biology 103 lecture 6
TRANSCRIPT
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Frog Gastrulation
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Results of gastrulation
Dorsal lip
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Lecture 6
Organogenesis, Limb
Development and Apoptosis
Plate from Vesalius' De Humani Corporis Fabrica (1543)
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From Gastrulation to Organogenesis
• Organogenesis is the formation of organs during development.
• Gastrulation involves massive cell movements that:
- produce three primary germ layers ( , ,)
- place cells from various regions of the blastula into new
associations with one another.
• Inductive signaling is vital for neurulation and organogenesis,e,g, the nervous system is induced by the notochord; limboutgrowth is induced by the AER; A-P axis of the hand by theZPA.
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Neurulation: Initiating the Nervous System
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Neurulation: Initiating the Nervous System
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Human embryo8
Neurulation in
vertebrates
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Birth defects due to improper
neural tube closure
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Spina bifida• anencephaly
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Sonic Hedgehog (SHH ) is required forsignaling during neural tube formation
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The strange case of the one-eyed lamb
Cyclopia
Due to sheep eatingcorn lilies which havehigh levels of naturallyoccurring cyclopamine 12
Organogenesis follows neurulation
through a series of inductive events
Initially expressedin notochord
interrupts SHH
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Limb development demonstrates
examples of inductive signaling
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Limb axes
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Dorsal to ventral = knuckles to palms
Thumb
Pinkie
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Limb development is sensitive to
thalidomide at very early stages
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Hox genes provide
the positional
information for limb
development
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Expression of Fibroblast Growth Factor 10
(FGF10 ) demarcates incipient limbs
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FGF10 loss of function mice are limbless
Min H et al. Genes Dev. 1998;12:3156-3161
Ectopic FGF10 causes ectopic limbs
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FGF signaling initiates and maintains
limb formation
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The AER is the site of inductive signals:
FGF8 expression in limb buds
22 Apical epidermal ridge
The zone of polarizing activity (ZPA)
controls the A-P limb axis
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Sonic hedgehog is
expressed in the
zone of polarizing
activity (ZPA)
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Cll, Vol. 75, 1401-1416, December 31, 1993, Copyright 0 1993 by Cll Press
Sonic hedgehog Mediates the
Polarizing Activity of the ZPA
Robert D. Rddle, Randy L. Johnson, Ed Laufer,
and Ciff Tabin
Department of Gnetics
Harvard Medical School
Boston, Massachusetts 02115
Summary
The zone of polarizing acivty ZPA) isa region at the
posterior margin of the lim bud that induces mirror-
image duplications whe n grafted to the anterior of a
second lim. We have isolated a vertebrate gene, Sonic
hedgehog, related to the Drosophila segment polarit y
gene hedgehog, wh i c h i s ex pres s ed s ec f i c l l y i n the
ZPA and in other regions of the embryo, that iscapable
of polarizing lims in grafting experiments. Retinoic
acid, which can convert anterior lim bud tissue into
tissue wth polarizing acivty concomitantly induces
Sonic hedgehog expression in the anterior lim bud.
Imlanting c llsthat express Sonic hedgehog into an-
terior lim buds issffic ent to cause ZPA-like lim
duplications. Like the ZPA, Sonic hedgehog expres-
sion leads to the activation of Hox genes. Sonic hedge-
hog thus appears to function as the signal for antero-
posterior patterning in the lim.
Introduction
When tissue from the posterior regi on of the lim bud is
grafted to the anterior border of a second lim bud, the
resultant lim w ll develop wth additional digitsin a mirror-
image sequence along the anteroposterior axis Saunders
and Gsseling, 1968; Figure 1). This finding has led to a
model that the zone of polarizing acivty ZPA) isresponsi-
ble for normal anteroposterior patterning in the lim. The
ZPA has been hypothesized to function by releasing a
sgnal, termed a morphogen, which forms a gradient
across the early embryonic bud. According to this model,
cll fate at different distances from the ZPA isdetermined
by the locl concentration of the morphogen, w th specific
thresholds of the morphogen inducing successive struc-
tures Wolpert, 1 969). The idea that the signal from the
ZPA isconcentration-dependent issupported by the find-
ing that the extent of digit duplication isproportional to
the number of implanted ZPA clls Tckle, 1981).
