The development of the tetrapod limb involves specification of the limb field and induction of the early limb bud through FGF10 signaling. The proximal-distal axis is established by the AER, which secretes FGF8 to maintain the progress zone. The anterior-posterior axis is specified by SHH expression in the ZPA. The dorsal-ventral axis forms through Wnt7a expression on the dorsal side. Cell death regulated by BMPs then separates the digits and forms joints.
2. PATTERN FORMATION
process by which embryonic cells form
ordered spatial arrangement of
differentiated tissues.
How is it that the embryo is able not only
to generate all the different cell types of
the body but also to produce them in a
way that forms functional tissues and
organs.
2
4. Many recent studies show that the basic
morphogenetic rules for forming a limb appear to
be the same in all tetrapods.
Fallen & Crosby (1997) showed that grafted
pieces of reptile/mammalian limb buds can
direct the formation of chick limbs
Sessions et al (1989) found that regions of
frog limb buds can direct the patterning of
salamander limbs and vice versa.
4
5. Development of the tetrapod limb has to function
in a three-dimensional coordinated system
Proximal-distal axis------ Fibroblast Growth
Factor family protein and Hox Genes
Anterior-posterior axis----- Sonic hedgehog
protein
Dorsal-ventral axis------ Wnt7a protein
5
6. 1. Formation of the Limb Bud
2. Generating the Proximal- Distal Axis of the
Limb
3. Specification of Anterior- Posterior Limb Axis
4. Generation of the Dorsal- Ventral Axis
5. Cell Death and the Formation of Digits and
Joints
6
DEVELOPMENT OF THE TETRAPOD LIMB
7. 1. LIMB BUD FORMATION
1.A Limb Field Specification
regulated by Hox Genes
and Retinoic Acid
Limb field position is determined with
respect to the level of Hox gene expression
along the anterior –posterior axis
Retinoic acid is also
critical in limb bud
outgrowth. 7
8. 1.B INDUCTION OF EARLY LIMB BUD:
FIBROBLAST GROWTH FACTORS
• Limb muscle precursors +
limb skeletal precursors-----
LIMB BUD
• Signal for limb bud
formation comes from the
lateral plate mesoderm cells
that later will become
PROSPECTIVE LIMB
MESENCHYME CELLS
•FGF10 = capable of initiating
the limb –forming interactions
between ectoderm and
mesoderm.
8
10. 1.C SPECIFICATION OF FORELIMB OR
HINDLIMB: TBX4 AND TBX5
The gene encoding the Tbx5 transcription factor is
transcribed in forelimbs of embryo while the gene
encoding the Tbx4 transcription factor is expressed
in hindlims.
10
12. 2. GENERATING THE PROXIMAL –DISTAL AXIS
OF THE LIMB: FIBROBLAST GROWTH FACTOR
& HOX GENES
AER (Apical Ectodermal Ridge)--- major signaling center for
the developing limb
• maintaining the mesenchyme
beneath it in a plastic, proliferating
phase that enables the proximal-distal
growth of the limb
•Maintain the expression of molecules
that generate anterior-posterior axis
•Interacting with the proteins
specifying the anterior-posterior and
dorsal-ventral axes so that each cell is
given instructions on how to
differentiate.
12
13. THE PROGRESS ZONE (PZ)
13
Molecules that are
secreted from AER
(FGF8) keep the PZ
mesenchyme cells
dividing.
14. AER NECESSARY FOR LIMB BUD
OUTGROWTH
Removal of AER at various times prematurely truncates limb
14
15. 15
HOX GENES AND THE
SPECIFICATION OF THE PROXIMAL-
DISTAL AXIS
PROXIMAL DISTAL
POSTERIOR
ANTERIOR
16. 16
3. SPECIFICATION OF THE ANTERIOR-
POSTERIOR LIMB AXIS: SONIC
HEDGEHOG PROTEIN
Zone of Polarizing Activity
(ZPA) --- block of
mesodermal tissue near the
posterior junction of the
young limb bud.
