Results of gastrulation

Frog Gastrulation

Dorsal lip

Lecture 6
Organogenesis, Limb
Development and Apoptosis

4
Plate from Vesalius' De Humani Corporis Fabrica (1543)

Neurulation: Initiating the Nervous System

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; limb
outgrowth is induced by the AER; A-P axis of the hand by the
ZPA.
6

Neurulation: Initiating the Nervous System

Human embryo

Neurulation in
vertebrates

Sonic Hedgehog (SHH) is required for

signaling during neural tube formation

Birth defects due to improper

neural tube closure
Spina bifida
anencephaly

Initially expressed
in notochord

The strange case of the one-eyed lamb

Cyclopia
Due to sheep eating
corn lilies which have
high levels of naturally
occurring cyclopamine

interrupts SHH

11

10

Organogenesis follows neurulation

through a series of inductive events

12

Limb development demonstrates

examples of inductive signaling

Limb axes
Thumb

Pinkie

Dorsal to ventral = knuckles to palms

13

14

Limb development is sensitive to

thalidomide at very early stages

15

16

Hox genes provide

the positional
information for limb
development

Expression of Fibroblast Growth Factor 10

(FGF10) demarcates incipient limbs

17

FGF10 loss of function mice are limbless

18

Ectopic FGF10 causes ectopic limbs

Min H et al. Genes Dev. 1998;12:3156-3161

19

20

FGF signaling initiates and maintains

limb formation

The AER is the site of inductive signals:

FGF8 expression in limb buds

Apical epidermal ridge

21

22

Sonic hedgehog is
expressed in the
zone of polarizing
activity (ZPA)

The zone of polarizing activity (ZPA)

controls the A-P limb axis
Cell, Vol. 75, 1401-1416,

December

31, 1993, Copyright

0 1993 by Cell Press

Sonic hedgehog Mediates the

Polarizing Activity of the ZPA
Robert D. Riddle, Randy L. Johnson,
and Cliff Tabin
Department of Genetics
Harvard Medical School
Boston, Massachusetts 02115

Ed Laufer,

Summary
The zone of polarizing activity (ZPA) is a region at the
posterior margin of the limb bud that induces mirrorimage duplications when grafted to the anterior of a
second limb. We have isolated a vertebrate gene, Sonic
hedgehog, related to the Drosophila segment polarity
gene hedgehog, which is expressed specifically in the
ZPA and in other regions of the embryo, that is capable
of polarizing limbs in grafting experiments. Retinoic
acid, which can convert anterior limb bud tissue into
tissue with polarizing activity, concomitantly induces
Sonic hedgehog expression in the anterior limb bud.
Implanting cells that express Sonic hedgehog into anterior limb buds is sufficient to cause ZPA-like limb
duplications. Like the ZPA, Sonic hedgehog expression leads to the activation of Hox genes. Sonic hedgehog thus appears to function as the signal for anteroposterior patterning in the limb.
Introduction

23

When tissue from the posterior region of the limb bud is

grafted to the anterior border of a second limb bud, the
resultant limb will develop with additional digits in a mirrorimage sequence along the anteroposterior axis (Saunders
and Gasseling, 1968; Figure 1). This finding has led to a
model that the zone of polarizing activity (ZPA) is responsible for normal anteroposterior patterning in the limb. The
ZPA has been hypothesized to function by releasing a
signal, termed a morphogen, which forms a gradient
across the early embryonic bud. According to this model,
cell fate at different distances from the ZPA is determined
by the local concentration of the morphogen, with specific
thresholds of the morphogen inducing successive structures (Wolpert, 1969). The idea that the signal from the
ZPA is concentration-dependent is supported by the finding that the extent of digit duplication is proportional to
the number of implanted ZPA cells (Tickle, 1981).
A candidate for the putative ZPA morphogen was identified 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 limb bud (Tickle et al.,
1982; Summerbell, 1983). The response to exogenous
retinoic acid is concentration dependent as the morphogen model demands (Tickle et al., 1985). Moreover, a differential distribution of retinoic acid exists across the limb
bud, with a higher concentration in the ZPA region (Thaller
and Eichele, 1987).
Recent evidence, however, has indicated that retinoic

