Final Exam - Limb Development

Question 1

Specification of limb bud region

Answer 1

• Is specified in the lateral plate mesoderm
• The overlying ectoderm aids in patterning and outgrowth
• Hox genes are involved in positioning the limb region and identity
• Hox-b5: When function is missing there is a shift in the placement of the forelimb, the shoulder girdle towards the neck
• Pitx1: Transcription factor that is expressed only in the hind limb, is necessary and sufficient for hindlimb identity

-specified: describes the developmental status of a group of cells that when isolated and cultured in a minimal medium will developing according to their normal fate

• limb development initiates as small buds on the side of the embryo
• Anterior buds give rise to forelimbs (wings in chick)
• Posterior limbs give rise to hind limbs
• The limb bud consists of mesoderm (originating from the lateral plate mesoderm) and an overlying layer of ectoderm

Question 2

Lateral plate mesoderm

Answer 2

• where the limb bud region is specified
• mesoderm in vertebrate embryos that lies lateral and ventral to the somites and gives rise to the tissues of the heart, kidneys, gonads, blood and limb connective tissue

Question 3

Hox genes

Answer 3

• involved in positioning the limb region and identity
• a family of homeobox-containing genes that are involved in patterning the antero-posterior axis
• are clustered on the chromosomes in one or more gene clusters or complexes
• combinatorial expression of different Hox genes characterizes different regions or structures along the axis

Question 4

Hoxb5

Answer 4

• involved in positioning the forelimb region and identity
• When function is missing there is a shift in the placement of the forelimb
• The shoulder girdle towards the neck

Question 5

Pitx1

Answer 5

• Transcription factor that is expressed only in the hind limb
• Is necessary and sufficient to specify hindlimb identity
• Pitx 1 mutants in hindlimbs show a transition to forelimb identity (take it away, you get two forelimbs)
• A major consequence of Pitx 1, Tbx 5 and Tbx 4 expression is the upregulation of Fgf10 and Fgf8

Question 6

Tbx5

Answer 6

• in the forelimb
• Are T-box transcription factors (related to brachyury)
• limb bud development through control of Fgf production
• The placement and expression of Tbx 5 and 4 are under the control of the Hox genes
• A major consequence of Pitx 1, Tbx 5 and Tbx 4 expression is the upregulation of Fgf10 and Fgf8

Question 7

Tbx4

Answer 7

• in the hindlimb
• are T-box transcription factors (related to brachyury)
• limb bud development through control of Fgf production
• The placement and expression of Tbx 5 and 4 are under the control of the Hox genes
• A major consequence of Pitx 1, Tbx 5 and Tbx 4 expression is the upregulation of Fgf10 and Fgf8

Question 8

Apical ectodermal rigde (AER)

Answer 8

• Essential for limb bud outgrowth and proximal-distal patterning of the limb
• The AER is a ridge of ectoderm that runs along the dorsal-ventral boundary of the limb
• Maintains a region of cells just proximal to it in the mesoderm, the Zone of proliferating and undifferentiated cells
• As cells leave this zone, they begin to differentiate
• Differentiation occurs in the proximal to distal direction as the limb extends along this axis
• A key signal produced by the AER is Fgf 8
• Fgf 8 is sufficient for limb bud growth
• Beads that have been soaked in Fgf 8 and applied to the limb region can mimic AER function
• Fgfs are necessary for limb bud outgrowth
• AER is required for outgrowth and if removed, outgrowth stops
• Cells behind it stopped proliferating
• Cells leave the proliferating zone, they differentiate and proximal structures form first then progressively more distal
• Removal of the AER at later stages of limb growth result in formation of more distal structures

-a thickening of the ectoderm at the distal end of the developing chick and mammalian limb bud, which is essential for limb bud outgrowth and for correct patterning along the proximo-distal axis of the limb

Question 9

Zone of proliferating undifferentiated cells

Answer 9

• a region of cells maintained by the apical ectodermal ridge (AER) and is just proximal to it
• these cells are proliferating (dividing) and are undifferentiated

