Chapter 1 Models and Methods for Studying Neural Development

What is neural development

• process by which the nervous system grows from its first beginnings in the embryo to its completion as a mature functioning system.

What are the Invertebrate model organisms (4)

• Fruit Fly (Drosophila)

• Worm

• Sea Urchins

• Sea Squirts

Why is the fly model organism useful (4)

• short life cycle (2 weeks)

• Cheap and easy to breed

• Large number of eggs(collected easily)

• Embryogenesis only takes 24 hours

What is the Fly model organism history (3)

• mutant flies with abnormal phenotypes

• The analysis of these mutant lines led to the discovery of the genes that caused theses genetic defects and so researches gained knowledge on the function of these critical genes that cause the abnormal phenotypes

• Used for both forward and reverse genetics approaches

What is the forward genetic approach

• genetic approach in which the aim is to identify a mutated gene responsible for a phenotype of interest.

What is the reverse genetic approach

• a genetic approach in which genes are selected for study, for example based on their sequence, and then manipulated to discover their function.

What method is used for studying fruit fly and why

• GAL4/UAS system

• allows specific genes to be activated in a spatially and temporally controlled manner and it can be used in a variety of ways. Eg gain of function or loss of function approach

What is the gain of function and loss of function approach

• For example, genes normally found in the Drosophila genome can be activated by the experimenter to discover what they do (called a gain‐of‐function approach).

• Alternatively, the method can be used to activate genetic inhibitors manufactured by the experimenter to produce molecules that block the actions of a specific Drosophila gene (called a loss‐of‐function approach).

Why is the worm (Caenorhabditis elegans) model organism useful (5)

• easy to maintain in the laboratory and viable organisms can be stored frozen.

• rapid development within 2–3 days

• transparent

• its anatomy is known in precise detail eg all of its neurons and the connections between them are known.

• Detailed knowledge of cell lineage from zygote to adult worm which is unusual and valuable as most model species lineage is incomplete

Why is the worm (Caenorhabditis elegans) model organism cell lineage useful (2)

• for any cell at any point in normal development it is possible to know what that cell will do and what it will become, that is its fate.

• Against this background of precise morphological knowledge, it is relatively straight- forward to study gene function by forward or reverse genetic methods.

What is hermaphrodite and what is the relationship between hermaphrodite and the worm

• hermaphrodite = an organism with both male and female sexual characteristics and organs.

• Since one of the sexes of C. elegans is hermaphrodite (the other is male), mutant worms that are severely defective and would be unable to mate can still be bred via self‐fertilization.

What are the vertebrate model organisms (8)

• Frog (Xenopus Laevis)

• Chick

• Zebrafish

• Mouse

• Human

• Ferret

• Cat

• Monkey

Why is the model organism frog (Xenopus Laevis) useful (3)

• large number of eggs

• Embryos are easily accessible as they develop outside of the body eg perform microsurgery

• Reverse genetic approach as the size, accessibility and robustness of Xenopus eggs and embryos does make them favourable targets eg an injection of molecules designed to raise or lower levels of specific gene products.

Why is the model organism frog (Xenopus Laevis) not useful (2)

• not ideal for forward genetics because it takes many months for females to reach maturity, which would make the breeding required to establish mutant lines difficult.

• They also have four copies of many genes (allotetraploid), complicating the study of inheritance.

Why is the model organism Chick useful (4)

• Its easy for the eggs to be obtained and stored.

• the embryo has a short incubation time eg the nervous system is well developed after only a few days

• it is relatively easy to observe and manipulate the embryo by opening a small window in the shell. Eg micro-manipulation

• Recently been used for testing the functions of developmental genes using mRNA‐mediated reverse genetic methods

Why is the model organism Zebrafish useful (3)

• embryos develop externally, they are readily accessible for experimental manipulation.

• Its embryos develop rapidly and are translucent, allowing morphogenesis to be visualized and recorded relatively easily.

• Can be used for both forward and reverse genetic approaches

Why is the model organism Mouse useful (2)

• It breeds relatively quickly and easily with large numbers of offspring and it is genetically tractable.

• Similarities between mice and humans extend to the DNA level: a study reported in 2002 that 99% of mouse genes have homologues in humans and 96% of these genes in the two species are arranged on the chromosomes in the same order.

Why are methods such as the CRISPR/Cas9 system used (2)

• improve the efficiency of the genetic changes in the ES cells

• CRISPR/Cas9 has been used effectively in a wide range of other vertebrates and invertebrates.

How does CRISPR/Cas9 method work for mouse model organism (5)

• CRISPR/Cas9 Method allows for targeted, highly efficient alterations of genome sequence.

• Once modified the ES cells can then be injected into early mouse embryos to generate chimeras (a chimera is an individual created when cells of different genotypes come together to form an embryo)

• these chimeras, some of the cells derived from ES cells with modified genomes will form germ cells.

• therefore genetic alterations made in the ES cells can be transmitted through the germ line to subsequent generations .

• This forms transgenic mice with specific additional DNA sequences (known as knock‐in mice) or with loss‐of‐function mutations in specific genes (known as knock‐out mice) have been established and studied.

What are conditional transgenic mice and what are their advantage’s (4)

• Animal in which only some cells in the organism are mutated.

• This is useful because the experimenter can focus on the effects of disrupting a gene only in specific cells of interest.

• It also minimizes complications arising from the possibility that if all the organism’s cells are mutated, the cells of interest will also be affected by secondary, collateral consequences of the gene’s loss in other regions.

• The conditional transgenic approach can also be used to mark specific cells early in development so that their progeny can be followed in subsequent development.

How are conditional transgenic mice created

• cre‐loxP system

How can human embryos be examined

• MRI = investigate their structure and molecular constitution in great detail in specimens obtained post‐mortem.

Why are Ferrets used as model organisms

• ferrets are born more immature than many other mammalian species commonly used in research, allowing easier access to the developing nervous system at an earlier stage.

Why are Cats used as model organisms

• Cats, unlike mice, have relatively high resolution binocular vision and have been subjects of research on the development of the visual system

Why are monkeys used as model organisms

• Monkeys have the advantage of being closely related to humans. They share many primate‐specific speciali- zations that cannot be studied in other species. They have been used particularly to study the development of neural structures, connections and functions in the nervous system.

What 2 approaches are used for studying molecular genetics of development (2)

(i) forward genetics, where one seeks to find the genes responsible for a particular aspect of an organism’s phenotype

(ii) reverse genetics, where one starts with a gene and manipulates it to discover its functions.