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Lecture 1

Gap Genes.


Which genes regulate segmentation of the embryo?

Kruppel; Knirps.


What is the Kruppel gene?

Zygotically active gap gene.


What is meant by gap gene?

Mutants of gap genes show defects/gaps in patterns.


What is the effect of mutation of Kruppel?

Lack of posterior segments.


What is the effect of mutation of Knirps?

Lack of anterior segments.


Describe how Kruppel is regulated.

Transcription activated by bcd and medium concentration of hb. Repressed by high hb concentration.


What are the effects of hb on Kr in terms of expression pattern in the embryo?

Kr is expressed within a concentration window of hb. Band of Kr in the middle of embryo.


What is meant by cross-regulation? Give examples of the drosophila embryo.

Regulation not within but between pathways. Kr is repressed by other gap proteins: hb protein at anterior boundary and Knirps/Tailless proteins at posterior boundary.


What is the evidence for Kr repression by Knirps at posterior boundary?

Knirps mutants show Kr expression extending towards the posterior.


What are gap proteins?

Transcription factors. Involved in cross-regulation including regulation of pair-rule gene expression.


What are pair rule genes? Give an example.

Group of zygotic genes with related phenotype. Even skipped and fushi tarazu genes.


What is the function of pair rule genes in drosophila embryo?

Definition of body segments.


What is the function of even-skipped gene?

Definition of odd numbered parasegments.


What is the function of the fushi tarazu gene?

Definition of even numbered parasegments.


Describe the structure of segments in terms of parasegments.

Each segment is made up of the anterior part of one parasegment and the posterior of another.


How many parasegments exist in the drosophila embryo?



Describe the expression regulation of eve leading to the formation of the 3rd parasegment (2nd stripe).

Each eve stripe is defined separately. DNA footprinting determined that low concentration of bcd and a high concentration of hb promote transcription of eve.Eve also regulated by dissection of sequence upstream of eve gene.Eve expression repressed in 4th parasegment (2nd stripe of ftz) by the giant and kruppel genes.


Lecture 2

Homeotic Genes.


What is the function of homeotic genes?

Regulation of genes involved in morphological organisation.


What regulates homeotic genes?

Gap genes; Pair-rule genes; Other homeotic proteins.


Give examples of 3 homeotic gene mutants in drosophila.

Antennapedia; Bithorax; Postbithorax.


Describe the Antennapedia mutant phenotype.

Change of appendage. Legs instead of antennae.


Describe the bithorax mutant phenotype.

Transformation of the anterior part of a haltere to a wing.


What is a haltere?

A non-vestigial devolved wing, used as a balance organ.


Which segment of dipterae body have a wing and which have a haltere?

Wing - T2; Haltere - T3.


Describe the postbithorax mutant phenotype.

Posterior part of the haltere becomes a half-wing.


What is HOM-C?

Homeotic Complex. Genomic region containing many homeotic genes which are conserved between species.


Describe the conserved sequence of HOM-C.

180bp - 60aa protein - Homeodomain - Transcription factor.


Describe the structure of the homeodomain.

Helix-turn-Helix. Conserved between distant species.


Describe the Drosophila homologues in mammals.

Homeobox genes - contain a homeobox. (20 other gene families contain a homeobox but aren't hox genes).


Describe the antennapedia complex.

5 genes: labial (lb); Proboscipedia (pb); Deformed (Df); Sex-combs reduced (Scr); Antennapedia (Antp). Contains 3 others: bcd; ftz; zrk. Affects segments of head and thorax.


Describe the bithorax complex.

3 genes: ultrabithorax (ubx); Abdominal-A (Abd-A); Abdominal-B (Abd-B).


Explain the importance of the order of genes in a homeotic complex.

Order of genes follows order of the regulated structures. Genes expressed in overlapping domains which means order must be conserved.


BX-C mutants show abnormal segmentation. What does this suggest?

Action of all 3 needed for correct segmentation.


How many Hox gene clusters are there in mammals?



Describe the evolution of the hox gene loci in vertebrated. Support.

Evolved as a result of gene duplication. 10/13 loci of vertebrates are homologous to 10/10 loci of Cephalocordates.


How are hox genes studied in mice?

ISH to mRNA; Transgenic knock-out/-in experiments.


What are knock-in experiments?

Insertions of novel loci into the genome.


Describe the pattern of vertebrate hind brain development.

Combinations of hox gene expressed determine the structures that form. Hox gene sequences give positional information about the structures they code (A-P positional information).


Give an example of where positional information of hox gene expression can be observed.

A-P pattern of the neural tube and somites of mouse embryo.


Describe the effect of a mutation in the Hox c8 gene in vertebrates.

Transformation of the 1st lumbar vertebrae into an extra thoracic vertebrae.


Describe the positional information given by hox genes for the structures of an arm.

