Neurobiology Notes

Drosophila as a Model for Neurodevelopment

  • Understand the use of Drosophila as a model to understand neuronal development and maintenance.

  • Understand the use of primary neurons to study axon outgrowth.

  • Apply lecture content to explain a paper Figure.

  • Discuss and interpret experimental evidence that supports the key concepts of neuronal development.

Today’s Session

  1. Background – Drosophila as a model for neurodevelopment.

  2. One data analysis and two mock exam questions.

  3. Q&A

Drosophila Embryo Development

  • All embryos are in lateral view (anterior to the left).

  • Includes endoderm, midgut, mesoderm, central nervous system, foregut, hindgut, and pole cells (marked in yellow).

CNS Development in Drosophila

  • CNS precursors derive from specialized parts of the ectoderm, specifically the neurogenic regions (purple stripes).

  • The ventral neurogenic region gives rise to the neuroblasts of the ventral nerve cord (aVenNC), which is part of the CNS belonging to the segmented germ band.

  • Shortly after segregation, neuroblasts undergo eight waves of mitosis (Hartenstein et al. 1987).

  • Their progeny, called ganglion mother cells (gmc), are located between the neuroblasts and the mesoderm (ms).

  • Ganglion mother cells and neurons form an irregular layer of increasing thickness on top of the neuroblasts.

  • Neuronal differentiation begins at stage 13.

  • A population of identifiable neurons lays down a scaffold of fibers on the dorsal surface of the CNS.

  • Starting at stage 14, the ventral nerve cord condenses.

Comparison Between Vertebrate and Fly Patterning

  • Drosophila: Dpp, Sog, Vnd.

  • Vertebrates: BMP4, Chd, Msx1, Pax6, Ind, Nkx2.2, Notochord.

Drosophila Primary Neuron Cultures

  • Embryo Collection, Dechorionation, and Sorting:

    1. Let flies lay eggs.

    2. Remove flies.

    3. Add sodium hypochlorite.

    4. Incubate for 90 seconds.

    5. Decant embryos into a sieve.

    6. Wash with water.

    7. Transfer to agar plate.

    8. Sort embryos by fluorescence and stage (pCenBr, pVisSys, gmc, mp, pVenNC, nb).

Studies Using Primary Neuron Cultures

  • Growth cone dynamics.

  • Actin dynamics.

  • Microtubule dynamics

Data Interpretation Task

  • Experimental Setup: Filopodial length phenotypes in: DMSO-treated wild-type primary neurons, neurons treated with drugs, or mutant neurons.

  • Cells are double-labeled for actin (green) and tubulin (magenta).

  • Drug Treatments: 800 nM Cytochalasin D (CytD) for 4 hours; 200 nM Latrunculin A (LatA) for 1 hour (both diluted in DMSO).

  • Quantifications: Filopodia length caused by drug treatment or mutations are normalized and compared with DMSO-treated controls.

  • Numbers above the bars indicate the number of filopodia analyzed in each experiment.

  • p values were calculated using the Mann–Whitney rank sum test (n.s., p > 0.05; ***p < 0.001; red indicates higher than wild type).

  • Scale bar: 10 μm.

Key Reagents and Their Functions:

  • Cytochalasin D (CytoD) and Latrunculin A (LatA): Drugs that prevent actin polymerization.

  • Chickadee (chic): Protein involved in actin polymerization.

Task Questions:

  1. Summarize the key finding as a figure title.

  2. Explain the outcome using knowledge from the lectures.

Task 1: Data Interpretation Summary

Key Finding:

  • Preventing actin polymerization in Drosophila primary neurons leads to a reduction in filopodia length.

Explanation:

  • Filamentous actin is required to shape filopodia.

  • Actin polymerization is essential to generate a force that pushes outgrowth of filopodia through the ‘clutch mechanism’.

  • Blocking actin polymerization through drugs (CytoD or LatA) or genetically by removing a protein required for polymerization therefore blocks the growth of actin filaments.

  • As a result, there is no force generated to push the membrane, and filopodia do not grow out.

Filopodia Growth Through the “Clutch” Model (or Horizontal Rock Climbing)

  • No adhesion = no advance.

  • Adhesion = advance.

Mock Exam Question 1

You are analysing the role of Ephrin receptor transcriptions factors, Ephrin receptors and ligands during limb development innervation.
a) What would happen to innervation of the developing limbs in Limx1b mutants? (2 marks)
b) Explain this outcome using your knowledge from the lecture (4 marks)

Answer:

a) Motorneurons originating from the LMCm (1 mark) will grow into the dorsal developing limb (1 mark).
b) Limx1b is a transcription factor that promotes the expression of ephrin-B2 (1 mark). Without Limx1b ephrin-B2 is not expressed (1 mark) in the dorsal limb (1 mark). Therefore, motorneurons that express the EphB1 receptor are not repelled and grow towards the dorsal side (1 mark).

Mock Exam Question 2

You are studying the development of retinal ganglion neurons using in vitro cell culture of neurons isolated from Xenopus retina. In the culture dish you add small, immovable beads that are coated with the extracellular matrix protein laminin.
a) Explain what happens when the axonal growth cone encounters the bead. (3 marks, 5 lines)
b) What effect does this have on growth direction of the axons and why? (2 marks, 4 lines)

Answer:

a) A focal adhesion forms between the bead and the growth cone membrane (1 mark): Laminin binds the Integrin receptor and thereby activates integrin signalling (1 mark). Activated integrins recruit components of the integrin complex which in turn recruit and link to actin filaments (1 mark).
b) The axons will turn/grow towards the bead direction (1 mark). Actin filaments are attached to focal adhesions, but continue to polymerise. This leads to a generation of force/push against the cell membrane which leads to an extension of the the growth cone membrane in bead direction (1 mark).