Question 10

Limb bud outgrowth

Answer 10

• Fgfs are necessary for limb bud outgrowth
• A key signal produced by the AER is Fgf 8 and is sufficient for limb bud growth
• AER is required for outgrowth and if removed, outgrowth stops
• Cells behind it stopped proliferating

Question 11

Fgf-8

Answer 11

• A major consequence of Pitx 1, Tbx 5 and Tbx 4 expression is the upregulation of Fgf10 and Fgf8
• a key signal produced by the apical ectodermal ridge (AER) that is sufficient for limb bud growth
• Beads that have been soaked in Fgf 8 and applied to the limb region can mimic AER function

Question 12

Fgf-10

Answer 12

-A major consequence of Pitx 1, Tbx 5 and Tbx 4 expression is the upregulation of Fgf10 and Fgf8

Question 13

AER removal experiment

Answer 13

• AER is required for outgrowth and if removed, outgrowth stops
• Cells behind it stopped proliferating
• Cells leave the proliferating zone, they differentiate and proximal structures form first then progressively more distal
• Removal of the AER at later stages of limb growth result in formation of more distal structures

Question 14

Progress zone model

Answer 14

• Cells interpret their position based on the amount of time they are present in the region behind the AER
• Progress zone=undifferentiated and proliferating cells
• Evidence?
• AER outgrowth is tightly linked to proximal-distal patterning
• Fgf functions to maintain the progress zone (removing this, they stop dividing, know where they are in the limb)
• When the AER is removed, cells can still interpret their position as though the full limb had formed
• If the progress zone is not maintained, more distal structures cannot form
• X-ray treatments were used to kill cells in the progress zone during limb bud outgrowth
• This results in loss of proximal but not distal structures
• Cells in the progress zone after treatment do not have time to replenish the zone until later outgrowth (used in cancer treatments)
• Cells that leave the zone are too few to generate mid proximal structures

-in the timing model of vertebrate limb development, an area at the tip of the limb bud where cells acquire positional values

Question 15

Two-signal model

Answer 15

• Diffusible signals in the early limb pattern the cells to specify proximal-distal values
• Elements differentiate progressively as they grow out
• Evidence?
• Fgf expressed in the AER signals distal identity
• Retinoic acid (RA) is expressed by the body wall muscles and opposes the Fgf gradient to signal proximal identity
• High gradient in the flank of the animal
• RA regulates a transcription factor called Meis in the proximal region
• Meis expression in distal regions is sufficient to create limb truncations
• Fgf mutations result in a truncated limb with proximal and distal structures but NO intervening structures
• Fgf and RA pattern early limb to generate most distal and proximal structures FIRST
• Limb outgrowth generates a region that can adopt an intermediate fate

Question 16

Killing cells of the progress zone (X-ray irradiation)

Answer 16

• used to prove the Progress Zone Model
• X-ray treatments were used to kill cells in the progress zone during limb bud outgrowth
• This results in loss of proximal but not distal structures
• Cells in the progress zone after treatment do not have time to replenish the zone until later outgrowth (used in cancer treatments)
• Cells that leave the zone are too few to generate mid proximal structures

Question 17

FGF

Answer 17

• used to prove the two signal model
• Fgf expressed in the AER signals distal identity
• Retinoic acid (RA) is expressed by the body wall muscles and opposes the Fgf gradient to signal proximal identity
• =OPPOSING GRADIENTS
• High gradient in the flank of the animal
• RA regulates a transcription factor called Meis in the proximal region
• Meis expression in distal regions is sufficient to create limb truncations
• Fgf mutations result in a truncated limb with proximal and distal structures but NO intervening structures
• Fgf and RA pattern early limb to generate most distal and proximal structures FIRST
• Limb outgrowth generates a region that can adopt an intermediate fate