A candidate for the putative ZPA morphogen wa s identi-
fied by the discovery that a source of retinoic acid can
result in the same type of mirror-image digit duplications
when placed in the anterior of a lim bud Tickle et al.,
1982; Summerbell, 1983). The response to exogenous
retinoic acid isconcentration dependent a s the morpho-
gen model demands Tickle et al., 1985). Moreover, a dif-
ferential disribution of retinoic acid exists across the lim
bud, wth a higher concentration in he ZPA region Thaller
and Eichele, 1987).
Recent evidence, however, has indicated that retinoic
acid isunlikly to be the endogenous factor responsible
for ZPA acivty reviewed by Brockes, 1991; Tabin, 1991).
One of the strongest challenges to retinoic acid as a andi-
date ZPA morphogen comes from the fact that exogenous
retinoic acid, at a oncentration that elictspatt ern duplica-
tions induces an endogenous retinoic acid-responsive
gene the retinoic acid receptor 8) to a much higher levl
than that normally seen in the posterior lim N ji et al.,
1991). This imlies that the ZPA contains less retinoic acid
than isrequired to induce lim bud duplications, and thus
retinoic acid isprobably not the ZPA sgnal. It isn ow be-
lieved that rather than directly mmcking an endogenous
sgnal, retinoic acid imlants act by inducing an ectopic
ZPA. The anterior lim tissue jus dis al to a retinoic acid
imlant and directly under the ectoderm has been demon-
s t ra ted to ac qu i re Z PA ac i v ty by s ri a l l y t rans p l anti ng
that tissue to another lim bud Summerbell and Harvey,
1983; Wanek et al., 1991). Conversely, the tissue next
to a ZPA graft does not gain ZPA acivty Smth , 1979).
Exogenous retinoic acid would thus appear to act up-
stream of the ZPA in lim patterning.
One approach that has been very successful in identi-
fying new signaling molecules important in patterning ver-
tebrate embryos ist o look for homologs of inductive sig-
nals from distantly related organisms. The segment
polarity genes are the firs to mediate intercllular commu-
nication in the developing Drosophila embryo, controlling
the patterning of cllswthin segmental units from which
the embryo isderived Ingham, 1988) . Several previously
isolated segment polarity genes, including armdillo, cubi-
tus interruptus, engrailed, gooseberry, zeste-white-3, and
wingless, are related to famliesof genes that are involved
in the regulation of vertebrate development reviewed by
Ingham, 1991).
The segment polarity gene, hedgehog, has recently
been cloned Mohler and Vani, 1992; Tabata et al., 1992;
Lee et al., 1992). hedgehog encodes a secreted protein
produced by a et of clls in the posterior of each segment
Mohler, 1988; Mohler and Vani, 1992; lngham and M rti-
nez-Arias, 1992). Moreover, there isenetic evidence that
thisprotein acts in a concentration-dependent manner to
instruct different cll fates across the developing segment
S. DiNardo, personal communication), thereby fufillin
the definition of a classic morphogen. The cloning of Dro-
sophila hedgehog provided the opportunity to determine
whether there are homologous genes in vertebrates and
whether, in particular, any play a role as inductive signals
during lim development.
Results
Islation of a Chicken Homolog
of Drosophila hedgehog
To identifyhedgehog homolog s expressed in the devel-
oping chick lim bud during chick embryogenesis, we de-
signed degenerate polymerase chain reaction PCR) prim-
ers corresponding to a sequence highly conserved
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FGF signaling initiates and maintains limb
formation, and Shh signaling from the ZPA
patterns the posterior-anterior axis
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A temporal and spatial gradient of Shh
signaling establishes the anterio-posterior
axis of the hand
Philip W.Ingham& MarysiaPlaczek,Nature ReviewsGenetics7, 841-850 (November2006)
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What would happen if you placed a source
of Shh in the anterior part of the limb bud?
Riddle et al, Cell 75: 1401 (1993)
From Gastrulation to Organogenesis
• Organogenesis is the formation of organs during development.
• Gastrulation involves massive cell movements that:
- produce three primary germ layers (endoderm, mesoderm,ectoderm)
-
place cells from various regions of the blastula into new
associations with one another.
• Inductive signaling is vital for organogenesis, e,g, the nervoussystem is induced by the notochord; limb outgrowth is inducedby the AER; A-P axis of the hand by the ZPA.
