Lecture 18 - How are the patterns of structures established

Question 1

What is FGF8?

Answer 1

Fgf8 is one of several genes encoded signalling molecules expressed in Apical Ectodermal Ridge (AER) & involved in proximal/distal patterning.

Dorsal epithelium expression Wnt7a, which is involved in D/V patterning (nails on top & pads under).

Question 2

Where is the AER found?

Answer 2

posterior distal part of limb bud

Question 3

What does the ZPA (zone of polarizing activity) specify?

Answer 3

A/P axis pattern

Question 4

What do limb buds appear as?

Answer 4

protrusions from the flank at precise positions along the A/P axis of the embryo.

Question 5

What is the role of Hox genes?

Answer 5

Hox gene expression in the lateral
plate mesoderm interpret positional information along the A/P axis.

Limb buds first appear as bumps across the A/P axis of embryo. Position of FORELIMBS & HINDLIMBS are defined by position of Hox genes.

Question 6

Where are Hox genes expressed?

Answer 6

Hox genes are expressed in overlapping domains. In lateral plate mesoderm & expressed in response to signals. LPM gives rise to limb buds. HOX genes expressed in a co-linear manner - relationship between order found on chromosome & spatial sequence in which they are expressed.

3’ Hox genes are expressed on anterior head regions. 5’ Hox genes in posterior regions.

Products of Hox4/5 genes interpret positional information to ensure that FORELIMBS always develop at the Cervical:Thoracic boundary.

Hox9-11 gene products interpret positional information to ensure that hindlimbs develop at the Lumbar:Sacral positioning.

The position of this boundary varies between species.

Question 7

What do Hox proteins do?

Answer 7

Hox proteins along & other factors induce LIMB-SPECIFIC transcription factors that specify limb type identity

Question 8

What TF establishes FORELIMB identity?

Answer 8

Tbx5

Question 9

What TFs establish HINDLIMB identity?

Answer 9

Tbx4 & Pitx1

Question 10

How do Hox genes interpret the positional information?

Answer 10

From the signalling gradients operating across the main body axis.

These include retinoic acid, Wnt & FGFs.

This ensures limb development in the right place & time.

THESE FACTORS, ALONGSIDE OTHER FACTORS SWITCH ON EXPRESSION OF LIMB SPECIFIC TFs THAT GOVERN LIMB TYPE IDENTITY.

Transcription factors also drive limb outgrowth - tbx5 in forelimbs and tbx4 & pitx1 in the hindlimb.

Question 11

What does overexpression of tbx5 lead to?

Answer 11

overexpression of tbx5 (normally expressed in wing, was overexpressed in leg) legs sprouted feathers.

Question 12

What does overexpression of tbx4 lead to?

Answer 12

overexpression of tbx4 (normally expressed in leg, overexpressed in wing) lead to 4 limbs instead of 3.

Question 13

What does expression of Pitx1 in forelimb result in?

Answer 13

Led to development of knee-like articulation, which is characteristic of a leg.

Question 14

What is the purpose of an Fgf10-Fgf8 feedback loop?

Answer 14

An Fgf10 - Fgf8 feedback loop is established that drives limb bud outgrowth.

Fgf10 –> Fgf8 –> Wnt3a

Tbx4 & Tbx5 both play a role in initiating limb outgrowth from flank of embryo - they both act through Wnt signalling - but with different Wnt ligands, to induce expression of important gene in limb development - FGF10. FGF10 important, expressed in mesenchyme & induces expression in AER via Wnt3a, which then feeds back onto Fgf8, which maintains fgf19 expression - feedback loop. Loss of Fgf10 leads to loss of limb.

Proximal distal action.

Question 15

What can an ectopic source of Fgf in the chick interlimb flank lead to?

Answer 15

An ectopic source of Fgf in the chick interlimb flank is sufficient for limb bud outgrowth.

it also expands the Tbx4 & Tbx5 expression domains

Ectopic bead induces apical external ridge.

Question 16

What does Tbx4/5 expression in the middle cause?

Answer 16

causes chimera development (wing & leg mix)

Question 17

How are skeletal elements specified in proximal to distal sequence?

Answer 17

Wing bud skeleton truncated distal to humerus. Earlier the limb bud was removed, the more proximal was truncation.

Only digits lost at later stages.

Specification takes around 72 hours.

Specification occurs in a proximal to distal sequence.

Question 18

Describe the PROGRESS-ZONE model of proximo-distal patterning

Answer 18

This model involves TIMING

Proximal distal positional information is specified by the length of time that cells spent in a zone of undifferentiated cells at the tip of the limb bud, called the progress zone. The cells are kept undifferentiated by signals produced AER - e.g. FGF8

As limb bud grows out, cells become displaced from the progress zone. Once they’ve left the zone, their position will be fixed, as they differentiate.

Cells leave early, with proximal positioning, cells that leave later will have more distal values.

Question 19

Describe the Hox genes involved in the development of limbs

Answer 19

Hox genes involved are homologues of HOXA & HOXD genes.

3’ genes are expressed first:
Hox10 - Humerus
Hox11 - Ulna & radius
Hox12 - Carpals
Hox13 - Digits

Hox paralogues are essential for limb segment specification & patterning.

Proximal structures specified first, distal; last.

Question 20

What is the 2-SIGNAL model of proximo-distal patterning?

Answer 20

RA signalling comes from flank to trunk. FGF from AER antagonise each other.

High RA specify proximal structures

High FGF specify distal structures

Model suggests that outgrowth of a limb would allow RA & FGF signalling to fall to lower levels in the middle - allowing intermediate structures to be specified.

This model wouldn’t rely on timing, but CONCENTRATION OF SIGNALS THE UNDIFFERENTIATED CELLS RECEIVE. This suggested proximal (stylopod), then distal (autopod), then middle (zeugopod) development.

