Limb Development

Question 1

Skeletal pattern formation from proximal to distal

Answer 1

Humerus - Stylopod
Ulna & Radius - Zeugopod
Metacarpals & Digits - Autopod

Question 2

Tissues developed by the following germ layer origins:

Somites (Sclerotome)
Somites (Myotome)
Somites (Dermatome)
Neural crest cells
Surface ectoderm

Answer 2

1. Cartilage -> Bone
2. Skeletal muscle
3. Dermis, connective tissues
4. Neurons
5. Skin epidermis and specializations

Question 3

STEPS IN TETRAPOD LIMB DEVELOPMENT

Answer 3

A. Specification
B. Determination
C. Induction (EMT)
D. Initiation
E. Outgrowth
F. Patterning

Question 4

Limb Disk -> Limb Bud

Question 5

Where is Hox6 - Hox9 found in an embryo

Answer 5

Thoracic region

Question 6

Where is Hox5 found in an embryo

Answer 6

Cervical region

Question 7

Where is Hox10 found in an embryo

Answer 7

Lumbar region

Question 8

Retinoic acid (RA) plays a crucial role in forelimb development, primarily by influencing the initiation of limb bud formation

Question 9

Steps in Tetrapod Forelimb Development

Specification = HH10
Determination = HH14
Induction (EMT) = HH15
Initiation = HH18
Outgrowth = HH18+
Patterning = ~HH25

Question 10

The _____ transcription factor is crucial for mesodermal differentiation and, specifically, somatic myogenesis (muscle development) in Drosophila

Answer 10

Twist

Question 11

Is a signaling molecule that plays a crucial role in anterior-posterior patterning. It helps position the forelimb field by repressing or limiting the expression of certain genes like Fgf8 in specific regions.

Answer 11

RA (retinoic acid)

Question 12

Shown at both the head (anterior) and tail (posterior) ends, ____ is a signaling molecule important for axis elongation and limb field specification. —- signaling can oppose RA and helps establish positional identity.

Answer 12

Fgf8

Question 13

differential gene expression in the lateral plate mesoderm (LPM)

Which TF is in somatic mesoderm and in splanchnic mesoderm

Answer 13

Somatic mesoderm = Irx3 - for limb development and patterning

Splanchnic mesoderm = FoxF1 - for visceral organ development and vascular formation

Question 14

Wing field

Tbx5 - Wnt2b - Fgf10
Wnt3a - Fgf8

Leg field

Pitx1 - Tbx4 - Wnt8c - Fgf10
Wnt3a - Fgf8

Question 15

Mesoderm (LPM) Ectoderm (AER)
Fgf10 ─► induces ─► Fgf8
▲ │
│<──── maintains ◄┘

Question 16

Pax3 → Tbx5/Tbx4 → Fgf10 → Fgf8 Loop

Pax3 = Regulates early limb muscle progenitor migration and limb field gene expression

Tbx5/Tbx4 = Initiates limb identity (forelimb or hindlimb)

Fgf10 = Stimulates ectodermal Fgf8

Fgf8 = Drives limb outgrowth and maintains Fgf10

Question 17

A signaling center at the distal tip of the limb bud, essential for limb outgrowth.

Answer 17

AER (Apical Ectodermal Ridge)

Question 18

Located in the posterior mesoderm of the limb bud; critical for anterior-posterior patterning.

Answer 18

ZPA (Zone of Polarizing Activity)

Question 19

Master regulator of chondrogenesis (cartilage formation)

Answer 19

Sox9

Question 20

Major structural component of cartilage extracellular matrix

Answer 20

Col2a1 (Collagen, Type II, Alpha 1)

Question 21

The Hox genes regulate positional identity along the proximo-distal (shoulder to digit) axis of the developing limb.

Hox9 = Stylopod (proximal limb: humerus/femur)

Hox11 = Zeugopod (intermediate limb: radius/ulna or tibia/fibula)

Hox13 = Autopod (distal limb: digits)

These genes work in a nested pattern: Hox9 → Hox11 → Hox13, from proximal to distal.

Question 22

Limb bud initiation

Limb arises as a condensation of cells from the lateral plate mesoderm and its ectodermal covering

Accumulation of cells create a bulge

Question 23

The limb fields have been defined by differential Hox expression and RA.

Mesenchyme cells from the limb field move from the lateral plate mesoderm and somites towards the outer ectodermal layer.

These cells accumulate and create a bulge under the ectoderm, this is the beginning of the limb bud.

Question 24

The mesenchyme cells are secreting a fibroblast growth factor,FGF10. TheFGF10activates the ectoderm.

The activated ectoderm secretes another fibroblast growth factor, FGF8 which serves to keep the mesenchyme cells in a state of constant proliferation, aiding the growth of the limb.

There is differential expression of the Hoxb8 gene occurring. Hoxb8 is being expressed below somite 19, helping to create an anterior-posterior boundary.

FGF8 also causes Shh to be expressed in the posterior region.

Shh goes on to cause yet another fibroblast growth factor, FGF4 to be made in the posterior region.

FGF4 travels back to influence the activated ectodermal region, helping to maintain expression of FGF8 and subsequently Shh.