New source of SHHintroduced: two pinkiesproduced, no thumbs
A is controlB is experimental
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Apoptosis: How death shapes life
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The Nobel Prize in Physiology or
Medicine 2002
H. RobertHorvitz
SydneyBrenner
John E.Sulston
“for their discoveries concerning genetic regulation of organ
development and programmed cell death”
Apoptosis is important in pattern formation
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Apoptosis shapes our own hands
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Cell Death:
homicide and suicide
• Cells may die by necrosis or may self-destruct by apoptosis, a geneticallyprogrammed series of events that includes:
• detachment of the cell from its neighbors
• cytoplasmic “blebbing” to form “apoptoticbodies”
• the fragmentation of its nuclear DNA
• engulfment by neighbors.
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Cells die with a characteristic
flair during apoptosis
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Apotosis in physiology
• White blood cells that can recognize our own tissues
• White blood cells that are no longer needed after aninfection
• Skin cells die by apoptosis and are sloughed off
• Uterine cells during menstruation
• Some cells harboring DNA damage
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Syndactyly in humans
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Koala: second and third digits fused
Apoptosis is essential for
normal development
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Caspase mutant
FYI-
Review article
How death shapeslife during
development
By E.H. Baehrecke
Nature Reviews Mol
Cell Biology 3:779
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Breakthroughs
in apoptosis
came from
genetics in C.
elegans
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Wild-type
ced-3 lossof function
mutant
What does this sayabout the normal
function of ced-3?required for apoptosis
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In ced-9 lof mutants, most cells die
by apoptosis
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What does this sayabout the normal
function of ced-9?
Discuss: how might you order the
ced-3 and ced-9 genes into a
pathway?
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Genetic analysis of developmental pathways
• In ced-9 mutants, all cells undergo apoptosis. So ced-9 normally protects cells from death
•
In ced-3 mutants, no cells undergo apoptosis. So ced-3
normally promotes cell death
• Is the characteristic cell death of ced-9 mutants dependent onthe activity of ced-3?
• Build a double mutant between ced-9 and ced-3 and now nocells die.
43ced-9 ced-3
Genetic control and conservation ofapoptosis pathways
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represses apoptosis
Is the characteristic cell death of ced-9 mutants dependent on the activity of ced-3?
create double mutant by knocking both out—> now no cells die
ced-9 must negatively regulate ced-3
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FYI Classes v2
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Cll, Vol. 76, 666-676, February 25, 1994, Copyright 0 1994 by Cll Press
C. ekgans Ce ll Survival Gene c&9
Encodes a Functional Homob g
of the Mamma)ian Proto-Onoogene M-2
Michael 0. Hengartner and H. Robert Horvitz
Howard Hughes Medical Ins itute
Depar tment of Biology
Massachusetts Ins itute of Technology
Cambridge, Massachusetts 02139
Summary
The ac iv ty of the C. elegans gene ted-9 is required
to protec t cells that normally surv ive from undergoing
programmed cell death. Here we describe the cloning
and molecular charac ter ixatlon of thisgene. ted-9 is
an element of a polyclstronic locus that also contains
the gene cyt-7, which encodes a protein
Slf I Il l f I r tO Cy t O
chrome bwo f complex II of the mltochondrlal resplra-
tory chain. ted-9 encode s a 280 amino acid protein
showing sequence and s truc tural sm lar it ies to the
mammalian proto-oncoge ne bcl-2. Overexpresalon of
bc l-2 can mmc the protec tive effec t of ted-9 on C.
elegans cell death and can prevent the ectopic cell
deaths that occur in ted-9 loss-of-function mutants.
These results suggest th at ted-9 and bcl-2 are homo-
logs and that the molacular mechan ism of progmmmed
cell death has been conserved f rom nematodes to
mammals .
Introduction
Programmed cell death plays an important role in animal
development and homeostasis and occurs in a wide va-
riety of tissues in both vertebrates and invertebra tes
(Gticksmann, 1950; Cohen, 1991; E liset al., 1991; Raff,
1992). In many tissues, cell death and cell proliferation
are precisely balanced to maintain the proper number and
types of cells and dis ruption of thisbalance can result in
disease ( rev iewed by Wlliams 1991) .
