Lecture 12: Organogenesis -Vertebrate and invertebrate limb development

Question 1

Organogenesis

Answer 1

• already learned about laying down the body plan and cell differentiation
• touch on development of specific organs
• cellular mechansims are essentially similar to those encountered in the early stages of development and are merely employed in different spatial and temporal patterns

Question 2

How many signalling pathways are there?

Answer 2

5 key developmental signalling pathways: 
Hedgehog pathway
Notch pathway
Wnt pathway
TGFB pathway
FGF pathway

• All 5 are essential during embryonic development
• All 5 are highly conserved from flies to humans.
• Aberrant activation is observed in a wide variety of cancers.

Question 3

The vertebrate limb

Answer 3

The limb is a great organ for studying cell interactions among a large number of cells and for studying intercellular signalling

The vertebrate limb is a classic dev. biology model system that has been studied at the tissue & molecular level for decades.

Which animal models? Mice - since they are amenable to transgenic manipulation but the basic principles of limb development have been mainly investigated using the chick.

The chick is very accessible for microsurgical manipulation as you can make a window in the eggshell, manipulate the developing limb bud and then reseal the shell and allow development to continue and monitor this process as it happens.

Question 4

What is essential for normal development of limbs to occur?

Answer 4

Temporal and spatial intercellular signalling is essential
for normal development to occur:
Timing and positioning of limbs

Question 5

The chick limb

Answer 5

-Limb development is initially visible 3 days after the egg has been laid and is fully formed by 10 days (7)

Question 6

What are the three orthogonal axes?

Answer 6

Proximodistal : from the base to the tip of the limb

Anteroposterior: parallel to the main body axis
(in the human hand it runs from the thumb –anterior –
to the little finger – posterior)

Dorsoventral: in the human hand it runs from the back of
the hand to the palm.
(8)

Question 7

What is the limb bud comprised of?

Answer 7

The limb bud is comprised of :

• a core of loose mesenchymal cells surrounded by an (sleeve) ectodermal epithelium.
• The mesenchymal cells give rise to the bones of the limb.
• The mesodermal cells that give rise to the muscles derive from the somites
• At the tip there is a thickening in the ectoderm called the apical ectodermal ridge
• Beneath are rapidly dividing undifferentiated mesenchymal cells: the progress zone
• Only when cells leave this region they start to differentiate.
• As the bud grows out cells start to differentiate and cartilagenous structures appear.
• The proximal part of the limb – the part nearest the body is the first to differentiate
• Differentiation proceeds distally as the limb bud grows out. (9, 10)

Question 8

Limb development time and space

Answer 8

Cartilage appears in a proximo to distal direction :
The humerus, radius, ulna, and then the digits

The chick limb bud at 3days is 1mm by 1mm but by 10 days has grown 10 fold in length but is still small compared to the size when the chick hatches

The AER disappears when the main elements of the limb are in place

During these later stages cartilage is replaced by bone and nerves only enter the bone at 4.5 days

Question 9

how does the limb bud develop at the appropriate position on the body?

Answer 9

• Limb buds develop at very precise locations along the AP axis from LPM.
• Transplantation experiments have shown specific regions of LPM are determined to form limb buds long before limb buds are visible .
• Hox genes regulate expression of genes required for limb development.
• FGF signalling is key to this
• a bead of FGF placed in the lateral plate mesenchyme between the fore and hind limb will initiate ectopic limb development. FGF10 knockouts develop no limbs (FGF crucial for where bud of limb dev occurs (12)
• Strikingly if the bead is placed in the anterior LPM a wing develops but in the posterior LPM a leg develops.
• This is due to TBX5 expression in the anterior LPM and TBX4 and PITX in the posterior LPM which are necessary for limb induction by FGF

SUMMARY
Limb bud induction depends on the signals being present (Hox) and the cells being competent to respond appropriately (FGF and TBX 4,5, PITX) (13)

Question 10

Once position is specified how do limb buds start to grow?

Answer 10

-Classical grafting experiments showed the importance of the AER for limb outgrowth.
-AER ablation leads to limb ablation
-The earlier the ablation the more severe the truncation
-Loss of AER leads to reduced proliferation and increased cell death
-FGF is the key signal here and an FGF bead can substitute for the AER
-Multiple FGFs are expressed in the AER: only a triple knockout causes complete truncation
(14, 15)

Question 11

How are skeletal elements laid down correctly along the proximodistal axis?

