1. BioSci 108 lecture 25 (Blumberg) page 1 © copyright Bruce Blumberg 2000. All rights reserved Bio 108 - 3/10/2000 Developmental control genes and cell behavior Contact information office hours W/F 3-4 phone 824-8573 blumberg@uci.edu (preferred contact mode) Lectures posted at http://blumberg-serv.bio.uci.edu/bio108-w2000/index.htm http://blumberg.bio.uci.edu/labtemp/bio108-w2000/index.htm

2. BioSci 108 lecture 25 (Blumberg) page 2 © copyright Bruce Blumberg 2000. All rights reserved Clarification from last lecture Chick limb grafting experiment (Fig 21-36) –to understand the experiment you must understand the question being asked are the signals that pattern the wing and leg different or are the signals the same but the response different? –signals different -> leg graft to the wing should produce something characteristic of wing –signals same but tissue response different - > the graft should produce a structure characteristic of leg the result was toes with claws on the wing, characteristic of the tip of the leg –interpretation - grafted tissue is committed to become leg but not a specific part of the leg because the structure formed was characteristic of the tip of the leg, we conclude that the nature of the signals in the wing and leg is the same –actually, we know which genes make the leg and wing different and it has been demonstrated that overexpression of these genes in the wing converts it to a leg.

3. BioSci 108 lecture 25 (Blumberg) page 3 © copyright Bruce Blumberg 2000. All rights reserved C. elegans and cell behavior We would like to know what are the rules that determine cell behavior and how are they encoded by genes –some of the steps that cells take are autonomous (self-determined) cell-autonomous behavior is intrinsic to a cell, does not depend on signals from other cells –other steps are influenced by signals from neighboring cells non cell-autonomous behavior is action taken under the influence of signals from another cell –one view of development is that it is like a computer program encoded in the genome –it is probably more correct to say that cells operate like an array of little computers (automata) extensive computer modeling has demonstrated that very complex forms can be generated from simple rules in cellular automata –Richard Dawkins, The Blind Watchmaker –Hans Meinhardt, The Algorithmic Beauty of Seashells.

4. BioSci 108 lecture 25 (Blumberg) page 4 © copyright Bruce Blumberg 2000. All rights reserved C. elegans and cell behavior (contd) it would help quite a bit in the study of development if we could trace the progeny of each cell and understand – the developmental choices each takes –the consequences of the choice –effects of altering the factors that control the choice most animals are far too complex for this –despite more than 100 years of study on each, Drosophila and amphibians are not suitable too many cells somewhat variable lineage the nematode worm, Caenorhabditis elegans is ideal (fig 21-39) –lineage of all cells is known! –small (1mm) and transparent –generation time is 3 days, egg -> adult many experiments can be performed on many animals in a small space at low cost –small genome (~10 8 bp) in six chromosomes –about 20,000 genes of these, mutations in about 3000 cause observable phenotypes - “essential genes” –complete genome is mapped and sequenced entire genome is available as cosmids allows any gene to be manipulated and reintroduced into the genome

5. BioSci 108 lecture 25 (Blumberg) page 5 © copyright Bruce Blumberg 2000. All rights reserved C. elegans and cell behavior (contd) –only about 1000 somatic cells and 1-2000 germ cells –anatomy is relatively simple (Fig 21-39,40) bilaterally symmetrical elongated nerve muscle, skin, gut mouth and brain anterior anus at posterior two layer body wall –outer layer, the hypodermis –inner muscular layer single tube of endodermal cells forms the intestine second tube of cells comprises the gonad, somatic cells outside, germ cells inside –two “sexes” hermaphrodite (a female that produces a small amount of sperm) –self -fertilization or cross-fertilization with a male –self-fertilization is good for genetic analysis »heterozygous parent -> homozygous progeny male - produces sperm and can mate with hermaphrodite

