Limb Development

Question 1

Define cell differentiation.

Answer 1

Process by which embryonic cells become
different from one another, resulting in the emergence of cell types such as muscle, nerve, skin and fat cells. It
is the achievement of a stable terminal state (not just transitory differences) and is characterized by the profile of proteins in that cell.

Question 2

Identify principles governing cell differentiation.

Answer 2

-Generative program: The embryo does NOT contain a description of the adult, rather it contains a generative program for making it (progressive series of instructions).

-Regulatory proteins work together as a “committee” to
control the expression of a eukaryotic gene

-Combinations of a few gene regulatory proteins can
generate many different cell types during development

Question 3

Identify the main cell stages following fertilisation.

Answer 3

1. Morulla (TOTIPOTENT cells, can become any tissues, including placenta)
2. Blastocyst (inner cell mass made of PLURIPOTENT cells, can become any tissue except placenta + trophoblast which form placenta)
3. (this only applies to cells derived from inner cell mass) Multipotent blood stem cells (can develop into more than one cell type like RBCs and WBCs, but are more limited than pluripotent cells) and other stem cells (e.g. for muscle, or for bone, etc.)

Question 4

Define Potency, totipotent, pluripotent, multipotent.

Answer 4

Entire repertoire of cell types a particular cell can give rise to in all possible environments.

• Totipotent (toti= whole).
Identical and unrestricted; can give rise to any cell of the body (EMBRYONIC) (e.g. Cells of the very early mammalian embryo)

• Pluripotent (pluri= more).
Less potent; can give rise to many cell types but not all (EMBRYONIC) (e.g. Inner cells of the blastocyst)

• Multipotent (multi= many)
Give rise to cells that have a particular function (ADULT) (e.g. Blood stem cells;)

Question 5

Define cell fate.

Answer 5

The fate of a cell describes what it will become in the course of normal development.
When a cell “chooses” a particular fate, it is said to be determined, although it still “looks” just like its undetermined neighbours. Determination implies a stable change - the fate of determined cells does not change.

Question 6

Describe two stages of commitment (and briefly what happens before that).

Answer 6

First, cell is naive.

1. SPECIFICATION (reversible)
   Capable of differentiating autonomously if placed in isolation BUT can be respecified if exposed to certain chemicals/ signals. How it becomes specified:
   -Intrinsic signal – cell autonomous signal tells the cell ‘who is it’
   -Extrinsic signal -a chemical or molecule in the environment gives the cell spatial information, tells the cell ‘where it is’
2. Determination (irreversible)
   Cell will differentiate (i.e. acquire cell specific gene expression) autonomously even when exposed to other factors or placed in a different part of the embryo (competence for alternative fates lost).

Question 7

Define competence of a cell. How can a cell lose competence.

Answer 7

Ability of a cell to respond to the chemical stimuli.

A cell can lose competence by changes in surface receptor or intracellular molecules

Question 8

Explain how gene expression changes underlie

cell differentiation/the mechanistic basis of fate decision.

Answer 8

“1. Bivalent (poised or paused) chromatin comprises activating and repressing histone modifications at the same location. This combination of epigenetic marks at promoter or enhancer regions keeps genes expressed at low levels but poised for rapid activation (genes both active and silenced)

2. When an ES cell receives a signal to differentiate into a specified cell lineage, activation of the specific developmental genes are needed for differentiation. The developmental genes needed will be activated and the other genes that are not required for that cell lineage will be repressed (silenced) through their bivalent domains.
3. After the differentiation has taken place, genes needed for differentiation may be silenced (because no longer needed).”

• Developmental regulatory genes such as HOX, SOX, T-box are associated with transcription factors (which can be stimulatory or inhibitory, balance of the two determines how strongly the gene is expressed) which bind upstream to promoter regions and act as developmental regulators. They bind to these regulatory sequences and help differentiate some cells down particular lineage.
• Combinations of a few regulatory proteins can generate many cell types

Question 9

At which cell stage can see some features change within cells to do with cell date decision ?

Answer 9

4 cell stage

Question 10

Identify a situation where it is possible to turn determined state back.

Answer 10

Experimentally, can reverse that locked in pattern, take terminally differentiated cells and turn them back
into embryonic stem cells. Through Somatic Cell Reprogramming (by defined factors or therapeutic cloning)

Question 11

Explain the embryonic development of the somites, myotomes and dermatomes.

Answer 11

Somite formation occur as paraxial mesoderm becomes segmented.

• Cells of paraxial mesoderm have an internal clock
• They go through cycles every 90 minutes defined by notch signalling clock
• Wave of signal passes through the embryo
• When the wave passes cells, they are programmed to change into part of a somite
• If the wave passes cells early in the cycle, they become the front end of the segment (head end)
• If the wave passes cells late in the cycle, they become the tail end of the segment
• Process repeated over and over
• myotome is the part of a somite that develops into the muscles
• dermatome is the part that develops into skin

Question 12

Identify and define the processes of growth and ossification of fetal bones taking place in fetal development.

Answer 12

1. Endochondrial Ossification
   - Uses hyaline cartilage as the model for long bone formation.
   - Radiologists can determine the skeletal age of a patient by examining the development of epiphyseal plates
2. Intramembraneous Ossification
   - Formation of bone in fibrous connective tissue (which is formed from condensed mesenchyme cells)
   - The process occurs during the formation of flat bones such as the mandible and flat bones of the skull

Question 13

Define mesenchyme.

