Chapter 19: Using Genetics to Study Development

DEVELOPMENT

• An organism arises from a fertilized egg as the result of three related processes– Cell division– Cell differentiation– Morphogenesis

DIFFERENTIATION

• Cells may initially remain undifferentiated– Embryonic stem cells

• Cells ultimately differentiate– Become specialized in

structure and function

DIFFERENTIATION

• Virtually all cells within a multicellular organism are genetically identical

• Differences between cells are due to differences in gene expression– Different subsets of genes are “on” and “off”– Different cell types make different proteins

GENE EXPRESSION

• Much of the regulation of gene expression occurs at the level of transcription

• Transcriptional regulation of gene expression is directed by– Maternal molecules in the

cell’s cytoplasm– Signals from other cells

PATTERN FORMATION

• The development of a spatial organization in which an organism’s tissues and organs are all in their characteristic places

• In animals, it begins in early embryo– Basic body plan is established– Major axes are established early

Most commonly used model organisms

Yeast (S. cerevisiae) Worms (C. elegans) Fruitfly (D. melanogaster)

Zebrafish (D. rerio) Mustard Weed (A. thaliana)Mouse (M. musculus)

Two important feature of Model Organism

• The availability of powerful tools of and study the organism genetically

• Ideas, methods, tools, and strains could be shared among scientists investigating the same organism, facilitating rapid progress.

The choice of a model organism depends on what question is being asked.

一、 Saccharomyces cerevisiae

• BAKER’S YEAST, the best studied unicellular eukaryote is the budding yeast S. cerevisiae.

• Unicellular eukaryotes offer many advantages as experimental model systems.

S. cerevisiae exists in three forms.

• Two haploid cell types, a and α

• The diploid product of mating between these two.

The lifecycle of the budding yeast S. cerevisiae

• These cell types can be manipulate to perform a variety of genetic assays.

• Genetic complementation can be performed the two mutations whose complementation is being tested.

• If the mutations complement each other, the diploid will be a wild type for mntations can be made in haploid cells in which there is only a single copy of that gene.

Generating precise mutations in yeast is easy

• The genetic analysis of S. cerevisiae is further enhanced by the availability of techniques used to precisely and rapidly modify individual genes.

Recombinational transformation in yeast

• The ability to make such precise changes in the genome allows very detailed questions concerning the function of particular genes or their regulatory sequences to be pursued with relative ease.

S. cerevisiae has a small, well-characterized genome

• Because of its rich history of genetic studies and its relatively small genome, S. cerevisiae was chosen as the first eukaryotic ( nonviral ) organism to have its genome entirely sequenced. This landmark was accomplished in 1996.

• The availability of the complete genome sequence of S. cerevisiae has allowed “genome-wide” approaches to studies of this organisn.

S. cerevisiae cells change shape as they grow

• As S. cerevisiae cells progress through the cell cycle. They undergo characteristic changes in shape.

The mitotic cell cycle in yeast

• Simple microscopic observation of S. cerevisiae cell shape can provide a lit of information about the events occurring inside the cell.

• A cell that lacks a bud has yet to start replicating its genome. A cell with a very large bud is almost always in the process of executing chromosome segregation.

二、 NEMATODE WORM,

caenorhabditis elegans

• In 1965 Sydney Brenner settled on the small nematode worm caenorhabditis elegans to study the important questions of development and the molecular basis of behavior, because it contained a variety of suitable characteristics.

• And due to its simplicity and experimental accessibility, it is now one of the most completely understood metazoan.

WHY?

it can be handled like a microbe –

very amenable to genetic analysis -

on agar plates

in liquid medium

as frozen stocks

self-fertilization

crosses with males

short life cycle

genome sequence known Dec 11 1998 Vol 282: 5396 C. elegans: Sequence to Biology

• easy to observe under microscope-

•easy to make mutants-

small size (1 mm)

transparent body

invariant cell number

mutagenesis

DNA microinjection

RNA interference

http://130.15.90.245/photos.htm

C.elegans H.sapiens

Chromosomes 5 + 1 22 + 2Genome Size 97 million 3000 millionEncoded Proteins 19,099 ~30,000Life Span 0.06 years ~80 yearsSexes male, herm. male, femaleSomatic cells 1031, 959 ???Neurons 381, 302 100,000,000,000Synaptic connections 5,000 100,000,000,000,000Body size 1 mm ~170 cmBody weight 5 g ~75 kgFood E.coli Omnivore

Scorecard

C. elegans has a very rapid life cycle

• At 25 fertilized embryos of℃ C. elegans complete development in 12 hours and hatch into free-living animals capable of complex behaviors.