6. BioSci 108 lecture 25 (Blumberg) page 6 © copyright Bruce Blumberg 2000. All rights reserved C. elegans and cell behavior (contd) C. elegans has invariant development –egg gives rise to a small worm with 558 cells –after hatching, four larval stages follows results in further growth and sexual maturation –lineage has been mapped movements and progeny of all cells is completely understood –lineage does not vary, unlike most other organisms random variations do not occur, lineage and cell division are predictable position of a cell in the lineage tree will predict its ultimate fate Fundamental question can be answered with C. elegans –embryo is formed from a larger group of cells that can be grouped into fewer differentiated cell types –Are all cells of a particular type descended from a single “founder cell”? lineage analysis shows that this is not true - for C. elegans or most other organisms each class of cells is derived from several founder cells originating in separate branches of the lineage tree cells of similar fate may not be “close relatives” very different cells may be closely related by lineage (neurons and muscle)

7. BioSci 108 lecture 25 (Blumberg) page 7 © copyright Bruce Blumberg 2000. All rights reserved Genes that control development Problem: how does one understand the rules that operate in each branch of the lineage tree to generate an array of cell types, each in the appropriate numbers Mutations in genes can have various effects, depending on the gene affected. –housekeeping genes are those that every cell needs to survive and proliferate control metabolic pathways, DNA synthesis, etc mutations are frequently lethal –other genes may be required to produce proteins that particular differentiated cell types require mutations may cause loss of function but will not affect overall body plan –developmental control genes are specifically required for correct developmental choices mutations will alter the body plan normal cells may be in abnormal pattern normal cells in abnormal numbers developmental control genes affect the lineage tree –may be classified according to which part of the tree is affected –can be associated with rules of cell behavior that generate the specific part of the lineage tree –allows genetic analysis of developmental mechansims

8. BioSci 108 lecture 25 (Blumberg) page 8 © copyright Bruce Blumberg 2000. All rights reserved Genes that control development (contd) mutations come in two flavors –loss-of-function mutations reduce or abolish gene activity typically recessive organisms can usually survive and function normally with one wild-type copy of a gene –gain-of-function increases the activity of a gene produces a new activity usually dominant one mutant copy can cause a phenotype how does one identify for genes in a particular developmental pathway? –search for mutations that disrupt the process –screen progeny of a large population of animals that have been exposed to mutagens chemical, X-ray, etc –after genes are identified, test pairwise for complementation - identifies genes that are allelic to each other (two or 1 figure) –next, search for mutations in other genes that will suppress the effects of the original mutation extragenic suppressors typically encode proteins that interact with the already identified gene

9. BioSci 108 lecture 25 (Blumberg) page 9 © copyright Bruce Blumberg 2000. All rights reserved C. elegans vulva development an excellent model system to study genetic interactions in development is induction of the vulva (egg laying orifice in a hermaphrodite) (Figure 21-41) –ventral opening in the hypodermis that is formed by 22 cells that arise in specific lineages from three precursor cells in hypodermis –anchor cell (does not divide) responsible for generating the passage from the gonad to the hypodermis laser ablation studies show that vulva will not form if anchor cell is lost. all other gonadal cells can be deleted and anchor cell will still induce a vulva if the anchor cell is shifted in position, the vulva shifts as well how to identify genes involved in this process? –mutagenize and screen for changes in the vulva vulvaless - appear to lack the signal multivulva - all six hypodermal cells capable of responding to the anchor cell signal behave as though they have received it. –bottom line - more than 30 distinct genes are involved in regulating vulva development

10. BioSci 108 lecture 25 (Blumberg) page 10 © copyright Bruce Blumberg 2000. All rights reserved C. elegans vulva development (contd) focus on five genes, mutations in any of these will affect vulva phenotype –lin3, let23, sem-5, let 60 and lin 45 –loss-of-function mutations result in vulvaless –gain-of-function -> multivulva –data suggest that these belong to a single genetic pathway how to order genes in a pathway? –create double mutations and test the phenotypes when they are combined –A is upstream of B if its product acts by regulating the product or activity of B –typically the mutants tested are a gain of function in one gene crossed with a loss of function in another –observing which phenotype dominates orders the genes in the pathway genetic analysis shows that lin3 -> let 23 -> sem-5 -> let 60 -> -> lin 45. –lin-3 is required in the anchor cell –others act in hypodermal cells mutation in one will cause multivulva phenotype even when the anchor cell has been destroyed genes have been cloned, sequenced and identified lin-3 (EGF) -> let-23 (EGF receptor) -> sem-5 (GRB-2) -> let-60 (ras) -> lin-45 (raf)

