Drosophila melanogaster

     • Notch signaling is crucial for neurogenesis.

2. Back to Mammals

   • Neurogenesis in the Ventricular Zone of the Neural Tube

   • Development of the Cerebral Cortex

     • Radial glia and formation of cortical layers.

     • Ganglionic eminence.

3. Neurogenesis in the Neural Crest

Early Development in C. elegans

• C. elegans characteristics:

  • Simple structure with about 1000 cells.

  • Rapid regeneration time.

  • Transparency aiding studies.

  • Simple nervous system composed of 302 neurons and 56 glial cells.

• Blastomeres in Development:

  • AB (Large somatic cell): AB blastomere.

  • P1 (Smaller germ line cell):

  • AB derived cells spread out, completing gastrulation.

  • P1 subdivides into different blastomeres (MS, E, C, P3) migrating to the embryo's interior.

  • P4 blastomere develops into germ line cells.

  • P0 is the zygote.

The Next Developmental Phase of C. elegans

• Cell Migration and Functions:

  • AB blastomeres spread across the external surface.

  • Gastrulation leads MS, E, C, and D blastomeres to migrate interior.

  • Neurons originate primarily from the ventrolateral surface from AB progeny cell divisions.

• Nerve Ring Formation:

  • C blastomeres migrate interior, forming the nerve ring.

  • The nerve ring is horseshoe-shaped, containing neuronal processes of sensory (ASH) and interneurons that form synapses.

  • Descendant of AB (Abarpa) undergoes 5 rounds of division to generate 9 neurons and 10 hypodermic cells.

Summary of C. elegans Development

• AB Blastomere Dynamics:

  • First Division: Asymmetric division produces AB and P1 blastomeres, with AB being larger and anterior.

  • Subsequent Divisions: Further divisions produce daughter cells ABa and ABp; their positions shift with ABa more anterior than ABp.

  • Descendants: Include ABarp, ABpla, and ABpra, generating various cells (neurons, supporting cells, hypodermal, pharyngeal, and body mesoderm).

  • Asymmetry: Cell types produced by multiple AB descendants contribute to complexity.

  • Life Cycle: Includes embryonic stage, four larval stages, and adulthood.

Questions for Reflection

• Question 1: Which of the following are true in C. elegans development?

  • A. Life cycle of C. elegans includes the embryonic stage, four larval stages, and adult stages.

  • B. Nervous system develops from AB blastomeres.

  • C. Hypodermis develops from P blastomeres.

  • D. Both A and B.

Drosophila Gastrulation

• Gastrulation Site: On the ventral surface.

• Dorsal Protein Gradient: Found throughout the syncytial blastoderm, which becomes localized to nuclei in the ventral region, forming a gradient from ventral to dorsal.

Neurogenesis in Drosophila Larvae

• Ventral Neurogenic Region: Specified by short gastrulation (sog).

• Neuroblast Formation: Neuroblasts delaminate from ectoderm during gastrulation.

  • “Blast” indicates a dividing progenitor cell.

  • Delamination: Physical separation of cells.

  • Neuroblasts divide to form ganglion mother cells (GMC), with each producing two neurons.

Cell Lineage in Drosophila

• Gene Expression: Neuroblasts express proneural genes via the achaete-scute (AS-C) complex.

  • Proneural Genes: Encode transcription factors to promote neuronal differentiation.

  • AS-C Complex: Comprises four proneural genes - achaete, scute, asense, and lethal of scute.

  • Result of Deletion: Absence of neuroblasts in the fly.

  • Relation to Gene Families: A part of the larger basic helix-loop-helix (bHLH) transcription factor family that drives cell-specific differentiation in various tissues, including muscle and nerve cells.

Proneural Clusters in Drosophila

• Achaete Immunostaining:

  • Identifies segmental proneural clusters, bilaterally symmetrical, two clusters per hemisegment.

• Cell Specification: Initially, a cluster expresses AS-C genes; later, one per segment continues.

• Neuroblast Selection:

  • Laser ablation studies indicate that a delaminating neuroblast inhibits nearby cells from becoming neuroblasts.

Proneural Gene Mutants

• Types of Mutants:

  • Achaete-scute loss-of-function.

  • Neurogenic gene mutants: Notch or Delta loss-of-function.

• Research Methods: Forward genetic studies reveal mutations linked to neuronal differentiation suppression.

Delta and Notch Cell-Cell Signaling

• Mechanism:

  • Components: Delta and Notch signaling mediated through the action of gamma secretase, a protease that cleaves the intracellular domain of the Notch receptor.

• Signaling Pathway:

  1. Ligand Binding: Notch ectodomain activation through Delta binding.

  2. Cleavage by Gamma Secretase: Cleaves Notch, releasing the Notch intracellular domain (NICD).

  3. Nuclear Translocation and Activation: NICD translocates to the nucleus, activates transcription by binding transcription factors.

• Signaling Summary: Delta-Notch signaling and signal transduction influence neuroblast determination.