Question 18

Retinoic acid

Answer 18

• used to prove the two signal model
• Fgf expressed in the AER signals distal identity
• Retinoic acid (RA) is expressed by the body wall muscles and opposes the Fgf gradient to signal proximal identity
• =OPPOSING GRADIENTS
• High gradient in the flank of the animal
• RA regulates a transcription factor called Meis in the proximal region
• Meis expression in distal regions is sufficient to create limb truncations
• Fgf mutations result in a truncated limb with proximal and distal structures but NO intervening structures
• Fgf and RA pattern early limb to generate most distal and proximal structures FIRST
• Limb outgrowth generates a region that can adopt an intermediate fate

-small non-protein secreted signalling molecule with many roles in development

Question 19

Meis transcription factor

Answer 19

• Retinoic acid regulates a transcription factor called Meis in the proximal region
• Meis expression in distal regions is sufficient to create limb truncations

Question 20

Zone of polarizing activity (ZPA) / polarizing region

Answer 20

• is located in the posterior region of the limb bud and is necessary for anterior to posterior patterning
• Is mesoderm tissue
• Sonic hedgehog (Shh) is a ligand secreted protein in the ZPA (ie. Morphogen)
• There is a concentration gradient of Shh in the limb (High in the posterior, Low in the anterior)
• [Shh] specifies A-P positional identity
• If ZPA is transplanted within a new position in the bud, this is able to induce a new A-P pattern
• Is a true organizer
• Able to induce surrounding tissue of the host to change fate
• ZPA produces a morphogen (Shh) that forms a gradient
• Cells interpret [Shh] to develop a particular structure (e.g. digit 2)
• The graft of ZPA (donor) would form a mirror image if placed in the anterior
• A weaker signal (i.e. less cells in the graft) lowers [Shh] and induces predictable structures in intermediate to posterior identity
• Similar results are seen with a shorter period of time that the transplant is left in place

-in developing chick and mouse limb buds, an area at the posterior margin of the bud which produces a signal specifying position along the antero-posterior axis

Question 21

Sonic hedgehog (Shh)

Answer 21

• a ligand secreted protein in the Zone of Polarizing Activity (ie. Morphogen)
• There is a concentration gradient of Shh in the limb (High in the posterior, low in the anterior)
• [Shh] specifies A-P positional identity
• ZPA produces a morphogen (Shh) that forms a gradient
• Cells interpret [Shh] to develop a particular structure (e.g. digit 2)
• a weaker signal (i.e. less cells in the graft) lowers [Shh] and induces predictable structures in intermediate to posterior identity

Question 22

Organizer

Answer 22

-a signalling center that directs development of the whole embryo or of part of the embryo such as a limb
the zone of polarizing activity is a true organizer, able to induce surrounding tissue of the host to change their fates

Question 23

Morphogen

Answer 23

• any substance active in pattern formation whose spatial concentration varies and to which cells respond differently at different threshold concentrations
• Sonic hedgehog (Shh) secreted ligand and protein from the zone of polarizing activity is an example of a morphagen due to cells interpreting the different concentration levels of Shh

Question 24

ZPA grafting experiments

Answer 24

• If ZPA is transplanted within a new position in the bud, this is able to induce a new A-P pattern
• Is a true organizer
• Able to induce surrounding tissue of the host to change fate
• ZPA produces a morphogen (Shh) that forms a gradient
• Cells interpret [Shh] to develop a particular structure (e.g. digit 2)
• The graft of ZPA (donor) would form a mirror image if placed in the anterior
• A weaker signal (i.e. less cells in the graft) lowers [Shh] and induces predictable structures in intermediate to posterior identity
• Similar results are seen with a shorter period of time that the transplant is left in place

Question 25

Hox gene expression

Answer 25

- hox gene expression reflects positional information along the proximal-distal and anterior-posterior axis - Anterior-Posterior axis: curved concentration with Hoxd9 (most anterior), Hoxd9-11, middle and Hoxd9-13, (most posterior - Proximal-distal axis: vertical lines with Hoxa9 (most posterior), Hoxa9-11 (middle) and Hoxa9-13 (most distal)

Question 26

Bone identity

Answer 26

- Hox9=humerus - Hox10=humerus , radius, ulna and carpus - Hox11=radius, ulna, carpus and phalanges - Hox12=carpus - Hox13=palanges