• The first stage juvenile(L1) passes through four juvenile stages(L1-L4) over the course of 40 hours to become a sexually mature adult.

The life cycle of the worm, C.elegans

• Under stressful conditions, the L1 stage animal can enter an alternative developmental stage in which it forms what is called a dauer.

• Dauers are resistant to environmental stresses and can live many months while waiting for environmental conditions to imptove.

C. elegans has a simple body plan. Its lineages is relatively few and well studied.

The body plan of the worm

THE WORM

In case of self-fertilization there are ~ 0.1 - 0.3% male worms in the population.

http://www.wormatlas.org/handbook/contents.htm

Males

Males (5AA;X0) arise from fusion of nullo-X gametes and normal X-bearing gametes. Nullo-X gametes are generated by spontaneous non-disjunction of the X chromosome during meiosis in the germ line.

Nervous system

Worm and neurobiological studies

• Depression• Neurodegeneratio

n• Schizophrenia• Insomnia• Addiction• Memory• Learning • etc.

The cell death pathway was discovered in C. elegans

• The most notable achievement to date in C. elegans research has been the elucidation of the molecular pathway that regulates apoptosis or cell death.

• Analysis of the ced mutants showed that, in all but one case, developmentally programmed cell death is cell autonomous, that is, the cell commits suicide.

Nobel Prize in Physiology or Medicine

2002

Sydney Brenner

John Sulston

Robert Horvitz

"for their discoveries concerning genetic regulation of organ development and programmed cell death"

Apoptotic pathways of C. elegans and vertebrates are

conserved

RNAi was discovered in C. elegans

• In 1998 a remarkable discovery was announced. The introduction of dsRNA into C. elegans silenced the gene homologous to the dsRNA. It significant in two respects.

Nobel Prize Nobel Prize in Physiology or Medicinein Physiology or Medicine

20020066

"for their discovery of RNA interference - gene silencing by double-stranded RNA"

Andrew Z. Fire Craig C. Mello

One is that RNAi appears to be universal since introduction of dsRNA into nearly all animal, fungal, or plant cells leads to homology-directed mRNA degradation.

• The second was the rapidity with which experimental investigation of this mysterious process revealed the molecular mechanisms.

Long term storage of the worm

C. elegans can be stored indefinitely at very low temperature (-70 ~ -100 °C freezer)

Freezing solution:S Buffer [129 ml 0.05 M K2HPO4, 871 ml 0.05 M KH2PO4, 5.85 g NaCl] + 30% glycerin 1:1 with M9 containing worms (preferably starved L1)

In the dauer larval stage, it canalso be kept at 16 °C for months

三、 FRUIT FLY,

Drosophila melanogaster

• Drosophila has a raid life cycle• The salient features of the Drosophila life

cycle are a very rapid period of embryogenesis, followed by three period of larval growth prior to metamorphosis.

The Drosophila life cycle

• One of the key processes that occurs during larval development is the growth of the imaginal disks, which arise from invaginations of the epidermis in mid-stage embryos.

• Imaginal disks differentiate into their appropriate adult structures during metamorphosis (or putation).

• Imaginal disks in Drosophila

The first genome maps were produced in Drosophila

• genes are located on chromosomes;• each gene is composed of two alleles that

assort independently during meiosis; • genes located on separate chromosomes

segregate independently, whereas those linked on the same chromosome do not.

Genetic mosaics permit the analysis of lethal genes in adult flies

• Mosaics are animals that contain small patches of mutant tissue in a generally “normal” genetic background.

• The analysis of genetic mosaics provided the first evidence that Engrailed is required for subdividing the appendages and segments of flies into anterior and posterior compartments.

• The most spectacular genetic mosaics are gynandromorphs.

• Gyandromorphs

• These are flies that are literally half male female. The X instability occurs only at the first division.