Wnt7a is also being expressed, but only in the dorsal region of the ectoderm. Wnt7a also helps maintain expression of Shh

Question 25

THE ORGANIZER REGIONS OF THE LIMB BUB

Answer 25

APICAL ECTODERMAL RIDGE (AER) : PD AXIS ZONE OF POLARIZING ACTIVITY (ZPA): AP AXIS

Question 26

LIMBS HAVE POLARITY

Question 27

ROLE OF ZPA

Answer 27

ANTERIOR-POSTERIOR AXIS

Question 28

What would happen if you transplant ZPA to anterior side of the limb bud? THE SAME THING HAPPENS WHEN YOU BUILD A ZPA BY MAKING CELLS EXPRESS SHH

Answer 28

Induces a mirror image limb to form

Question 29

role of AER

Answer 29

PROXIMAL-DISTAL AXIS

Question 30

Apical ectodermal ridge essential in limb development ! What would happen when apical ectodermal ridge (AER) is removed?

Answer 30

Limb development ceases

Question 31

Apical ectodermal ridge essential in limb development ! What would happen when there is an extra apical ectodermal ridge (AER)?

Answer 31

Wing is duplicated

Question 32

What would happen when AER from forelimb mesenchyme is transplanted into a leg mesenchyme?

Answer 32

Normal limb (leg) development

Question 33

What would happen when AER from forelimb mesenchyme is transplanted into a non-limb mesenchyme?

Answer 33

AER regresses, limb development ceases

Question 34

What would happen when AER is replaced by FGF bead?

Answer 34

Normal wing development FGF4 or FGF8 can replace AER

Question 35

AER directs and maintains development of limb mesoderm: The earlier the AER is removed, fewer components formed (proximal components only)

Question 36

Limb Polarity Anterior/Posterior Axis Hox genes establish patterning along the anterior/posterior axis The ZPA (via Sonic hedgehog) help cells to interpret their anterior/posterior coordinates. Tbx5 and Tbx4 determine anterior vs. posterior limb development.

Question 37

Limb Polarity Proximal/Distal Axis Fibroblast growth factors mediate interactions between the AER and PZ involved in limb growth and differentiation. Hox genes are also instrumental in specifying proximal/distal polarity.

Question 38

Limb Polarity Dorsal/Ventral Axis Wnt7a is expressed only in the dorsal portion of the limb. Wnt7a also causes expression of Lmx1 which is instrumental in dorsalizing cells.

Question 39

Hox code of the limb 3' Hox genes = expressed earlier, more anterior 5' Hox genes = expressed later, more posteriorly

Question 40

Hox cluster genes (HoxA-D) - Exhibit spatial and temporal colinearity Hox a & d = forelimb; Hox c & d = hindlimb

Question 41

Hox paralogue groups

Answer 41

9 = Scapula 10 = Humerus 11 = Ulna & Radius 12 = Metacarpals 13 = Digits

Question 42

human with ______ resulting from homozygous HOXD-13 null mutation

Answer 42

synpolydactyly

Question 43

expressed only in the anterior portion of the embryo, arising from the lateral plate mesoderm. ----specifies forelimb development.

Answer 43

Tbx5

Question 44

is expressed only in the posterior regions of the lateral plate mesoderm, causing development of a hindlimb.

Answer 44

Tbx4

Question 45

If anFGF bead (aqua)is placed in the anterior region, within the area of Tbx5 expression, it will induce the formation of a new anterior limb. In this situation, the FGF bead mimics the activity of the mesenchyme cells that form the limb bud. These mesenchyme cells normally produce FGF10 which activates the overlying ectoderm to form the Apical Ectodermal Ridge (AER).

Question 46

If an FGF bead laced in the posterior region where Tbx4 is being expressed results in the formation of another hindlimb. Again, the FGF bead is mimicking the activity of the mesenchyme cells, but under the influence of Tbx4, a posterior type limb bud is formed instead.

Question 47

Differentiation of the limb: PCD armpit and digits Chick: posterior and anterior necrotic zone abnormalities: webbed digits (zygodactyly)

Question 48

SCULPTING THE AUTOPOD IN CHICK AND DUCK: Both show BMP4 expression in the interdigital webbing; BMP4 induces apoptosis. The duck foot (but not the chicken foot) expresses the BMP4-inhibitory protein, _____ in the interdigital webbing. Thus, the chicken foot undergoes interdigital apoptosis (as seen by neutral red dye accumulation in the dying cells), but the duck foot does not.

Answer 48

Gremlin

Question 49

intracellular mediator of BMP signal transduction

Answer 49

SMAD

Question 50

SKELETON DIFFERENTIATION Skeletal elements arise from mesenchymal cells of limb bud. Mesenchymal condensation in central region of proximal limb bud cells differentiate into cartilage cells and form the cartilage model for the future bone.

Question 51

SKELETON DIFFERENTIATION - the order of appearance: is scapula, humerus, ulna and digits V and IV, then radius and digits III, II, and I. established; ossification begins later. Limb remains cartilaginous until the gross form of the limb is well established

Question 52

MUSCLE DIFFERENTIATION All muscle precursors arise from the myotomes. 1. Formation of extensor and flexor muscle masses 2. Formation of individual primordia within masses 3. Reconstruction of the primordia from phylogenetically primitive to species-specific 4. Development of definitive form; muscle joining with tendons Formation of muscle begins to occur just after skeletal elements begin to form

Question 53

FINAL PRODUCTS Functional limb composed of multiple differentiated components:

Answer 53

* Stylopod - Humerus in forelimb - Femur in hindlimb * Zeugopod - Radius & Ulna in forelimb - Tibia & Fibula in hindlimb * Autopod - Metacarpals and Digits in forelimb - Metatarsals and Digits in hindlimb