17. 17
SONIC HEDGEHOG IS EXPRESSED IN
ZPA
SHH works by initiating and sustaining a cascade of other proteins,
such as BMP2 and BMP7. A gradient of BMPs expressed from
ZPA specify the digits.
However, transplantation of ZPA to the anterior portion of limb
bud leads to the formation of mirror-image digit pattern.
18. 4. GENERATION OF THE DORSAL-
VENTRAL AXIS: WNT7A
18
• Wnt7a gene is expressed
only in dorsal portion of
limb bud.
• It induces activation of
the Lmx1 gene in dorsal
mesenchyme and this gene
encodes a transcription
factor that appears to be
essential for specifying
dorsal fates in the limb.
19. 5. CELL DEATH AND THE FORMATION
OF DIGITS AND JOINTS
19
• Signal for apoptosis in the autopod
region is provided by the Bone
Morphogenetic Proteins.
• BMP2, BMP4 and BMP7 are each
expressed in the interdigital mesenchyme
•Nogging proteins supressed BMPs
•Noggin proteins is synthesized in
developing digits.and perichondrial cells
surrounding them.
•However Blocking BMP signaling
between digits prevents interdigital
apoptosis.
20. FORMING JOINTS
20
• Bone Morphogenetic Proteins
are responsible in forming
joints.
•BMP7 is synthesized in the
perichondrial cells sorrounding
the condensing chondrocytes
and promotes cartilage
formation.
•Other BMPs, BPM2 and GDF5,
are expressed at the regions
between the bones, where joints
will form.
21. SUMMARY: THE DEVELOPMENT OF
TETRAPOD LIMB
The places where limbs emerge from the body axis
depend upon Hox gene expression.
The specification of the limb field into a hindlimb or
forelimb bud is determined by Tbx4 and Tbx5
expression.
The proximal-distal axis of the developing limb is
determined by the induction of the ectoderm at the
dorsal-ventral boundary to form the apical
ectodermal ridge (AER). This induction is caused by
an FGF10. The AER secretes FGF8, which keeps the
underlying mesenchyme proliferative and
undifferentiated. This mesenchyme is called
progress zone
21
22. SUMMARY: THE DEVELOPMENT OF
TETRAPOD LIMB
As the limb grow outward, the stylopod forms first, then
the zeugopod, and the autopod is fomed last. Each of
these phases involves the expression of Hox genes, and
the formation of the autopod involves a reversal of Hox
gene expression that distinguishes fish fins from
tetrapod limbs
The anterior-posterior axis is defined by the expression
of Sonic hedgehog in the posterior mesoderm of the limb
bud. This region is called the zone of polarizing activity
(ZPA). If the ZPA or Sonic hedgehog-secreting cells or
beads are placed in the anterior margin, they establish a
second, mirror-image pattern of Hox gene expression and
a corresponding mirror-image duplication of the digits
22
23. SUMMARY: THE DEVELOPMENT OF
TETRAPOD LIMB
The ZPA is established by the interaction of FGF8
from the AER and mesenchyme made competent to
express Sonic hedgehog by its expression of
particular Hox genes. Sonic hedgehog acts, probably
in an indirect manner, to change the expression of
Hox genes in the limb bud
23
24. SUMMARY: THE DEVELOPMENT OF
TETRAPOD LIMB
The dorsal-ventral axis is formed, in part, by the
expression of Wnt7a in the dorsal portion of the
limb ectoderm. Wnt7a also maintains the expression
of Sonic hedgehog in the ZPA and FGF4 in the
posterior AER. FGF4 and Sonic hedgehog
reciprocally maintain each other’s expression.
Cell death in the limb is necessary for the formation
of digits and joints. It is mediated by BMPs. The
effects of BMPs can be regulated by the Noggin
protein and the BMPs can be involved both in
inducing apoptosis and in differentiating the
mesenchymal cells into cartilage 24
Editor's Notes
Good morning class. For today I’ll be discussing the Development of the tetrapod limb.
By the way, tetrapods are four footed or four-limbed animals or vetebrates. One example are us humans and mostly mammals.