• And the “line” separating the male and female tissues is random. Its exact position depends on the orientation of the two daughter nuclei after the first cleavage.

• This method is quite efficient. In fact, short pulse of heat shock are often sufficient to produce enough FLP recombinase to produce large patches of zˉ/zˉ tissue in different regions of an adult fly.

It is easy to create transgenic fruit flies that carry foreign DNA

• P-elements are transposable DNA segments that are the causal agent of a genetic phenomenon called hybrid dysgenesis.

• Hybrid dysgenesis

• P-element excision and insertion is limited to the pole cells, the progenitors of the gametes (sperm in males and eggs in females).

• P-elements are used as transformation vectors to introduce recombinant DNAs into otherwise normal strains of flies.

P-element transformation

• This method of P-element transformation is routinely uses to identify regulatory sequences such as those governing eve stripe 2 expression.

• In addition, this strategy is used to examine protein coding genes in various genetic backgrounds.

Drosophila melanogaster

• Bilaterally symmetric segmented body– Head– Thorax– Abdomen

Drosophila melanogaster

• Cytoplasmic determinants are present in the unfertilized egg– Provide positional information for placement of

axes prior to fertilization– Establishes number and orientation of segments– Ultimately trigger formation of specific

structures within each segment

Drosophila melanogaster• Egg develops in mother’s ovary

– Surrounding cells with nutrients, etc.

• Mitosis begins following fertilization– First ten divisions include no growth,

cytokinesis– Single multinucleate cell results

Drosophila melanogaster

• Nuclei migrate to periphery of embryo at tenth division

• Plasma membranes finally partition ~6,000 nuclei into separate cells at the thirteenth division– Basic body plan already

determined at this time• Body axes and segment

boundaries

Drosophila melanogaster

• Subsequent embryonic events create clearly visible segments– Initially look very similar

• Some cells move to new positions– Organs form

• Wormlike larva hatches– Eats, grows, & molts

Drosophila melanogaster

• Larva eventually forms a pupa– Enclosed in a case

• Metamorphosis occurs– Change from larva to adult fly

• Adult fly emerges from case– Each segment is anatomically

distinct

Drosophila melanogaster

• Each segment in the adult fly is anatomically distinct– Characteristic appendages

Drosophila melanogaster

• Gradients of maternal molecules in the early embryo control axis formation– Cytoplasmic determinants already present in

unfertilized egg– Encoded by mother’s maternal effect genes

• a.k.a., “Egg-polarity genes”

• Encode proteins or mRNAs that are placed into the egg while still in the mother’s ovary

Drosophila melanogaster

• One group of maternal effect genes establishes the anterior-posterior axis of the embryo

• Another set of maternal effect genes establishes the dorsal-ventral axis

• Female flies possessing mutations in maternal effect genes appear phenotypically normal, but produce offspring with mutant phenotypes

Drosophila melanogaster

• Bicoid is an egg-polarity gene– “Two-tailed”

• Mothers defective in bicoid produce embryos lacking the front half of their body– Duplicate posterior structure at both ends

Drosophila melanogaster

• Bicoid gene product is concentrated at anterior end of fly embryo– Gradient of gene product– Essential for setting

up anterior end of fly

• Gradients of other proteins determine the posterior end and the dorsal-ventral axis

Drosophila melanogaster

• The bicoid protein and the products of other egg-polarity genes are transcription factors– Regulate the expression

of some of the embryo’s genes

Drosophila melanogaster

• Segmentation genes– Genes of embryo– Expression regulated by products of egg-

polarity genes– Direct the actual formation of segments after

the embryo’s major axes are defined

Drosophila melanogaster

• Three sets of segmentation genes are activated sequentially– Gap genes

– Pair-rule genes

– Segment polarity genes

• The activation of these sets of genes defines the animal’s body plan– Each sequential set regulates increasingly fine

details

Drosophila melanogaster

• Gap genes– Map out basic subdivisions along the embryo’s

anterior-posterior axis– Mutations cause “gaps” in the animal’s

segmentation

Drosophila melanogaster

• Pair-rule genes– Define pattern in terms of pairs of segments– Mutations result in embryos having half the

normal number of segments

Drosophila melanogaster

• Segment polarity genes– Set the anterior-

posterior axis of each segment

– Mutations produce segments where part of the segment mirrors another part of the same segment