Answer 13

Generalised embryonic connective tissue derived from mesoderm

Question 14

Describe the location and function of HOX genes.

Answer 14

-where: expressed along the long axis of the embryo from head to tail
-function: determine the body axis + the position of the limbs along the body axis + the shape of bone
(products are transcription factors which bind to DNA, and thereby regulate the transcription of other genes (e.g. TBX5, TBX4))

Question 15

Describe the process of development of limbs (not the role of genes in it, the process itself).

Answer 15

• Upper Limb buds appear on approximately day 24 between somites C5-T1
• Lower limb buds appear on approximately day 28 between somites L1-S2

-In week 7 the forelimbs rotate 90° laterally and the hind limbs rotate 90 ° medially.
• Results in the flexor compartments being anterior in the upper limb and posterior in the lower limb
• The sole of the foot is equivalent to the palm of the hand and big toe (hallux) is equivalent to the thumb (pollux)

-By week 8 all major components of the limbs are present and medial rotation of the LL is complete

Question 16

Describe the components of limb bud.

Answer 16

• Limb Bud consists of:

• Core of mesenchyme derived from parietal layer of lateral plate mesoderm
• Ectoderm which forms the outer covering of the limb (epidermis)
• Ectoderm is thickened at the ‘apex’ of the developing limb to form the Apical Ectodermal Ridge (AER, organizer of tissue structures)

Question 17

Describe the development of the Proximo-distal.

Answer 17

• AER controls proximo-distal development

• It induces the underlying tissue to remain as a population of undifferentiated, rapidly proliferating cells- known as the PROGRESS ZONE (of proliferating mesenchyme)
• As cells move further away from the AER they will begin to differentiate into cartilage and muscle
• This differentiation results in proximo-distal development

1) HOX-8 controls the position of the limb on the long axis of the body
2) Initiation of outgrowth of the fore limb is controlled by the TBX5 gene and FGF-10
3) AER secretes FGF4 and FGF8 to maintain the progress zone and the further development of the proximo- distal axis
4) As growth progresses, mesenchymal cells are left behind the advancing ridge (and its influence) and so they begin to differentiate

Question 18

Describe the development of the antero-posterior axis.

Answer 18

• The antero-posterior axis is regulated by the Zone of Polarizing Activity (ZPA, formed by a cluster of cells near the posterior border of the limb)
• It ensures that the thumb grows on the cranial (anterior) side of the limb bud
• ZPA expresses the protein sonic hedgehog (SHH). ZPA moves distally with the AER
• Adding a ZPA to the limb bud results in mirror image duplication of digits (in chicks)

Question 19

Describe the development of the Dorso-ventral axis.

Answer 19

• BMPs (Bone Morphogenic Proteins) in the ventral ectoderm induce EN1 protein
• EN1 represses WNT7 restricting its expression to the dorsal limb ectoderm
• WNT7 induces LMX1 which then specifies the cells to be dorsal

Question 20

What are some proteins which affect expression of the HOX genes ?

Answer 20

Expression of the HOX genes is dependent on SHH, FGFs and WNT7a

Question 21

What is the consequence in the variations in the combinations of HOX gene ?

Answer 21

Variations in the combinations of HOX genes ensure that the:
• Upper and lower limbs are different TBX5 (upper limb), TBX4 (lower limb)
• Patterns for the proximal (arm) middle (radius and ulna) and distal (hand) are defined

Question 22

What is the consequence of failure of apoptosis in between digits ?

Answer 22

Syndactyly (webbing)

Question 23

Identify the most common non- chromosomal malformation.

Answer 23

Congenital heart defects

Question 24

Which of UL or LL abnormalities are more common ?

Answer 24

UL abnormalities more common that LL

Question 25

Identify some abnormalities associated with Limb defects.

Answer 25

Abnormalities affecting CVS, GU system and craniofacial structures

Question 26

Identify and briefly define the main limb defects.

Answer 26

* Amelia = complete absence of the limbs * Meromelia = partial absence of the limbs * Phocomelia = absence of long bones * Micromelia = segments are abnormally short The following ay involve either the hands or feet or both: • Brachydactyly = short digits • Syndactyly = fused digits - failure of apoptosis • Polydactyly = extra digits • Cleft foot = lobster claw deformity.

Question 27

What are the main causes of limb defects ? How ?

Answer 27

-Hereditary -Environmental (teratogens), Affects (just teratogens?) the progress zone with failure of cell division (weeks 4&5)

Question 28

Explain the cause and effects of thalidomide.

Answer 28

-Thelidomide is a chemical teratogen - Mechanism: a) SHORT EXPOSURE leads to loss of blood vessels, uniform or localised cell death, loss or partial loss of ZPA and AER signaling which later recovers to distalise remaining tissue. b) PROLONGED OR EARLY EXPOSURE leads to total loss of vessels, widespread cell death, and all signaling lost. -Effect is phocomelia (flipper-like arms or legs, if short exposure), or amelia (if prolonged or early exposure), associated also with intestinal atresia and cardiac abnormalities

Question 29

Explain the cause and effects of Holt Oram Syndrome.

Answer 29

Cause: TBX5 mutations Effect: Defects in limb development: Upper limb deformities, heart defects

Question 30

Once the the cranio-caudal position is set, limb growth is regulated along what three axes ?

Answer 30

1. Proximo-distal axis 2. Antero-posterior axis 3. Dorso-ventral axis