Question 21

What is the SIGNAL-PROGRESS zone model?

Answer 21

This model suggests that RA do specify the proximal part, but before the progress zone is established. It suggests that RA signalling prevents establishment of the progress zone. FGFs instead of specifying distal structures directly, antagonize RA to allow progress zone to be established in the distal part of limb. Once this occurs, timing defines middle & distal parts

Question 22

What are the 3 models of proximo-distal patterning?

Answer 22

1st model - progress zone model involves timing
2nd model - 2 opposing gradients of signals
3rd model - signals followed by timing

Question 23

Describe the discovery of the Zone of Polarizing Activity (ZPA) or polarizing region in the chick wing bud

Answer 23

It was predicted that the ZPA produces a signal or morphogen which specifies AP position & digit identity.

ZPA grated & mirror image duplication of pattern was clear. Affects wing development.

ZPA produces a morphogen which species AP position & digit identity.

Homogenous population of cells, concentration of morphogen forms over cells & gives cells positioning on A/P axis - source of morphogen on left (where ZPA is) - high posterior, low anterior.

High levels = digit 3
Middle levels - digit 2
Low levels = digit 1

Question 24

What morphogen is produced by the ZPA?

Answer 24

Sonic hedgehog (Shh) - Ssh encodes a protein that fulfils the criteria for a polarizing area in the limb. Shh grafted to chick wing bud - concentration dependent duplication.

ZPA grated to anterior & led to limb duplications.

Shh signalling species digit patterning in 12 hours in the chick wing

Question 25

What does cyclopamine do?

Answer 25

Inhibits shh pathway. Leads to low shh & 1 digit formation. Later, higher concentration of Shh and digit 1 & 2 formation. After 12 hours, Shh fully working & normal pattern occurs.

Question 26

How to make a pattern of more than 3 digits?

Answer 26

Digit 1 to 3 - concentration Digit 4 - timing Chick leg - 4 digits Normal & duplicated pattern - ZPA gives rise to 4th digit. 1,2 & 3 are specified by Shh signalling, but unlikely that Shh specify digit 4, but more likely ZPA cells become digit 4.

Question 27

What does ZPA give rise completely to?

Answer 27

Digit 4 & 5

Question 28

How does Shh signalling operate through Gli transcription factor?

Answer 28

No Shh signalling - Ptch1 inhibits Smoothened allowing Gli activators (A) to be processed into Gli repressor (R) - preventing transcription of target genes. Shh signalling - Shh relieves the Pthc1 inhibition of Smoothened preventing Gli activators being processes into repressors. This results in a Gli3A/R gradient across the limb bud & activation of target genes including Ptch1, Gremlin1, Tbx2/2.

Question 29

What are the 3 Gli genes' role?

Answer 29

Gli3 important in limb. Shh allows conversion of Gli-A to activator from repressor. Shh works via a gradient.

Question 30

How is digit identity determined downstream of Shh signalling?

Answer 30

- Tbx3 determines digit 3 - Tbx2 determines digit 4 Works with 5' HOX genes.

Question 31

What is the feedback loop coordinates A/P & P/D patterning at early stages?

Answer 31

- The Gremlin gene encodes a BMP antagonist. - It is expressed in limb bud mesenchyme - It is known as the Apical Ectodermal Ridge (AER) maintenance factor (AERMF). Existence of the AERMF was first predicted. - AER-FGF4/8 are required for Shh expression - Reinforces the principal that genetic signalling loops are important in Developmental Biology. Grem inhibits BMP, which prevents inhibition of AER, which activates Shh, which reinforces Grem.

Question 32

How is limb bud specific expression controlled by the ZPA Regulatory Sequence?

Answer 32

Limb specific expression of Shh is controlled by distal regulatory sequence, mega-base away from Shh gene - Lmbr1. This is ZPA regulatory sequence. TFs - e.g. Hox & Han2, bind to ZRS, switching on Shh expression. TFs can also suppress ZRS & no Shh expression.

Question 33

What is D/V patterning determined by?

Answer 33

Ectoderm (epithelium) Experiment to show ectoderm shows signals which specifical D/V pattern. Removal of ectoderm & flipped 180, and grafted, reversed D/V patterning of mesoderm. The ectoderm produces signalling molecules that induce transcription factors Dorsal - Wnt7a induces TF called lmx1b. Ventral - BMPs induced TF - engrailed1 in ventral ectoderm. Engrailed prevents expression of Wnt7a.

Question 34

What do mutations in mouse DV patterning result in?

Answer 34

Dorsalisation & ventralisation Wnt7a mutant - double ventral Lmx1b mutant - double ventral Engrailed1 mutant - double dorsal

Question 35

Summarise how patterns of structures are established?

Answer 35

1. The products of Hox genes determine the A/P position that limbs emerge from the embryonic trunk. 2. Tbx4, Tbx5 & Pitx1 are involved in determined limb-type identity: forelimb (Tbx5), hindlimb (Tbx4 & Pitx1). 3. Tbx4 & Tbx5 trigger limb bud initiation by activating an FGF10/FGF8 feedback loop between the mesenchyme & AER. 4. The AER is essential for limb outgrowth along the proximo-distal (PD) axis. 5. PD patterning has been described by the progress zone, two-signal & signal-progress zone-models. 6. A concentration gradient of Shh from the ZPA specifies A/P position & digit identity. 7. A Shh-Gremlin1-Fgf feedback loop between the ZPA & AER coordinates AP & PD patterning at early stages. 8. Shh transcription is controlled by a distant sequence called the ZPA Regulatory Sequence (ZRS). 9. D/V patterning is specified by signals produced by the ectoderm (epithelium) - excluding the AER.