DEVELOPMENT
1. BONE DEVELOPMENT
-Provides support which facilitates movement, protective, metabolism (hematopoiesis,
mineral store for calcium).
-Types:
Long bones- limbs
Short bones- hands and feet
Irregular bones- vertebrae, innominate
Flat bones- sternum, skull
Sesamoid bones- surrounded by tendinous tissue such as patella.
-Made of organic and inorganic compounds:
1) Calcium phosphate-based hydroxyapatite provides hardness and rigidity.
2) Interwoven mesh of collagen fibers provides resilience and flexibility.
> Extracellular matrix secreted, monitored and maintained by bone cells.
-Types of bone:
1) Cortical/ compact
Solid outer layer, withstands compressive forces
Tightly packed units, osteons.
Osteons arranged around central/ haversian canal (blood, nerve).
Primary cells are osteocytes which monitor.
2) Trabecular/ spongy/ cancellous
Mesh of loosely arranged trabeculae.
No central canal, surrounded by bone marrow.
-Ossification
1) Intramembranous- bones form directly from soft tissues of embryo (mesenchyme).
2) Endochondral- cartilage template built upon by osteoblasts.
-Stages of bone formation
> Primary ossification center forms diaphysis, develops in utero.
> Secondary ossification center around joint surfaces.
> Growth plates separate these two regions, cartilaginous allowing bone growth.
-Growth plate
> Resting chondrocytes (cartilage cells) -> proliferative -> maturing (lay down cartilage) ->
hypertrophic phase (apoptosis) -> osteoblasts transform edges of former chondrocyte into
bone.
> Maturity in skeleton is reached when cartilage formation rate decreases and bone
formation increases, ultimately catching up.
,2. DEVELOPMENT OF THE LIMB
2.1 Tetrapod limb development
-The tetrapod limb comprises three parts (prox to distal):
> Stylopod- humerus, femur
> Zeugopod- radius and ulna/ tibia and fibula
> Autopod- carpals and phalanges/ tarsals and phalanges
> The thumb is anterior and the pinky posterior, the palm is ventral and the back dorsal.
-Limb development in mice:
> E9.5= start to see limb buds on flank of trunk
> E11.5= limb bud forms paddle like structure
> E12.5= condensation of cartilage cells within paddle, forming individual digits
> E18.5 day before birth. See individual figures, cells between cartilage condensations have
apoptosed.
> E... denotes days after fertilization.
-Axis of the developing limb bud in the chick embryo
> The proximo-distal axis is specified by the apical ectodermal ridge (AER) seen as a
thickening of the ectoderm.
> Experiments demonstrating this:
AER removed, no limb. AER necessary for outgrowth of limb.
AER added, mirror image duplication of digits.
Mesenchyme from leg put in limb resulted in formation of leg. Mesenchyme has
information necessary to specify type of limb. AER= outgrowth, mesenchyme= type
of limb.
Replacement with non-limb mesenchyme stops limb development.
Fgf8 is strongly expressed in AER. Bead soaked in Fgf8 results in normal limb
formation, keeps underlying mesenchyme proliferative.
> The anterior-posterior axis is specified by the zone of polarizing activity (ZPA), a
condensation of mesenchymal cells on the posterior end. Also specifies number of digits.
> Experiments demonstrating this:
ZPA transplanted to anterior end results in mirror image duplication of digits. Mirror
polydactyly in humans. Also specified radius, ulna, tibia, and fibula.
Sonic hedgehog (Shh) is expressed in ZPA. It is a secreted protein with lipid
molecules meaning it is difficult to soak in beads. Transfect chick embryo fibroblasts
with Shh expression vector. Cells harvested and pelleted, and pellet transplanted to
anterior of limb bud. Resulted in mirror image duplication.
> Both ZPA and AER duplications result in mirror image duplication of digits. ZPA and AER
form positive feedback loop leading to axis outgrowth and specification of digits.
, -Limb bud induction in the chick embryo
1) Making mesoderm permissive for limb formation
> Somites specify position and size of limb field, where limb bud is formed. Transplanting
somites changes limb field size.
> Limb field is the area of mesoderm competent to form limb bud. Splitting the limb field
also results in multi-limbed animals. Smaller limb field is still able to give fully formed limb.
> Antagonistic actions of Fgf8 and retinoic acid control where the forelimb is formed,
making mesoderm permissive for limb formation.
Fgf8: expressed in heart and tail region
RA: expressed in middle. Lipophilic, diffuses into cells and binds transcription factor
in cytoplasm to control gene expression. Loss results in failure to form forelimb buds.
Controls Tbx5.
2) Specifying forelimb and hindlimb
> Fgf10 induces limb bud formation; expressed in forelimb and hindlimb. Soaked beads
result in development of additional limbs.
Fgf10 bead between forelimb and hindlimb fields results in chimera (half arm/ wing,
half leg).
> Tbx5 is strongly expressed in forelimb field, specifies identity.
> Tbx4 is strongly expressed in hindlimb field, specifies identity.
3) Inducing epithelial to mesenchymal transition
> Tbx5 induces ETM transition.
> Lateral plate is important, at beginning mesoderm is epithelial. Becomes epithelial sac
covering mesenchymal cells inside.
4) Establishing feedback loops for limb bud formation
> Forelimb: Tbx5 controls fgf10 expression through Wnt2b intermediate.
First positive feedback loop- Fgf10 controls proliferating cells which activates
expression of Tbx5 and Wnt2b. Lateral plate mesoderm.
Second positive feedback loop- Fgf10 activates Fgf8 expression in ectoderm via
Wnt3a. Fgf8 in turn activates Fgf10. Leads to formation of AER and growth of
mesenchymal cells.