Proximal-Distal information: Hox9 - Scapula; Hox10 - Humerus; Hox11 - Ulna + Radius; Hox12 - Metacarpals; Hox13 - Digits.


Describe, using an example, the effect of hox gene mutations on human arm development.

LOF mutations of Hox a11 and Hox d11 cause fusion of wrist and humerus (no ulna/radius).


How many years ago did the common ancestor of all organisms with hox genes live?

550MYA (Before Cambrian).


What is a Paralogue?

Pair of genes that derive from the same ancestral gene.


What is a Homologue?

One of a group of similar DNA sequences sharing common ancestry.


What is an Orthologue?

One of 2+ homologous sequences found in different species.


Lecture 3

Body Plans.


Give examples of repeating units in different animals.

Segments in annelids/arthropods. Vertebrae in vertebrates. Hands in tetrapods.


How are variations in body plans expressed?

Changes in number and character of segments.


What effect does modular organisation have on body plan variation?

One segment can change without affecting others.


What are the legacy approaches used to study genes involved in body plans?

Comparative molecular embryology; Developmental Biology.


What is the modern approach used to study genes involved in body plans?

Interference RNAs - Decrease expression by interrupting transcription/translation.


How can body plans be altered epigenetically?

Alteration of the expression site of hox genes involved. Changes in target genes (?).


Compare/contrast the vertebral patterns in birds and mammals. What is the cause of the difference?

Same total number of vertebrae. Mammals have 7 cervical vertebrae. Birds have ~14. Difference caused by change in hox expression in somites.


What are somites?

Segments of mesoderm of vertebrate embryos which form vertebrae.


Describe the genetic basis of different number of cervical vertebrae of birds and mammals.

Anterior boundary of Hox C6 expression is different between the two. Anterior boundary shows a sharp gradient. Posterior is more gradual.


How can hox gene regulation be compared?

Comparine Cis-Regulatory Elements by fusing them to reporter genes. Observing the effect of other genes on the reporter genes with hox CREs.


What is the evidence supporting the repeated loss of legs in snakes?

2 types of snake exist, one with pelvic bone, one without. Suggests 2 different mechanisms of leg loss.


How have snakes lost their forelimbs?

Comparison of Hox c6 in snakes and mice shows that in mice, both genes span the flank (space between fore and hind limbs), in snakes the genes are expressed in the flank as well as the forelimb region. Loss of forelimbs therefore caused by expansion of Hox c6 domain causing most vertebrae to become thoracic.


Compare segmental organisation of brine shrimp and the grasshopper.

Brine shrimp - Exen expression of hox genes in thoracic segments - Segments very similar; Grasshopper - Hox gene expression spatially separated - Segments show different characteristics.


Which genes are responsible for segmental organisation?

Antp; Ubx; Ubx+Abd-A; Abd-B.


Which gene regulates insect hindwing morphology?



Describe the features of modern insects which support the theory of the common ancestor being a 4-winged insect.

Diptera - Forewings + halteres; Lepidoptera - Forewings + Hindwings - Camouflage/mimicry adaptation; Coleoptera - Hard case + Hind wing. All 3 groups have 4 wings with different adaptations.


What is the genetic basis of evolution of the haltere?

Ubx represses genes needed for hindwing growth and flattening.


What is the genetic basis for the adaptations of lepidopterian hind wing?

Ubx regulates genes involved in pigmentation of the hindwing and shape of the scales.


What is the function of the distal-less gene? Support.

Promotes limb outgrowth. Mutants have truncated limbs.


Suggest limb morphology of common ancestor of bilaterians based on Dll expression in modern clades.

Common ancestor must have had primitive limbs promoted by Dll since Dll is expressed with the same function in all 3 clades of bilaterians.


Describe the interaction between Dll and Ubx.

Dll sequence contains binding sites for Ubx. Ubx represses Dll (hence haltere in diptera).


Using modern examples, describe the abdominal limb morphology of common ancestor of arthropods.

Insects have no limbs - repressed by Ubx. Centipedes do. Ancestor must have had primitive limbs on all segments. Removal of Ubx repression may be the novel trait in centipedes however lepidoptera larvae show prolegs on abdomen - no legs in adult which further proves the former theory (ancestors had legs on all segments).


Describe the genetic basis of proleg expression in butterfly larvae.

Ubx expressed in abdominal segments. Ubx should repress Dll causing no legs to be expressed. Presence of prolegs suggests modulation of Dll expression by Ubx.


Lecture 4

Flexibility and Constraints.


What is pleiotropy? Gvie an example.

Control of many traits by a single gene. Hox genes.


What is genetic flexibility?

Ability to acquire existing regulatory motifs.


Describe genetic flexibility in freshwater sticklebacks.