Delta-Notch Mediated Lateral Inhibition

• Mechanism of Inhibition:

  • Proneural cluster with AS-C bHLH transcription factors activates Delta.

  • A single cell in the cluster randomly expresses more AS-C proteins and Delta, triggering Notch in surrounding cells, inhibiting their AS-C gene expression and preventing their neuroblast differentiation.

• Stochastic Variation: Levels of AS-C, Notch, and Delta proteins vary among cells, leading to neuroblast determination.

Neurogenesis Feedback Mechanisms

• Cell Communication: The cell expressing more Delta activates Notch signaling in neighboring cells, inhibiting their ability to form neuroblasts.

• Feedback Amplification: The increase in AS-C protein expression drives Delta production, leading to neuroblast formation.

Questions for Reflection

• Question 2: Which of the following shows higher expression in neuroblast during Drosophila neurogenesis?

  • A. Delta

  • B. Achaete scute proteins

  • C. Notch

  • D. Both A and B.

Overview of Neurogenesis in Mammals

1. Neurogenesis in the Neural Tube:

   • Contains progenitors for all neurons and glia.

   • Bipolar Morphology: Progenitor cells show in-and-out movement during the cell cycle.

   • Cell Cycle Dynamics:

     • Cells move inward during G2 phase, undergo M-phase at the inner surface (ventricular surface), and migrate outward during S-phase.

2. Cell Cycle Phases:

   • G1 Phase: Growth prior to DNA synthesis.

   • S Phase: DNA synthesis occurs.

   • G2 Phase: Preparation for mitosis.

   • M Phase: Mitotic phase includes mitosis and cytokinesis, resulting in two daughter cells.

Fate Mapping in Neural Tube Development

• Procedure:

  • Retrovirus injected into the neural tube carries a gene for β-galactosidase.

  • This enzyme cleaves a substrate producing an insoluble blue product.

  • Progenitors in S-phase can incorporate viral DNA into their genomes, and progeny inherit the incorporated viral DNA, detectable through staining.

Fate Mapping with Viruses

• Viral Specificity: Retroviruses only integrate into genomes of dividing cells during the cell cycle.

  • Modified genomes can carry genes coding for easily detectable proteins (e.g., GFP).

• Multipotent Progenitors: Capable of differentiating into motor neurons, astrocytes, and oligodendrocytes (glial cells).

Neural and Glial Fate Decisions

• Control Mechanism: Oscillations in proneural genes influence neural or glial fate decisions in progenitor cells. Higher probability of glial fate later in neurogenesis.

Neuron Birthdating Techniques

• Incorporation of Labeled Nucleotides:

  • Nucleotides (3H-thymidine, BrdU, EdU) are incorporated during DNA replication in S-phase.

  • Label availability is limited, hence only dividing cells at that time are labeled.

  • When cells stop dividing, they retain the label; progeny may dilute the label with continued division.

• Pioneering Work: Richard Sidman and Joseph Altman (1960s) pioneered the method for determining neuron birthdates.

Cerebral Cortical Histogenesis

• Birth-Dating Studies: Show the inside-out pattern of cortical development.

  • Injection Timing Examples:

  • Injected at E11: Target subcortical white matter.

  • Injected at E13: Found in deep cortical layers (layers 5 and 6).

  • Injected at E15: Found in superficial cortical layers (layers 2, 3, and 4).

Immature Neuron Migration

• Pathway:

  • Immature neurons migrate from the ventricular zone to the pial surface into the cortical plate.

  • Migration involves passing previous neurons and is guided by radial glia.

Composition of the Cerebral Cortex

• Layers of the Cortex:

  • Layer I: Few neuronal cells, primarily intracortical axons and synapses.

  • Layers II/III: Neurons for intracortical and callosal projections.

  • Layer IV: Neurons receiving thalamocortical projections.

  • Layers V/VI: Neurons projecting away from the cortex; layer V targets vary by modality while layer VI is corticothalamic.

  • Neuronal Origins: Excitatory neurons derive from the dorsal ventricular zone, while inhibitory interneurons arise from the ventral ganglionic eminence.

White Matter and Gray Matter Dynamics

• White Matter: Composed of myelinated axons, functions primarily to transmit signals across the brain, spinal cord, and body.

• Gray Matter: Composed of neuron cell bodies, primarily involved in receiving and processing information.

Temporal and Spatial Gradients in Neurogenesis

• Neuron Birth Patterns:

  1. Neurons are “born” at different times and locations within the CNS.

  2. Gradients Observed:

  • Reverse rostro-caudal gradient: Caudal (spinal cord) develops first, rostral later.

  • Lateromedial gradient: Lateral develops first, medial later.

  • Ventro-dorsal gradient: Ventral develops first, dorsal later.

• Complex Gradients: Certain structures, such as the hippocampus, may exhibit very intricate gradient patterns.

Questions for Reflection

• Question 1: Which of the following are true during development of the cerebral cortex?

  • A. Deeper layers of the cortex, such as layer V and VI, develop first.

  • B. Deeper layers of the cortex develop later.

  • C. Layers I and II develop first.

  • D. Both B and C.