1. BONE DEVELOPMENT
-Provides support which facilitates movement, protective, metabolism (hematopoiesis,
mineral store for calcium).
-Types:
Long bones- limbs
Short bones- hands and feet
Irregular bones- vertebrae, innominate
Flat bones- sternum, skull
Sesamoid bones- surrounded by tendinous tissue such as patella.
-Made of organic and inorganic compounds:
1) Calcium phosphate-based hydroxyapatite provides hardness and rigidity.
2) Interwoven mesh of collagen fibers provides resilience and flexibility.
> Extracellular matrix secreted, monitored and maintained by bone cells.
-Types of bone:
1) Cortical/ compact
Solid outer layer, withstands compressive forces
Tightly packed units, osteons.
Osteons arranged around central/ haversian canal (blood, nerve).
Primary cells are osteocytes which monitor.
2) Trabecular/ spongy/ cancellous
Mesh of loosely arranged trabeculae.
No central canal, surrounded by bone marrow.
-Ossification
1) Intramembranous- bones form directly from soft tissues of embryo (mesenchyme).
2) Endochondral- cartilage template built upon by osteoblasts.
-Stages of bone formation
> Primary ossification center forms diaphysis, develops in utero.
> Secondary ossification center around joint surfaces.
> Growth plates separate these two regions, cartilaginous allowing bone growth.
-Growth plate
> Resting chondrocytes (cartilage cells) -> proliferative -> maturing (lay down cartilage) ->
hypertrophic phase (apoptosis) -> osteoblasts transform edges of former chondrocyte into
bone.
> Maturity in skeleton is reached when cartilage formation rate decreases and bone
formation increases, ultimately catching up.
,2. DEVELOPMENT OF THE LIMB
2.1 Tetrapod limb development
-The tetrapod limb comprises three parts (prox to distal):
> Stylopod- humerus, femur
> Zeugopod- radius and ulna/ tibia and fibula
> Autopod- carpals and phalanges/ tarsals and phalanges
> The thumb is anterior and the pinky posterior, the palm is ventral and the back dorsal.
-Limb development in mice:
> E9.5= start to see limb buds on flank of trunk
> E11.5= limb bud forms paddle like structure
> E12.5= condensation of cartilage cells within paddle, forming individual digits
> E18.5 day before birth. See individual figures, cells between cartilage condensations have
apoptosed.
> E... denotes days after fertilization.
-Axis of the developing limb bud in the chick embryo
> The proximo-distal axis is specified by the apical ectodermal ridge (AER) seen as a
thickening of the ectoderm.
> Experiments demonstrating this:
AER removed, no limb. AER necessary for outgrowth of limb.
AER added, mirror image duplication of digits.
Mesenchyme from leg put in limb resulted in formation of leg. Mesenchyme has
information necessary to specify type of limb. AER= outgrowth, mesenchyme= type
of limb.
Replacement with non-limb mesenchyme stops limb development.
Fgf8 is strongly expressed in AER. Bead soaked in Fgf8 results in normal limb
formation, keeps underlying mesenchyme proliferative.
> The anterior-posterior axis is specified by the zone of polarizing activity (ZPA), a
condensation of mesenchymal cells on the posterior end. Also specifies number of digits.
> Experiments demonstrating this:
ZPA transplanted to anterior end results in mirror image duplication of digits. Mirror
polydactyly in humans. Also specified radius, ulna, tibia, and fibula.
Sonic hedgehog (Shh) is expressed in ZPA. It is a secreted protein with lipid
molecules meaning it is difficult to soak in beads. Transfect chick embryo fibroblasts
with Shh expression vector. Cells harvested and pelleted, and pellet transplanted to
anterior of limb bud. Resulted in mirror image duplication.
> Both ZPA and AER duplications result in mirror image duplication of digits. ZPA and AER
form positive feedback loop leading to axis outgrowth and specification of digits.
, -Limb bud induction in the chick embryo
1) Making mesoderm permissive for limb formation
> Somites specify position and size of limb field, where limb bud is formed. Transplanting
somites changes limb field size.
> Limb field is the area of mesoderm competent to form limb bud. Splitting the limb field
also results in multi-limbed animals. Smaller limb field is still able to give fully formed limb.
> Antagonistic actions of Fgf8 and retinoic acid control where the forelimb is formed,
making mesoderm permissive for limb formation.
Fgf8: expressed in heart and tail region
RA: expressed in middle. Lipophilic, diffuses into cells and binds transcription factor
in cytoplasm to control gene expression. Loss results in failure to form forelimb buds.
Controls Tbx5.
2) Specifying forelimb and hindlimb
> Fgf10 induces limb bud formation; expressed in forelimb and hindlimb. Soaked beads
result in development of additional limbs.
Fgf10 bead between forelimb and hindlimb fields results in chimera (half arm/ wing,
half leg).
> Tbx5 is strongly expressed in forelimb field, specifies identity.
> Tbx4 is strongly expressed in hindlimb field, specifies identity.
3) Inducing epithelial to mesenchymal transition
> Tbx5 induces ETM transition.
> Lateral plate is important, at beginning mesoderm is epithelial. Becomes epithelial sac
covering mesenchymal cells inside.
4) Establishing feedback loops for limb bud formation
> Forelimb: Tbx5 controls fgf10 expression through Wnt2b intermediate.
First positive feedback loop- Fgf10 controls proliferating cells which activates
expression of Tbx5 and Wnt2b. Lateral plate mesoderm.
Second positive feedback loop- Fgf10 activates Fgf8 expression in ectoderm via
Wnt3a. Fgf8 in turn activates Fgf10. Leads to formation of AER and growth of
mesenchymal cells.