Let us have a brief introduction.
One of the most dramatic properties of developing organisms is the PATTERN Formation. And PATTERN FORMATION is the process by which embryonic cells form ordered spatial arrangement of differentiated tissues. Or it is the generation of complex organizations of cell fates in space and time.
So in this process, one question usually ask is how is it that the embryo is able not only to ….. . . . . . . .
Let’s find out as we go deeper to this chapter.
Let us have a brief review on the different parts and regions of the tetrapod limb.
Vertebrate limb is an extremely complex organ with an asymmetrical arrangement of parts.
When we say ASSYMETRICAL the 2 sides or 2 halves of an organ or organism are not the same in terms of their morphology.
There are 3 main axes to consider in vertebrate limb.
One of which is the PROXIMAL-DISTAL axis or the shoulder to finger in forelimbs and the hip to toe in hindlimbs
The second is the ANTERIOR-POSTERIOR axis or the thumb to pinky axis.
Third is the DORSAL-VENTRAL axis or the knuckle and palm axis.
The bones of the limb, be it wing flippers foot or hand consist of 3 regions
First is the PROXIMAL STYLOPOD region consist of the humerus bone in forelimb or femur bone in hindlimb
Second is the middle region the ZEUGOPOD consist of radius and ulna for forelimb or tibia and fibula bones for hindlimb
And the third region is DISTAL AUTOPOD consists of carpals and fingers in forelimb and tarsals and toes in hindlimb.
Many researchers…….
In the work of Fallen and Crosby they Were able to shoW that the ………………………..
Development of the tetrapod limb has to function ……………….
When We say 3-dimensional cordinated system the 3 axes of the vertebrate limb are all interrelated and coordinated. The formation or development of one axis mutually supports the formation of the other 2 axis.
We Will be discussing the coordination of these 3 axes later.
Researchers Were able to identify particular proteins and genes that play a role in the formation of the 3 axes
The proximal-distal axis appears to be regulated by fibroblast groth factor family protein
The anterior-posterior axis seems to be regulated by sonic hedgehog protein
The dorsal-ventral axis is regulated by Wnt7a protein
Here is the outline of the Whole Tetrapod Limb Development
First is the formation of the limb bud…………………..
Let us proceed to the first process, the LIMB BUD FORMATION
There are 4 limb buds per embryo and they are located opposite to each other With respect to the midline
The first step in limb bud formation is the LIMB FIELD SPECIFICATION and it is regulated by HOX GENES and retinoic acid
By the Way Limb field is Where limb bud Will emerge.
Limb field position is determined…….
And also retinoic acid is critical in limb bud outgroWth since blocking OF Retinoic acid Synthesis prevents limb bud initiation
After limb field specification, INDUCTION OF EARLY LIMB BUD folloWs and it begins When mesenchyme cells such limb muscle precursors and limb skeletal precursors proliferate and accumulate underneath epidermal tissue creating a circular bulge called limb bud.
Recent studies on the earliest stage of limb formation have shoWn that the signal for limb bud formation……..
And this PROSPECTIVE LIMB MESENCHYME CELLS secrete the paracrine factor FGF10
And this FGF10 is capable of……………
In this experiment, beads containing fgf10 are placed ectopically beneath the flank ectoderm and then extra limb emerges.
Thus, fgf10 is responsible in Induction of early limb bud.
Any question? If none let us proceed hahaa
The next process is the Specification of forelimb or hindlimb
The limb buds have to be specified as being a forelimb or a hindlimb.
So hoW are this distinguised?
There are a lot of researches conducted to ansWer this question. And they all agreed to only one correlation and it says that……..
Many researchers like Ohuchi et al, Logan et al, Takuchi et al and Rodriguez-Esteban et al provided a GAIN OF FUNCTION EVIDENCE that Tbx4 and Tbx5 specify hindlimb and forelimb respectively.
So in this experiment this bud induced by placing bead containing fgf10 close to the forelimb portion expressed Tbx5 and become forelimb.