acid is unlikely to be the endogenous factor responsible

for ZPA activity (reviewed by Brockes, 1991; Tabin, 1991).
One of the strongest challenges to retinoic acid as a candidate ZPA morphogen comes from the fact that exogenous
retinoic acid, at a concentration that elicits pattern duplications, induces an endogenous retinoic acid-responsive
gene (the retinoic acid receptor 8) to a much higher level
than that normally seen in the posterior limb (Noji et al.,
1991). This implies that the ZPA contains less retinoic acid
than is required to induce limb bud duplications, and thus
retinoic acid is probably not the ZPA signal. It is now believed that rather than directly mimicking an endogenous
signal, retinoic acid implants act by inducing an ectopic
ZPA. The anterior limb tissue just distal to a retinoic acid
implant and directly under the ectoderm has been demonstrated to acquire ZPA activity by serially transplanting
that tissue to another limb bud (Summerbell and Harvey,
1983; Wanek et al., 1991). Conversely, the tissue next
to a ZPA graft does not gain ZPA activity (Smith, 1979).
Exogenous retinoic acid would thus appear to act upstream of the ZPA in limb patterning.
One approach that has been very successful in identifying new signaling molecules important in patterning vertebrate embryos is to look for homologs of inductive signals from distantly related organisms. The segment
polarity genes are the first to mediate intercellular communication in the developing Drosophila embryo, controlling
the patterning of cells within segmental units from which
the embryo is derived (Ingham, 1988). Several previously
isolated segment polarity genes, including armadillo, cubitus interruptus, engrailed, gooseberry, zeste-white-3, and
wingless, are related to families of 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 set of cells in the posterior of each segment
(Mohler, 1988; Mohler and Vani, 1992; lngham and Martinez-Arias, 1992). Moreover, there is genetic evidence that
this protein acts in a concentration-dependent manner to
instruct different cell fates across the developing segment
(S. DiNardo, personal communication), thereby fulfilling
the definition of a classic morphogen. The cloning of Drosophila 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 limb development.
Results
Isolation of a Chicken Homolog
of Drosophila hedgehog
To identify hedgehog homologs expressed in the developing chick limb bud during chick embryogenesis, we designed degenerate polymerase chain reaction (PCR) primers corresponding to a sequence highly conserved

24

FGF signaling initiates and maintains limb

formation, and Shh signaling from the ZPA
patterns the posterior-anterior axis

A temporal and spatial gradient of Shh

signaling establishes the anterio-posterior
axis of the hand

Philip W. Ingham & Marysia Placzek, Nature Reviews Genetics 7, 841-850 (November 2006)

25

What would happen if you placed a source

of Shh in the anterior part of the limb bud?

26

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.

Riddle et al, Cell 75: 1401 (1993)

Inductive signaling is vital for organogenesis, e,g, the nervous

system is induced by the notochord; limb outgrowth is induced
by the AER; A-P axis of the hand by the ZPA.

New source of SHH

introduced: two pinkies
produced, no thumbs
A is control
B is experimental
27

28

Apoptosis: How death shapes life

The Nobel Prize in Physiology or

Medicine 2002!
!
!
for their discoveries
concerning genetic regulation of organ
development and programmed cell death!

Sydney
Brenner

H. Robert
Horvitz

John E.
Sulston

29

Apoptosis is important in pattern formation

31

30

Apoptosis shapes our own hands

32

Cell Death:
homicide and suicide

Cells die with a characteristic

flair during apoptosis

Cells may die by necrosis or may selfdestruct by apoptosis, a genetically

programmed series of events that includes:
detachment of the cell from its neighbors
cytoplasmic blebbing to form apoptotic
bodies
the fragmentation of its nuclear DNA
engulfment by neighbors.
33

Apotosis in physiology

White blood cells that can recognize our own tissues

White blood cells that are no longer needed after an

infection

Skin cells die by apoptosis and are sloughed off

Uterine cells during menstruation

Some cells harboring DNA damage

34

Syndactyly in humans

35

36

Apoptosis is essential for

normal development

Koala: second and third digits fused

Caspase mutant

37

38

Breakthroughs
in apoptosis
came from
genetics in C.
elegans

FYIReview article
How death shapes
life during
development
By E.H. Baehrecke
Nature Reviews Mol
Cell Biology 3:779

Wild-type
ced-3 loss
of function
mutant

39

What does this say

about the normal
function of ced-3?
required for apoptosis

40

10

In ced-9 lof mutants, most cells die

by apoptosis

Discuss: how might you order the

ced-3 and ced-9 genes into a
pathway?

What does this say

about the normal
function of ced-9?

42

41

represses apoptosis

Genetic analysis of developmental pathways

Genetic control and conservation of

apoptosis 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 on the activity of ced-3?
create double mutant by knocking both out> now no cells die
Is
the characteristic cell death of ced-9 mutants dependent on
ced-9 activity
must negatively
ced-3
the
ofregulate
ced-3?

