Invertebrate Development

Question 1

What fates do the three embryo layers acquire during development?

Answer 1

Ectoderm: Outer surface, CNS, neural crest
Mesoderm: Notochord, Bone tissue, tubule cell of kidney, RBC, facial muscle
Endoderm: Digestive tissue, pharynx, respiratory tube

Question 2

Why are fruit flies used as a model for nervous system development and function?

Answer 2

Humans and flies diverged more than 500 million years ago but 75% of disease-related genes in humans have functional orthologues in the fly
Smaller NS than human including bisymmetrical brain
Fruit flies can learn
Have segmental nerves similar to ours and a ventral nerve chord (VNC) instead of a spinal cord

Question 3

Describe the fruit fly life cycle with nervous system details.

Answer 3

Embryogenesis (lasts about 24 hours) - NS established
1st instar larva (motile creature so needs to be able to coordinate movement e.g. crawl towards food)
2nd instar larva
3rd instar larva
Pupa (still has NS even though not a motile part of the life cycle - allow adult to crawl out)
Adult
Refined and added to later on

Question 4

How does the nerve cord differ in position in invertebrates and vertebrates?

Answer 4

In invertebrates, the nerve cord is on the ventral side and the circulatory system is on the dorsal side
Vertebrates is on the opposite side

Question 5

Describe the method of using forward genetics to discover genes.

Answer 5

Using mutagenic agent to cause a mutation somewhere in the genome
Identify lines with developmental defects
Identify gene that has been mutated
Give it a name related to phenotype
e.g. hedgehog mutant results in short and stubby embryo with hairs

Question 6

What are the 4 steps in formation of the nervous system?

Answer 6

1. Neural Induction (regions of ectoderm endowed with neurogenic capabilities: D-V axis)
2. Neural patterning (subdivision along D-V and A-P axes)
3. Segregation of neural progenitor cells from the epidermal progenitor cells
4. Division and differentiation: CNS (neuroblasts into neurons and glia) and PNS (SOPs into sensilla - small sense organs)

Question 7

What happens in the early stages of the development of the drosophila embryo?

Answer 7

Early embryo is a syncytium
Nuclear division (not cellular) to produce many nuclei within cytoplasm
Nuclei migrate to the periphery of the cytoplasm
Forms syncytium blastoderm then a cellular blastoderm
Various TFs can diffuse between nuclei and set up AP patterning that wouldn’t be possible if they were cells

Question 8

Describe neural induction as the first step in the formation of the invertebrate nervous system.

Answer 8

Neural Induction = Process by which embryonic cells in the ectoderm decide to acquire a neural fate
Oocytes contain conc gradient of Dorsal protein (TF and morphogen)
High conc of Dorsal promotes Ventral fate
High levels of Dorsal activate production of Snail (TF, inducing mesoderm)

Low/no Dorsal leads to production of Decapentaplegic (DPP - extracell signalling)
Intermediate levels produces SOG (extracellular negative regulator of DPP and induces neuroectoderm)

DPP binds to serine-threonine kinase receptors on cells which communicate with nucleus using TFs (Mads), switch on epidermal genes and switch off neural genes - promoting epidermal fates

Question 9

Describe patterning the DV axis as part of the second step (neural patterning) in the formation of the invertebrate nervous system.

Answer 9

Gradients of DPP, SOG and Dorsal also initiate patterning of neuroectoderm by regulating expression of 3 homeodomain TFs:
- msh (muscle segment homeobox) - high DPP and low dorsal
- ind (Intermediate neuroblasts defective) - middle DPP and dorsal
- vnd (ventral NS defective) - low DPP and high dorsal
vnd inhibits ind which inhibits msh

Question 10

Describe determining neuroblast identities as part of the second step (neural patterning) in the formation of the invertebrate nervous system.

Answer 10

NS also divided in segments - simplifies
Within each segment, 800 neurons derive from fixed arrangement of 60 neuroblasts
Location confers specific identity, confers specific behaviour to each neuroblast

Rationale:
1. divide AP axis into reiterated series of domains
2. Use common local blueprint of neural cell arrangement in each segment

Identity conferred by intersection of genes expressed in segmental fashion along AP axis and genes expressed along DV axis

Question 11

Describe AP patterning as part of the second step (neural patterning) in the formation of the invertebrate nervous system.