In this bud induced by placing FGF bead near to the hindlimb portion expressed Tbx4 and developed as hindlimb
And those bud induced by placing FGF bead at the center or at the boundary of forelimb and hindlimb portion expressed Tbx5 anteriorly and Tbx4 posteriorly forming a chimeric structure With anterior resembling forelimb and posterior portion resembling hindlimb.
Thus Tbx4 and Tbx5 appear to be critical in instructing the limbs to become hindlimb and forelimb respectively.
Let us move on to the 2nd stage of limb development; the generation of the proximal-distal axis.
As mesenchyme cells enter the limb region they secrete factors that induce the overlying ectoderm to form a stucture called APICAL ECTODERMAL RIDGE.
This ridge runs along the distal margin of limb bud and Will become the major signalling center for the dev…….
It has a lot of function that explains Why limb development is a 3-dimensional coordinated system.
It maintains the mesenchyme……..
…
…
So apical ectodermal ridge has contributions in forming the 3 axes of the limb.
The proximal-distal axis is defined only after the induction of the apical ectodermal ridge by the underlying mesoderm.
The limb bud elongates by means of proliferation of the mesenchyme cells underneath the AER. This region of cell division is called the PROGRESS ZONE and it extends about 200 micrrometer from AER.
Molecules that are secreted from AER ……..
When AER is removed from an early limb bud, only most proximal parts are made.
As shoWn in letter B picture, AER is removed after Stylopod formation and only humerus bone is developed.
And here in letter D, AER is removed during the development of zeugopod or middle region of limb, only humerus and ulna bone are developed.
Thus (effects click) Removal of AER at vaious times prematurely truncates/shortens limb.
The type of structure formed along the proximal-distal axis is specified by the Hox genes.
A While ago We mention that hox genes played a role in specifying the place Where the limbs ill form of the specification of limb.
No they Will play a second important role in specifying Wether a particular mesenchymal cell Will become stylopod,zeugopod or autopod
The mechanism by Which Hox genes could specify the proximal-distal axis is not yet fully understand but it thought that as limb bud groWs outWard, the pattern of hox gene expression changes.
When the stylopod is forming Hoxd-9 and Hoxd-10 are expressed in the Progress Zone mesenchyme.
When the zeugopod bones are being formed, the pattern of hox gene expression shifts remarkably. The posterior portion expresses all the Hoxd genes from Hoxd9 to 13 While only Hoxd-9 is expressed anteriorly.
When autopod is forming there is a further redeployment of Hox gene expression. Hoxd-9 is no longer expressed rather Hoxa 13 and Hoxd 13 is expressed anteriorly While throughout the 2/3 of posterior portion hoxa 12 13 and Hoxd 10 11 12 and 13 are expressed.
Let us proceed to the third stage of limb development process; the specification of the Anterior-Posterior limb axis
The anterior-posterior Limb axis is specified by a small block of mesodermal tissue ………….
This region of the mesoderm has been called the ZONE OF POLARIZING ACTIVITY.
Through research studies they found out that the molecules conferring this ppolarizing activity on the ZPA is the SONIC HEDGEHOG protein.
Sonic hedgehog Works by ……………
HoWever, …………
(take note that the normal induction of ZPA occurs only in posterior portion)
Because this transplanted ZPA here in anterior portion duplicates the expression of Hox genes responsible in forming digits thus creating a mirror image digit structure.
One molecule that appears to be particularly important in specifying the dorsal-ventral polarity is Wnt7a.
……………………..
……………….
Research conducted by Parr and McMahon, they genetically deleted Wnt7a from mouse embryo. The resulting embryo had sole pads or ventral portion on both surfaces of their pas, showing that Wnt7a is needed for the dorsal patterning of the limb.
……………………..
……………………….
These BMPs are expressed throughout the Progress Zone mesenchyme. So hoW digits are formed When BMPs are expressed throughout PZ?
The suprression of BMPs come fom the Noggin proteins Which is synthesized in developing digits……………
……………………… that Will result to Webbed feet or hands.