Build a double mutant between ced-9 and ced-3 and now no

cells die.

ced-9

ced-3

43

44

11

Cell, Vol. 76, 666-676,

February 25, 1994, Copyright

0 1994 by Cell Press

C. ekgans Cell Survival Gene c&9

Encodes a Functional Homobg
of the Mamma)ian Proto-Onoogene M-2
Michael 0. Hengartner and H. Robert Horvitz
Howard Hughes Medical Institute
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139

Summary
The activity of the C. elegans gene ted-9 is required
to protect cells that normally survive from undergoing
programmed cell death. Here we describe the cloning
and molecular characterixatlon of this gene. ted-9 is
an element of a polyclstronic locus that also contains
the gene cyt-7, which encodes a protein SlfIIllfIrtO CytOchrome bwo of complex II of the mltochondrlal resplratory chain. ted-9 encodes a 280 amino acid protein
showing sequence and structural similarities to the
mammalian proto-oncogene bcl-2. Overexpresalon of
bcl-2 can mimic the protective effect 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 that ted-9 and bcl-2 are homologs and that the molacular mechanism of progmmmed
cell death has been conserved from nematodes to
mammals.
Introduction
Programmed cell death plays an important role in animal
development and homeostasis and occurs in a wide variety of tissues in both vertebrates and invertebrates
(Glticksmann, 1950; Cohen, 1991; Ellis et 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 disruption of this balance can result in
disease (reviewed by Williams, 1991).
In the nematode Caenorhabditis elegans, 131 of the
1090 somatic cells generated during hermaphrodite development undergo programmed cell death (Sulston and Horvitz, 1977; Sulston et al., 1983). Genetic studies have led
to the identification of 14 genes that are involved at various
steps of this process; these genes can be placed into a
genetic pathway for programmed cell death in C. elegans
(reviewed by Ellis et al., 1991; Driscoll, 1992). Three of
these genes are involved in the regulation and execution
of all 131 deaths. The activities of two of these three genes,
ted-3 and ted-4 (called ted for cell death abnormal), are
required for cells to die (Ellis and Horvitz, 1988). In ted-3or
ted-4 mutants, essentially all cells that usually die instead
survive, differentiate, and (in at least some cases) properly
function (Ellis and Horvitz, 1988; Avery and Horvitz, 1987;
White et al., 1991). Genetic mosaic analyses suggest that
these two genes must be expressed by the cells scheduled
to undergo programmed death for these cells to die (Yuan
and Horvitz, 1990). The ted-4 gene encodes a protein with
no significant sequence similarity to any other protein in

the data bases (Yuan and Horvitz, 1992). The CM-3 gene
encodes a homolog of the mammalian interleukin-lPconverting enzyme (Yuan et al., 1993), which suggests
that the CED3 protein acts as a prOteaSe to cause programmed cell death.
The third gene involved in the control of all programmed
cell deaths, ted-9, negatively regulates the pathway for
programmed cell death: a c&9 gain-of-function mutation
prevents the deaths of cells that normally die, while mutations that inactivate ted-9 cause cells that normally live to
undergo programmed cell death (Hengartner et al., 1992).
Thus, the function of ted-9 is to prevent cells that normally
survive from undergoing programmed cell death. The absence of ted-9 function results in maternal-effect lethality,
indicating that c&-9 function is essential for C. elegans
development.
The proto-oncogene bcl-2 appears to function in mammals as ted-9 functions in nematodes. bcC2 was discovered and molecularly cloned based on its involvement in
a t(14;18) translocation that is observed in the majority
of follicular lymphomas diagnosed in the United States
(Fukuharaet al., 1979; Yunis et al., 1982). This translocation fuses the b&P locus to the immunoglobulin heavy
chain gene, resulting in the overexpression of 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
significantly the programmed cell death (apoptosis) of a
large variety of cells under various conditions that usually
lead to cell death. For example, bcl-2 protects interleukindependent lymphoid cell lines from apoptosis induced by
interleukin withdrawal (Vaux et al., 1988; Nufiez et al.,
1990) and thymocytes from apoptosis induced by glucocorticoids or by y-irradiation (Sentman et al., 1991; Strasser et al., 1991). bcl-2 also can protect neurons from
apoptosis induced by trophic factor withdrawal (Garcia et
al., 1992; Allsopp et al., 1993; Batistatou et al., 1993) and
can prevent apoptosis induced by c-myc in Rat-l cells
(Fanidi et al., 1992) and CHO cells(Bissonetteet al., 1992).
In tissues characterized by cell turnover, bcl-2 is often
expressed in progenitor or long-lived cells (Hockenbery
et al., 1991). Moreover, signals that rescue lymph node
germinal center cells from susceptibility to apoptosis induce bcC2 expression (Liu et al., 1991). Based on these
observations, it has been suggested that bc/-2 expression
protects cells that should survive from apoptosis (Hockenbery et al., 1991).
Recently, a number of genes with some sequence similarity to b&P have been reported (Boise et al., 1993; Kozopas et al., 1993; Lin et al., 1993; Oltvai et al., 1993; reviewed by Williams and Smith, 1993). Two of these genes,
bax and b&x, have effects on the regulation of apoptosis.
The relatively low sequence similarities (30%-500/b identity between various members) among these bcl-2 homologs suggest that this gene family might be of ancient origin
and not restricted to vertebrates.
Here we report the molecular characterization of the C.

FYI Classes v2

Apoptosis and disease

How might you show

functional homology
between ced-9 and
bcl-2?

45

46

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.
47

12