Answer 11

Bicoid mRNA (TF) deposited into maturing egg and anchored at Anterior cytoplasm
Synthesised after fertilisation and forms gradient (A high, P low)
Bicoid activates Hunchback expression (TF) which also forms AP gradient
These switch on gap genes in diff regions e.g. knirps, tailless, kruppel, giant
Cross-repressive interactions sharpen boundaries between domains
Gap gene products activate pair-rule genes in a reiterated pattern of smaller domains - establish number/position of segments
Kruppel is activated by certain levels of Hunch

Hox genes determine identity in each segment
Encode TFs, homeodomain interact with DNA
Intersection of patterning along AP and DV axes provides each neuroblast with unique identity
Hox expression modified basic pattern

Pair rule genes co-operate to regulate genes
Activation of wg (wingless) and hh (hedgehog) in adjacent stripes in each segment
wg and hh encode extracell. sig. molecules
Act as local morphogens leading to expression of other segmentation genes in 1-2 cell wide stripes across each segment

Question 12

Describe the third step in the formation of the invertebrate nervous system (Segregation of neural progenitor cells from epidermal progenitor cells).

Answer 12

Ectodermal cells have potential to differentiate into neuroblasts or SOPs but only fraction become committed
Laser ablation experiments demonstrate lateral inhibition (kill neuroblast, one neighbouring ectodermal cell becomes new neuroblast)
Initially all proneural cluster cells express Delta which binds to Notch receptor on neighbouring cell
Notch activation causes expression of HES family of transcriptional repressors (block expression of proneural genes)
One cell escapes inhibition and continues to inhibit neighbours - these lose neural competence and revert to epidermal fate
Notch pathway conserved in vertebrates

Question 13

What is the difference between symmetric and asymmetric division?

Answer 13

Symmetric division is proliferative (produces 2 identical cells from one cell)
Asymmetric division permits differentiation (Produces 1 like parent and one to differentiate)

Question 14

Describe Division and Differentiation as the 4th step in the formation of the invertebrate nervous system.

Answer 14

SOP divides asymmetrically to give 3 support cells and a neuron
Neuroblast repeatedly divides asymmetrically to self-renew and generate ganglion mother cell (GMC) - displacement of earlier born GMC - GMC divide 1 to generate 2 neuron

Numb (notch regulator) becomes localised to one side of SOP (1 daughter gets lots)
Apical/basal ends have accumulations of diff proteins - set plane of division

Neuroblasts divide to produce diff types of neuron caused by strict sequential expression of TFs (Hunchback, Kruppel, PDM1, Castor)
GMC inherits factor expressed at production
Displacement causes 1st born inside NC

Question 15

What are the methods that axons can use to find their targets?

Answer 15

1. Follow existing tracts - as long as pioneer neurone is there, neighbours can grow along that path already created; Follower axons can fasciculate (bundle to form fascicle)
2. Intermediate targets - guidepost cells e.g. grasshopper leg - Axon of sensory neurone Ti1 grows to reach CNS guided by neurones Fe1, Tr1 and Cx1 - experiment ablating Cx1 means axon goes off course, showing importance
3. Contact Guidance - Short range - extracellular matrix or integral membrane components; growth cone changes direction via cytoskeleton; contact attraction or repulsion
4. Diffusible cues - chemotropism - long range

Question 16

What are the characteristics of ligand and receptor families?

Answer 16

Modular nature (certain domains e.g. slit)
Domains repeated and shared between different families (e.g. nephrin and slit)
Attraction can be positive or negative
Signals usually unidirectional but can be bidirectional if both are membrane bound

Question 17

What are the characteristics of Semaphorins?

Answer 17

Large family of cell surface and secreted proteins
Receptors are multimeric complexes of plexins and neuropilin
Grasshoppers - band of semaphorin-1 expressing epithelial cells between Tr1 and Cx1

Question 18

Describe the attraction to the ventral midline in the context of commissural axon pathfinding.