Answer 11

How position along the proximodistal axis (shoulder to tip of digits) is specified is a matter of debate – two models prevail:

The Timing Model and the Two signal Model

Question 12

The Timing Model

Answer 12

• Proximodistal patterning is governed by the length of time spent in the progress zone.
• The first cells to leave the zone develop into humerus(proximal) ; the last cells into digits (distal).
• FGF maintains the progress zone (once leave progress zone start to differentiate, longer stay in progress zone more distal structures can form)
• Cells “count” the time in the progress zone e.g. by number of cell divisions

-This specifies their positional identity along the Proximodistal axis progressively
(17)

Question 13

The two signal model

Answer 13

• Cells in the limb bud acquire positional identity from the presence of two opposing gradients - Retinoic acid from the proximal region specifying proximal structures (green) and FGF from the distal region specifying distal structures (red)
• As the bud grows, cells out of range of either signal acquire an intermediate positional value
  (orange) . This model holds that the proximodistal axis is specified from an early age. (18)

Question 14

How is the anteroposterior axis of the limb patterned?

Answer 14

-Patterning along this axis specifies the digits and gives them their ID.
-The ZPA (zone polarizing activity, very potent region) is the organising centre that patterns this axis.
-A ZPA graft to the anterior region of a host limb leads to a mirror image pattern – instead of the normal pattern 2,3,4 the pattern 4,3,2,2,3,4 develops
-graft from mouse ZPA to chick limb induces additional chick digits
-ZPA from a wing bud to the anterior region of a leg bud will induce ectopic toes
-this the signal is conserved but the interpretation depends on the responding cells
-the additional digits come from the host: The ZPA changes the fate of the host cells
(19)

Question 15

What is the signal released for the ZPA?

Answer 15

-The morphogen Shh patterns the AP axis
-Shh is expressed by the ZPA
-Beads soaked in different concentrations of SHH induces different digits:
-Highest Shh induces digit 4.
->digit 1: SHH independent, 2: low SHH, 3:brief SHH expression, high SHH conc, 4: moderate SHH expression, 5: extended SHH expression
(20)

Question 16

How does SHH expression become restricted to the ZPA?

Answer 16

• Early on Hoxd9 and 10 are expressed in the LPM as it thickens to become a bud.
• Hoxd11,12,13 are then initiated in rapid succession in progressively more restricted domains in the posterior distal region of the fore and hind limb buds.
• Hoxa9-13 simultaneously become expressed in restricted domains across the PD axis : later genes restricted to more distal domains
• Loss of all Hox A and D genes leads to loss of Shh and severe limb truncations whereas ectopic Hoxd13 through out the early bud leads to SHH in both anterior and posterior mesenchyme and duplications of limb patterns
• Mutations in hu Hoxd13 leads to polydactyly. (21)

Question 17

Gli3

Answer 17

Gli3 is the key regulator
GliR
|
Shh -> V
GliA
-Gli3 -/- have polydactyly – thus Gli3 repressor is required to prevent digits forming in the anterior limb bud where there is no SHH
-In the posterior limb bud Shh -> Gli3 activator allowing full digit pattern formation.
-In Shh -/- high levels of Gli3 R so no digits form
-Double ko resembles the Gli3 -/- : main function of SHH is to supress Gli3 R in the posterior bud
-Several mutations in human Gli3 are associated with limb abnormalities
(22)

Question 18

What is the DV axis of the limb bud regulated by?

Answer 18

-the ectoderm
-The genes involved have been identified mainly in mice as dorsoventral pattern is easily analysed since the ventral surface of the paw has no fur.
-Combining ectoderm from left limb buds with mesoderm from right limb buds so the DV axis of ectoderm is reversed with respect to mesoderm but AP axis is unchanged, changes the DV pattern of the muscles and tendons. But what are the molecules?
-Wnt7a is expressed on the dorsal ectoderm. The Wnt7A -/ gives a double ventral limb
The ventral ectoderm expresses Engrailed 1 –a homeodomain transcription factor. Loss of En1 leads to Wnt7a expression in dorsal and ventral ectoderm and a double dorsal limb.
-These DV signals also interplay with the AP and PD signals. (23)

Question 19

How do limb muscles arise?

Answer 19

• Limb muscles arise from cells that migrate into the bud from the somites.
• The cells multiply and form dorsal and ventral blocks of presumptive muscle that undergo a series of divisions to give rise to individual muscles(24)

Question 20

What is the separation of the digits a result of

Answer 20

• Separation of the digits is the result of programmed cell death.
• BMPS regulate this process. Loss of function mutation in BMP receptors inhibits cell death in the chick leg bud and the digits formed as a result are webbed.
• Ducks and waterfowl have webbed feet simply due to less cell death during limb development. It is the mesoderm that regulates cell death both in the mesoderm and the ectoderm
• Chick limb mesoderm replaced with duck limb mesoderm leads to webbed chicken feet due to reduced cell death

Question 21

Insect limb development

Answer 21

-The organs and appendages of Drosophila develop from imaginal discs – epithelial sheets set aside in the embryo
-Hox genes act in the parasegments to specify which appendage will form
-Wing and leg discs are specified when the segments are being patterned and are initially clusters of 20-40 cells (the discs invaginate as pouches of epithelium from the embryonic ectoderm and remain until metamorphosis) and these grow 1000 fold during larval development
-The mechanisms and genes involved in limb patterning show remarkable similarity to vertebrate limb development
-Imaginal discs are divided into anterior and posterior compartments .
-In the wing disc a second compartment boundary develops– the DV boundary.
WING AP axis
-Signalling centres are established along the compartment boundaries.
-En expression in the posterior, reflects its expression in the epidermis of the parasegment from which the disc derives.
-En cells also express hedgehog. At the boundary Hh morphogen induces dpp.
-Dpp morphogen gradient : induces Omb (TBX) at low & spalt (Sal) at high levels.

