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Bio 108 - 3/10/2000 Developmental control genes and cell
behavior
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Contact information
office hours W/F 3-4
phone 824-8573
[email protected] (preferred contact mode)
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Lectures posted at
http://blumberg-serv.bio.uci.edu/bio108-w2000/index.htm
http://blumberg.bio.uci.edu/labtemp/bio108-w2000/index.htm
BioSci 108 lecture 25 (Blumberg) page 1
©copyright
Bruce Blumberg 2000. All rights reserved
Clarification from last lecture
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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.
BioSci 108 lecture 25 (Blumberg) page 2
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans and cell behavior
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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.
BioSci 108 lecture 25 (Blumberg) page 3
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans and cell behavior (contd)
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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 (~108 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
BioSci 108 lecture 25 (Blumberg) page 4
©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
BioSci 108 lecture 25 (Blumberg) page 5
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans and cell behavior (contd)
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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)
BioSci 108 lecture 25 (Blumberg) page 6
©copyright
Bruce Blumberg 2000. All rights reserved
Genes that control development
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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
BioSci 108 lecture 25 (Blumberg) page 7
©copyright
Bruce Blumberg 2000. All rights reserved
Genes that control development (contd)
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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
BioSci 108 lecture 25 (Blumberg) page 8
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans vulva development
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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
BioSci 108 lecture 25 (Blumberg) page 9
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans vulva development (contd)
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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)
BioSci 108 lecture 25 (Blumberg) page 10
©copyright
Bruce Blumberg 2000. All rights reserved
C. elegans vulva development (contd)
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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
BioSci 108 lecture 25 (Blumberg) page 11
©copyright
Bruce Blumberg 2000. All rights reserved
heterochronic genes
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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
BioSci 108 lecture 25 (Blumberg) page 12
©copyright
Bruce Blumberg 2000. All rights reserved
Apoptosis
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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),
BioSci 108 lecture 25 (Blumberg) page 13
©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.
BioSci 108 lecture 25 (Blumberg) page 14
©copyright
Bruce Blumberg 2000. All rights reserved