Drosophila melanogaster

• The products of many of the segmentation genes are transcription factors– Directly activate the next set of genes

• Summary– Products of the egg-polarity genes regulate the regional

expression of the gap genes– Gap genes control the localized expression of the pair-

rule genes– Pair rule genes activate specific segment polarity genes

in different parts of each segment– Segment polarity genes activate homeotic genes

HOX Genes

• Hox genes were discovered in D. melanogaster

• Homeodomain proteins that bind DNA

• Organized in gene clusters

• See Figure 17-7

HOX mutations

• Hox gene mutations in mice have been shown to produce homeotic transformation of vertebral or spinal segments.

• HOX13 mutations cause synpolydactyly

• Semidominant causing interphalangeal webbing and extra digits in hands and feet.

• Figure 17-8

HOMEOTIC GENES

• Master regulatory genes

• Specify the types of appendages and other structures that each segment will form

• Mutations produce flies with structures in incorrect places

HOMEOTIC GENES

• Encode transcription factors

• Control the expression of genes responsible for specific anatomical structures– e.g., “Antennae go here”– e.g., “Legs go here”– ………Oops!!!

HOMEOTIC GENES

• Homeotic genes are master genes that regulate the expression of numerous other genes– Some of the regulated

genes are regulatory themselves

Drosophila DEVELOPMENT

Hierarchy of Gene Activity• Maternal genes• Segmentation genes of embryo

– Gap genes– Pair-rule genes– Segment polarity genes

• Homeotic genes of the embryo• Other genes of the embryo

HOMEOTIC GENES

• Homeotic genes of Drosophila all possess homologous segments– 180-nucleotide sequence = homeobox– Encodes 60-amino-acid homeodomain

• Homologous sequences have been found in many other animals– e.g., Insects, nematodes, mollusks, fish, frogs, birds,

humans, etc.– Related genes are even found in yeast, etc.– Hox genes

HOMEOTIC GENES

• Vertebrate genes homologous to the homeotic genes of Drosophila have maintained their chromosomal arrangement

HOMEOTIC GENES

• Not all homeobox-containing genes are homeotic genes– Some do not directly control the identity of

body parts– Most are associated with development

• e.g., Drosophila bicoid, several segmentation genes, etc.

HOMEODOMAIN

What is the role of the homeodomain?

• Polypeptide segment

• DNA-binding function– Can bind to any DNA sequence– Other domains of such proteins confer

sequence-specificity• Sequence-specificity determines which genes are

regulated by a particular homeotic protein

四、 House Mouse, Mus musculus

• The mouse enjoys a special status due to its exalted position on the evolutionary tree: it is a mammal and, therefore, related to humans.

• The mouse provides the link between the basic principles, discovered in simpler creatures like worms and flies, and human disease.

Mouse Embryonic Development Depends on Stem Cells

• Their small size prohibits grafting experiments of the sort done in zebrafish and frogs,

• Microinjection methods have been developed for introducing.

Overview of mouse embryogenesis

It Is Easy to Introduce Foreign DNA into the Mouse Embryo

• DNA is injected into the egg pronucleus, and the embryos are places into the oviduct of a female mouse and allowed to implant and develop.

• The injected DNA integrates at random positions in the genome

• Creation of transgenic mice by microinjection of DNA into the egg pronucleus

• In situ expression patterns of embryos obtained from transgenic mice

Homologous Recombination Permits the Selective Ablation of Individual

Genes

• The single most powerful method of mouse transgenesis is the ability to disrupt, or “knock out ，” single genetic loci. This permits the creation of mouse models for human disease.

• Gene disruption experiments are done with embryonic stem (ES) cells

Gene knockout via homologous recombination

Mice Exhibit Epigenetic Inheritance

• Studies on manipulated mouse embryos led to the discovery of a very peculiar mechanism of non-Mendelian, or epigenetic, inheritance.

• This phenomenon is known as parental imprinting.

• Imprinting in the mouse

• The basic idea is that only one of the two alleles for certain genes is active.

• It has been suggested that imprinting has evolved to protect the mother from her own fetus.