In the nematode Caenorhabditis elegans , 131 of the
1090 somatic cellsgenerated dur ing hermaphrodite devel-
opment undergo progra mmed cell death (Sulston and Hor-
v tz 1977; Suls ton et al., 1983) . Genetic s tudies have led
to the identification of 14 genes that are involved at various
s teps of thisprocess ; these genes can be placed into a
genetic pathway for programmed cell death in C. elegans
( rev iewed by Eliset al., 1991; Discoll, 1992) . Three of
these genes are involved in the regulation and execution
of al 131 deaths . The ac iv ties of two of hese three genes ,
ted-3 and ted-4 (called ted for cell death abnormal), are
required for cells to die (Ellisand Horv itz , 1988) . In ed-3or
ted-4 mutants , essentially al cells that usually die ins tead
survive, differenti ate, a nd (in at least some cases) properly
func tion (Ellisand Horv itz , 1988; Avery and Horv itz , 1987;
White et al., 1991). Genetic mosaic analyses suggest tha t
these two genes mus t be expressed by he cellsscheduled
to undergo programmed death for these cells to die (Yuan
and Horvitz, 1990). The ted-4 gene encodes a protein wth
no sgnificant sequence smlar ity to any other protein in
the data bases (Yuan and Horvitz, 1992). The CM-3 gene
encodes a homolog of the mammalian inter leukn- lP-
converting en zyme (Yuan et al., 1993), which suggests
that the CED3 prot ein acts as a
prOeaSe
to cause pro-
grammed cell death.
The third gene involved in the control of al programmed
cell deaths, ted-9, negatively regulates the pathway for
programmed cell death: a c&9 gai n-of-function mut ation
prevents the deaths of cells that normally die, while muta-
tions that inactivate ted-9 cause cellsthat normally live to
undergo programme d cell death (Hengartner et al., 1992).
Thus , the func tion of ted-9 is o prevent cells that normally
survive from undergoing programme d cell death. The ab-
sence of ted-9 function results in maternal-effect lethali ty
indicating that c&-9 func tion is essential for C. elegans
development.
The proto-oncogene b cl-2 appears to function in mam-
mals as ted-9 functions in nematodes. bcC 2 was discov-
ered and molecularly c loned based on its involvement in
a t(14;18) translocation that isobserved in the major ity
of follicular lymphomas dia gnosed in the United States
(Fukuharaet al., 1979; Yunis et al., 1982). This transloca-
tion fuses the b&P locus to the immunoglobulin heavy
chain gene, resulting in he overexpression o f normal Bcl-2
protein in 6 cells (Tsujimoto et al., 1984; Bakhshi et al.,
1985; Cleary et al., 1988; Tsujimoto and Croce, 1988 ; Seto
et al., 1988). Overexpression of bcl-2 prevents or delays
sgnificantly the programmed cell death (apoptos is ) of a
large var iety of cellsunder var ious conditions that usually
lead to cell death. For example, bcl-2 protects interleukin -
dependent lymphoid cell lines from apoptos is induced by
interleukin wi thdrawal (Va ux et al., 1988; Nufiez et al.,
1990) and thymocytes from apoptosis induced by gluco-
corticoids or by y-irradiation (Sentman et al., 1991; Stras-
ser et al., 1991). bcl-2 also can protect neuron s from
apoptosis induce d by trophic factor withdrawal (Garcia et
al., 1992; Allsopp et al., 1993; Batis tatou et al., 1993) and
can prevent apoptosis induce d by c-myc in Rat-l cells
(Fanidi et al., 1992) and CHO cells(Bissonetteet al., 1992).
In tissues character ized by cell turnover, bc l-2 isoften
expressed in progenitor or long- lived cells (Hockenbery
et al., 1991). Moreover, signals that rescue lymph node
germinal center cells from susceptibility to apoptosis in-
duce bcC2 expression (Li u et al., 1991). Based on these
observations, it has been suggested that bc/-2 expression
protects cells that should survive from apoptosis (Hocken-
bery et al., 1991).
Recently , a number of genes wth some sequence sm-
lar ity to b&P have been repor ted (Boise et al., 1993; Kozo-
pas et al., 1993; Lin et al., 1993; Otvai et al., 1993; re-
v iewed by Wlliams and Smth, 1993) . Two of these genes,
bax and b&x, have effects on the regulation of apoptosis.
The relatively low sequence smlar it ies (30 500/b iden-
titybetween var ious members ) among these bc l-2 homo-
logs suggest that thisgene famly mght be of anc ient or igin
and not restricted to vertebrates.
Here we report the molecular chara cterization of the C.
How might you showfunctional homologybetween ced-9 andbcl-2 ?
Apoptosis and disease
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Key Concepts•
Cell death is an essential part of
development and life
•
Apoptosis is a form of programmed cell
death.
•
A conserved pathway of genes controls
the decision for cells to die.
•
Defects in apoptosis result in
developmental defects and disease.
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