(26, 27)

Question 22

Outcomes of AP patterning in insects

Answer 22

Outcomes of AP patterning: Wing veins

Clone of cells ectopically expressing Hh in the anterior compartment leads to ectopic sites of Dpp expression with new gradients of Dpp and resulting in a wing with a mirror-repeated symmetric vein pattern
(28)

Question 23

Wing DV axis

Answer 23

• Notch plays its part
• Apterous expression specifies dorsal character.
• The DV boundary cells also act as an organising centre; Wingless is the signalling molecule. Where did this gene name come from?.
• Apterous induces the Serrate and Fringe in dorsal cells. Expression of the Notch ligand, Delta is restricted to ventral cells. Notch is expressed by both dorsal and ventral cells.
• Fringe inhibits Notch signalling via Serrrate and promotes via Delta. Only Notch/Fringe expressing cells at the interface of Delta expressing cells show high Notch signalling. The output is activation of wingless which induces Delta and Serrate at the boundary thus maintaining its own expression.
• Wingless acts as a morphogen to activate different genes at specific threshold concentrations which pattern the wing and are responsible for its outgrowth.

(listen to slide!!! 29)

Question 24

Leg Imaginal disc

Answer 24

• The insect leg is a jointed tube of epidermis.
• The epidermal cells secrete the hard exoskeleton.
• Inside there are muscles, nerves and connective tissue.
• The imaginal disc is like a collapsed cone. At metamorphosis the epithelial cells change shape causing the outward extension of the leg.
• The outermost ring forms the base of the leg and the inner rings form the more distal structures. (30)

Question 25

Leg AP and DV axes

Answer 25

Leg AP and DV axes.

-There are many similarites to what is seen in the wing:
BUT Hh induces Wg at the boundary of the ventral half
-Notch and Delta are required for joint formation. Delta and Serrate are expressed in concentric rings and activation of Notch at these points induces joint forming cells.
-Leg and wing discs are patterned by some of the same molecules but the outcome is very different. The interpretation of positional signals is under the control of Hox genes.

Question 26

(Extra slides not sure where from and what refer to?)

Wing

Answer 26

The Wg and Dpp morphogens provide cells with positional identity through their graded concentrations. These may be achieved via simple diffusion, or through cytonemes or via regulated expression of receptors (Dpp represses expression of one of its receptors – thick veins)

How the PD axis is patterned is less clear although a role for Wnt in both short and long range gene activation has been shown across this axis.

At metamorphosis, ptterning of the disc is complete and the invaginated pouch is turned inside out as its cells differentiate.and it extends and folds so that one surface comes to lie beneath the other. Thus, the adult wing is a structure where two epidermal layers – the dorsal and ventral layers – are close together
33

Question 27

Mutation in LMX1B

Answer 27

The dorsal and ventral mesenchyme are clonally restricted cell populations – this is the first mesenchyme to show this sort of cell lineage restriction. The Wnt7a target LMX1B is dorsally restricted and mutations in human LMX1B lead to loss of the dorsal structures such as nails.

Question 28

How does Gli3 repressor specify the different digits?

Answer 28

Gli3 targets: Sal1 TBX2 and Hoxd13
Maintenance and propagation of Shh expression requires AER-FGF signalling
as part of a positive epithelial–mesenchymal feedback loop between ZPA & AER.

Is that the whole story?……..No
SHH is crucial for initial specification of digit identity, BMPS (TGFB) regulated by Gli3, also play a role at several stages to pattern the limb.

Question 29

Initiation, propagation, termination

Answer 29

Broad initial BMP expression is required for initial AER development.
BMP up regulates its antagonist GREM1 which reduces overall BMP activity.
Shh in the ZPA also up regulates GREM.
GREM1 is crucial in the feedback loop between ZPA & AER
Descendants of Shh expressing cells cant up regulate GREM so a distance forms between SHH and GREM +ve cells breaking the feed back loop
the GREM1-mediated increase in AER-FGF eventually triggers a FGF/Grem1 inhibitory loop, which terminates outgrowth/patterning by shutting down Grem1

Question 30

Summary limb bud development

Answer 30

In summary, limb-bud development seems to progress from

BMP4-dependent initiation
via predominantly SHH-dependent specification and
proliferative expansion of the distal autopod primordia
to FGF-mediated self-termination .

These interconnected signaling feedback loops define a self-regulatory limb patterning system, mostly because of their differential regulatory impact on Grem1