Pitx1 gene is responsible for development. The coding sequence of Pitx1 is identical in freshwater and marine sticklebacks. The Cis-Regulatory fragment responsible for expression of Pitx1 in the "pelvic limb" has been inactivated in freshwater sticklebacks, causing a reduction of the pelvic fin.


What are genetic constraints?

Expression possibilities limited by ability to use only existing motifs.


Describe genetic constraints in butterflies.

Engrailed wing pigment pattern only expressed if the engrailed protein is bound to an enhancer region. Distal wing pigmentation pattern is only expressed if the distal protein is bound to an enhancer region.


Describe genetic constraints in human vertebrae.

7 cervical vertebrae in mammals. Alterations of hox gene function causing extra cervival ribs instead of vertebrae mostly causes death in utero. Children with extra ribs also have a high cancer incidence suggesting the function of the hox gene responsible is also associated to cell proliferation.


Lecture 5

Programmed Cell Death.


What are the 2 types of cell death? Describe their properties.

Pathological - Necrosis. Caused by burn/damage to tissue. Results in rupture of cells and inflammation of tissue due to escape of proteases. Apoptosis - Programmed cell death.


What is Caenovhabditis elegans?

A transparent nematode.


Why is C. elegans used to study PCD?

Transparency allows identification of every cell. Laser ablation of a specific cell is possible.


Where does PCD occur?

Edges of lesions.


Outline the cellular processes during PCD.

Nucleus and cytoplasm condense. DNA is fragmented. Proteins are cleaved. Cell fragments packaged/membrane-bound to prevent damage of surrounding tissue by e.g. proteases. Phagocytosis of remnants to avoid leakage/inflammation.


What suggests that there is only one mechanism for PCD?

Apoptosis shows similar phenotypes in different contexts.


How many cells are in adult C. elegans?



How many cells die during development of C. elegans?



Describe the normal cell death pattern in C. elegans.

One cell gives rise to 2, then 4 daughter cells. One of the 4 daughter cells (3rd gen) dies.


Describe the process and genetic control of PCD.

Ced-3 and ced-4 genes induce apoptosis. Ced-9 gene inhibits ced-3 and ced-4. If apoptosis is induced. Cell undergoes blebbing and is engulfed via phagocytosis.


Lecture 6

Roles of PCD.


What are the 4 roles of PCD?

Deletion of unwanted/redundant structures; Sculpting of developing structures; Removal of dangerous/injured cells; Regulation of cell number.


Give 2 examples of vestigial structures which are deleted by apoptosis.

Pronephric tubules; Notochord.


Describe the role of apoptosis in dimorphism.

Deletion of tadpole tail in temporal dimorphism of the frog. Deletion of either the mullerian of wolffian ducts, depending on the gender, in sexual dimorphism of mammals.


What causes mullerian apoptosis?

Induction by MIF gene and activation by testosterone.


Describe the role of apoptosis in sculpting of feet in chicks and ducklings.

Both duck and chick express BMP in the interdigital mesoderm (protein responsible for PCD). Ducks also express Gremlin - a protein which inhibits the action of BMP.


What evidence is there to show that PCD pattern is determined by the mesoderm?

Chick mesoderm surgically combined with duck ectoderm leads to separated digits. Duck mesoderm surgically combined with chick ectoderm results in webbed feet.


Describ the theory surrounding cell number regulation in the nervous system.

Neurons survive if they contain NGF - nerve growth factor. Limited numbers of NGF are produced in target cells. Nerves which proliferate correctly, making functional connections with target cells, are rewarded with NGF - keeping them alive.


Describe the experiments suggesting that extracellular signals are important in PCD regulation.

Cells cultured with NGF do not undergo cell death. Cells cultured in low density (signals don't reach other cells) do.


Give examples of local and systemic signals which activate PCD.

Local - TNF/MIF/BMP; Systemic - Thyroid hormones.


How is PCD regulated?

Removal of survival signals activated PCD. Provision of activating signals. Combination of both.


Describe the action of the vertebrate homologue of ced9.

bcl-2. Forms pores in intracellular membranes. Inhibits death. May dimerise with death promoting bax protein - Ratios determine probability of PCD.


Describe the action of the vertebrate homologue of ced3.

ICE. Cysteine protease (caspase). Cleaves Asp-X (nucleus/lamina; cytoskeleton; ER; Cytosol). Triggers proteolytic cascade if activated.


What is the evidence showing that caspase is the central component of all PCD?

Caspase inhibitors block PCD in all tested animals.


Describe the action of the vertebrate homologue of ced4.

Apaf-1. Binds to and activates caspases.


Draw the PCD pathway in vertebrates.

Growth factors; Bax; Bcl-2; Caspases; Apaf-1; (Ligand; FADD); Active caspases - Membrane blebbing/Cytoskeletal collapse/Genome degradation.