11. BioSci 108 lecture 25 (Blumberg) page 11 © copyright Bruce Blumberg 2000. All rights reserved C. elegans vulva development (contd) genes have been cloned, sequenced and identified (Fig 21-44) lin-3 (EGF) -> let-23 (EGF receptor) -> sem-5 (GRB-2) -> let-60 (ras) -> lin-45 (raf) –lin-3 encodes a protein similar to the vertebrate epidermal growth factor –let-23 encodes an EGF receptor - a membrane bound tyrosine kinase –sem-5 encodes a protein similar to GRB-2 proteins. These contain SH2 and SH3 domains interact with receptor tyrosine kinases to mediate their effects on other cellular components –let-60 is similar to ras and lin-45 to raf. These are serine/threonine kinases that relay signals from the cell surface to the nucleus. this example illustrates a remarkable conservation of signaling mechanism throughout evolution –Drosophila sevenless pathway is quite similar –virtually identical pathway operates to regulate skin development in vertebrates and mammals –mutations in this pathway are known to cause cancers

12. BioSci 108 lecture 25 (Blumberg) page 12 © copyright Bruce Blumberg 2000. All rights reserved heterochronic genes another important concept in the regulation of development is the concept of developmental timing –an important mechanism in morphological evolution –mutation in a single control gene can alter the entire developmental tree –heterochronic mutants cause cells to behave inappropriately for their position in the lineage tree daughter cell can behave like its parent or grandparent leading to a lineage duplication leads to the endless reiteration of the pattern lin-14 (fig 21-45) is a C. elegans mutant that alters developmental decisions made in the first stage larva. –Gene has been cloned - nuclear protein ubiquotously expressed in late embryo in first stage larva -> then disappears protein maintains the immature state –loss-of-function mutations cause cells to precociously develop mature phenotype animal reaches adulthood prematurely with an abnormally small number of cells protein levels are abnormally low –gain-of-function -> repetition of the pattern of choices normally made at the first larval stage result is immature form of cuticle protein levels remain high during larval stages

13. BioSci 108 lecture 25 (Blumberg) page 13 © copyright Bruce Blumberg 2000. All rights reserved Apoptosis Apoptosis - programmed cell death –an orderly process by which cells die and their contents are recycled without damage to nearby cells program is intrinsic to the cells may occur rapidly and not obviously genes identified that control this process –contrast with necrosis where cells just burst and spill their contents into the extracellular space cells contain powerful degradative enzymes that are normally sequestered in compartments if these are released, damage to other cells results, perhaps leading to runaway necrotic change (such as gangrene) –apoptosis is an important developmental process it must occur in the correct cells at the appropriate time for normal development many cells in an organism are fated to die, even a substantial fraction consider duck and chicken - ducks have webbed feet, chickens not –all vertebrates with limbs have webbing between digits. Apoptosis in most leads to its loss other examples - tadpole tail, neuronal number, loss of undesirable lymphocytes (autoantigens),

14. BioSci 108 lecture 25 (Blumberg) page 14 © copyright Bruce Blumberg 2000. All rights reserved Apoptosis (contd) Apoptosis and C. elegans development –C. elegans provides an important model for apoptosis during development since cell fate is known evidence that apoptosis program is intrinsic also comes from C.elegans –C. elegans hermaphrodite generates 1030 cells during development exactly 131 of these cells die –are there genes that control this process? Two mutations identified that prevent cell death ced-3 and ced-4. –Loss-of-function mutations - cells survive and differentiate –Gain-of-function - many cells die »gain of fx -> loss of ced-9 »ced-9 normally represses cell death ced-9 is similar to the mammalian protooncogene bcl2 –bcl2 normally functions to prevent cell death. It is mutated in many cancers –incredibly, human bcl-2 gene can rescue ced-9 mutants when transferred to worms pathways are highly conserved apoptosis is an inherent and fundamental property of cells.