Biology 103 Lecture 6

8/18/2019 Biology 103 Lecture 6

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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

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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

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Apoptosis is a form of programmed cell

death.

•

A conserved pathway of genes controls

the decision for cells to die.

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Defects in apoptosis result in

developmental defects and disease.

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Biology 103 Lecture 6