Neurobiology Notes

Drosophila as a Model for Neurodevelopment

  • Learning Outcomes:

    • 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

  • Background – Drosophila as a model for neurodevelopment.

  • Data analysis and mock exam questions.

  • Q&A.

Drosophila Embryo Development

  • All embryos are in lateral view, with anterior to the left.

  • Key structures:

    • Endoderm, midgut

    • Mesoderm

    • Central nervous system (CNS)

    • Foregut, hindgut

    • Pole cells

CNS Development in Drosophila

  • CNS precursors derive from neurogenic regions (purple stripes) of the ectoderm.

  • 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 SHh HNF3β

    • Nkx2.2 Nkx6.1

Drosophila Primary Neuron Cultures

  • Embryo Collection, Dechorionation, and Sorting:

    1. Collect embryos from food vials using a cotton/plug.

    2. Dechorionate embryos by adding sodium hypochlorite for 90 seconds.

    3. Wash with water using a sieve.

    4. Transfer embryos to an agar plate.

    5. Sort embryos by fluorescence and stage.

  • Primary Neuron Populations

    • pCenBr

    • pVisSys

    • gmc

    • mp

    • pVenNC

    • nb

Dynamics in Primary Neuron Cultures

  • Growth cone dynamics

  • Actin dynamics

  • Microtubule dynamics

Data Interpretation Task: Filopodial Length Phenotypes

  • Filopodial length phenotypes in:

    • DMSO-treated wild-type primary neurons

    • Neurons treated with drugs or being mutant

  • Cells are double-labeled for actin (green/white) 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 of filopodia length are normalized and compared with DMSO-treated controls.

  • Numbers above bars indicate the number of filopodia analyzed.

  • Filopodia were completely absent in all cases of CytoD and LatA treatment.

  • 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.

  • Cytochalasin D (CytoD) and Latrunculin A (LatA) are drugs that prevent actin polymerization.

  • Chickadee (chic) is a protein involved in actin polymerization.

Key Findings Summary

  • 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 blocks 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

  • Analyzing the role of Ephrin receptor transcription 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)

  • Motorneurons originating from the LMCm will grow into the dorsal developing limb.

  • Limx1b is a transcription factor that promotes the expression of ephrin-B2.

  • Without Limx1b, ephrin-B2 is not expressed in the dorsal limb.

  • Therefore, motorneurons that express the EphB1 receptor are not repelled and grow towards the dorsal side.

Mock Exam Question 2

  • Studying the development of retinal ganglion neurons using in vitro cell culture of neurons isolated from Xenopus retina.

  • In the culture dish, small, immovable beads are added, 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)

  • A focal adhesion forms between the bead and the growth cone membrane:

    • Laminin binds the Integrin receptor and thereby activates integrin signalling.

    • Activated integrins recruit components of the integrin complex, which in turn recruit and link to actin filaments.

  • The axons will turn/grow towards the bead direction.

    • Actin filaments are attached to focal adhesions but continue to polymerize.

    • This leads to a generation of force/push against the cell membrane, leading to an extension of the growth cone membrane in the bead direction.