Answer 18

Send axons to midline where ventral nerve cord is forming before they cross midline and send growth cones towards anterior of animal (opposite side to where cell body is, giving ladder-like structure)
Netrin expressed at midline as chemoattractant
(Netrin mutant - don’t see ladder like appearance as many aren’t attracted)

Question 19

Describe the repellant activity to the ventral midline in the context of commissural axon pathfinding.

Answer 19

Slit expressed at midline, repels axons
Robo (receptor for slit) expressed on axons - series of circles instead of ladder
Robo2 sufficient to tell them not to go up the midline but can’t prevent recrossing

Initially low robo receptor on growth cone, GC is attracted to Netrin of midline, levels of robo receptor increase and can be influenced by slit (acts as repellent to take away from midline) so migrates anteriorly without recrossing midline

Question 20

What changes in responsiveness can be made to slit?

Answer 20

Axons unable to cross midline – phenotype of two parallel lines
Early on (for some axons) comm is off, robo is high and never cross midline
For commissural axons, Commissureless (Comm) is on and sequesters/prevents Robo from reaching growth cone surface
Comm levels drop once axon has crossed midline
Robo reaches plasma membrane and axon is responsive to Slit repellent

Question 21

What mechanisms are apparent in the spatial patterning of dendritic branches in the PNS?

Answer 21

1. Self avoidance: Dendrites from same cell avoid crossing so evenly-spaced dendrites and minimal redundancy
   E.g. Down’s syndrome cell adhesion molecule (Dscam) is cell surface receptor and can have 38000 isoforms
   One dendrite from one part of the cell can tell if the one it comes in contact with is in the same state by the isoforms, same=no overlap
2. Tiling: Dendrites of neighbouring sensory neurons of same class avoid crossing resulting in contiguous but non-overlapping fields

Question 22

What are the functions of the distinct regions in the adult drosophila brain?

Answer 22

Antenna lobes are involved in olfaction.
Mushroom bodies are involved in learning and memory
Medulla and Lobula form part of the optic lobe which receive photoreceptor projections from the eye

Question 23

What is a connectome?

Answer 23

Comprehensive map of neural connections within an organism’s NS
Completed for C. elegans (only 302 neurons)
Still require functional relationships even if we know how it works
Current fly research involves mapping neuronal circuitry underlying vision, olfaction, learning and complex innate behaviours

Question 24

Describe using UAS/Gal4 systems for targeted misexpression of proteins as an example of a technique for elucidating neuronal circuits.

Answer 24

Fly1: Artificially induced Gal4 present downstream of genomic enhancer (nothing happens)
Fly2: Gal4 activating sequence upstream of gene X (nothing happens)
Cross Fly1 x Fly2
Offspring will have both components expressed in certain cell type and will express gene X in just the cells we want
Can be kept individually even if gene X is lethal

Question 25

Describe using optogenetics as an example of a technique for elucidating neuronal circuits.

Answer 25

Using idea of UAS/Gal4 inducible system
Put Channelrhodopsin from chlamydomonas into drosophila with neuron specific Gal4
Only neurones we want will activate
Blue light causes all-trans retinal to undergo a conformational change to 13-cis-retinal which opens the sodium channel, sodium goes into the axon and AP is generated

CHR in giant axon triggers burst of wing beats upon blue light
Experiment with headless flies to show reaction isn’t caused by light entering the eye

Question 26

Describe using thermogenetics as an example of a technique for elucidating neuronal circuits.

Answer 26

Transient Receptor Potential IC - non-selective cation channels, some temp sensitive
Can increase temperature by 10 degrees celsius and make fly unable to walk forward

Question 27

Describe the impact of fruitless as a master regulator of male courtship behaviour.

Answer 27

Present in males and females but differentially spliced
Males lacking FruM appear externally normal but defective in most courtship aspects
Females engineered to express FruM look normal but perform male initial stages
Identified by forward genetics - male mutants would court other males

Question 28

What is the french flag model of morphogens and positional information?

Answer 28

Lewis Wolpert (1960s)
In a hypothetical epithelium, signal is produced by cells at one side of the cell.
This signal makes cells become blue, white or red.
Cells closest receive a signal above the highest threshold and become blue
Further cells respond to a lower dose and become white cells.
Furthest cells do not receive enough of the signal and become red cells by default.

Immediate response is expression of specific TFs in domains which influence cell behaviour/fate through gene expression