Chapter 12: Paraxial and Intermediate

Mesoderm generates all organs between ectodermal wall and endodermal tissue

Mesoderm is divided into 5 regions

1.Chordamesoderm --> Notochord

2.Paraxial mesoderm (somitic dorsal mesoderm): --> Somites --> produces connective tissues (bone, muscle, cartilage, dermis)

3.Intermediate mesoderm: -->Urino-genital system

4.Lateral plate mesoderm: -->  heart, blood vessels and blood cells, lining of body cavities, extra-embryonic membranes (fetal nutrition)

5.Head mesenchyme: musculature of face

Mesoderm is specified in mediolateral axis

Different concentrations of BMP induce differential expression of Fox family transcription factors

Foxf1 (forkhead family of  transcription factors) is expressed in lateral plate mesoderm 

- Foxc1 and Foxc2 - paraxial mesoderm --> somites 

- Depletion of Foxc1 and Foxc2 --> expression of Pax2 --> intermediate mesoderm

- give segmental pattern to vertebrate embryo 

- determine the path of neural crest cells and spinal nerve axons 

- give rise to cells that form vertebrae, ribs, dermis and skeletal muscles

Number of somites is characteristic of each species: chick – 50, mice – 65 and snakes - 500

• Somite formation includes: 
• – periodicity 
• - epithelialization 
• - specification 
• - differentiation

• Paraxial cells condense into Somitomeres   
•     --> compact and bound together by epithelium  
•     -->separate into somites

•Expression of hairy1 gene in caudal half of each somite as well as posterior portion of presomitic mesoderm

•Caudal fissure (small arrow in B) begins to separate somite from presomitic mesoderm

•Posterior region of hairy gene expression extends anterior and progressively the band shortens

•Another band of hairy 1 expression starts --> moves anteriorly like a wave

•Band of hairy1 gene expression in pre-somitic mesoderm is the site of pinching-off of a new somite

• 
• Hairy1 gene --> targets ephrin genes (Ephrin B2) and its receptor (EphA4 – tyrosine kinase)

Formation of Somite boundary between EphrinB2 in the posterior of the somite and EphA4 on the anterior portion of presomite mesoderm

Budding off of somites coincide with the expression of EphA4

Somitogenesis progresses caudally

Epithelialization - N-cadherin helps in conversion of loose mesenchymal cells to epithelial somite

Axis specification:  

• Somites form different structures at different positions along A-P axis 
•     - Ribs in thoracic region 
•     - Vertebrae in other regions 

Somites that form cervical vertebrae cannot form ribs    

Axis specification is mediated by Hox genes

Somitogenesis – occurs simultaneously with the regression of primitive streak

Cells passing through Hansen’s node secrete FGF8 --> prevents Lunatic fringe protein

Regression of Hansen’s node --> FGF8 producing cells move posteriorly --> activation of Lunatic fringe protein --> somitogenesis

Periodicity - first somite appears in the anterior portion - new somites bud off at regular intervals

Notch signaling pathway – cells from presumptive somite boundary are transplanted into a region of unsegmented mesoderm   

•     --> activation of Lunatic fringe protein  
•     --> activation of notch pathway --> new somite boundary

Addition of Lunatic fringe protein to non-boundary cells --> formation of boundary --> somites

Mutations in Notch signaling --> aberrant vertebrae

DLL3 – delta-like3 gene encodes a ligand for Notch

Mutations of DLL3 in humans --> spondylocostal dysplasia (defects in vertebrae and ribs)

- give rise to cells that form vertebrae, ribs, dermis, skeletal muscles

Sclelorotome – precursor of bone

Myotome – precursor of muscle

• 
• – cells from two lateral regions --> divide --> produce a lower layer of myoblasts (muscle precursor cells)  

Epaxial (Primaxial) --> deep muscles of back 

Hypaxial (Abaxial) --> body wall, limbs and tongue

Central region of dermamyotome --> dermis of back skin

•BMP4 from dorsal epidermis induces Wnt. family of proteins in Somites

•Wnt + low conc. of Shh -->induce Primaxial myotome --> myogenic transcription factor Myf5

Neurotropin (NT3) from neural tube --> Dermatome

Wnt from ectoderm + BMP4 from lateral plate mesoderm --> Abaxial myotome

Wnt + high conc. of Shh --> Pax1 expression --> Sclerotome

Somites --> Primaxial and Abaxial myotome --> myoblasts (muscle cell precursors) --> respond to FGF --> multiply

Pax3 --> Myf5 and MyoD (muscle specific transcription factors) --> induce muscle specific genes (Creatine phosphokinase, Acetylcholine receptor and its own gene (MyoD)

Myotubes: Myoblasts stop division --> secrete fibronectin --> bind with integrin (in ECM) --> fuse together to form multinucleated myotubes (committed to become muscle fibers)

Alignment: myoblasts come together into chains (mediated by glycoproteins and cadherins)

• Fusion of cell membrane is activated by movement of Ca++, mediated by metalloproteases (meltrins)
• Maturation of muscle fiber - ready for muscular  contraction

Lineages to generate skeleton:  Somites --> axial skeleton. Lateral plate mesoderm --> limb skeleton. Cranial neural crest --> branchial arch and craniofacial bones.

• Modes of Osteogenesis: 
• Intramembrane ossification – direct conversion of mesenchymal cells into bone 

Endochondral ossification – mesenchymal cells differentiate into cartilage --> later replaced by bone

Osteogenic precursor cells proliferate --> condense into compact nodules --> some develop into capillaries and others into osteoblasts (bone precursor cells)

Osteoblasts deposit osteoid matrix --> osteoblasts arrayed along calcified region of matrix --> become trapped --> osteocytes (bone cells)

Intramembrane ossification is mediated by BMPs and CBFA1 proteins.

Mutations in CBFA1 – cleidocranial dysplasia (defects in skull and shoulders due to poor osteoblast differentiation)

1. Mesenchymal cells committed to become cartilagenous cells (facilitated by Pax1 and Scleraxis)

2. Mesenchymal cells condense --> Compact nodule --> differentiate into chondrocytes (cartilagenous cells) (initiated by N-cadherin; SOX9 gene)

3. Chondrocytes in the  center divide rapidly

4. Chondrocytes stop dividing and undergo hypertrophy and apoptosis --> mineralize their extra cellular matrix --> form bone marrow

5. Invasion of blood vessels

As cartilage cells die à cells surrounding cartilage model differentiate into osteoblasts -->  form bone matrix Slowly cartilage is replaced with bone

Osteoclasts: derived from same precursors of macrophages -->  enter bone  marrow through  blood vessels -->  pump H+ ions -->  acidify and hollow  out bone marrow  for erythropoiesis

Osteroporosis: Number of osteoclasts is tightly regulated - too much activity of osteoclasts --> too much bone is dissolved  A condition accelerated in women during   menopause (hormones)

Osteropetrosis: rare congenital disorder in which the bones become overly dense. This results from an imbalance between the formation of bone (osteoblasts) and the breakdown of the bone by osteoclasts

Intermediate mesoderm --> Urinogenital system (kidneys, gonads and respective duct systems)

Kidney: functional unit is nephron (12 cell types, 10,000 cells) – Glomerulus –coiled blood capillary  Bowman’s capsule – cup-shaped structure around glomerulus

Kidney function: 180 L blood  filtered @ 1.2 L /min  99% reabsorbed in  tubules (salts, water,  metabolites)  remaining concentrated     fluid is excreted

Stage 1: On day 22 (human) – Pronephric duct arises in intermediate mesoderm (just ventral to anterior somites)  - cells migrate caudally

Stage 2: As pronephric tubules degenerate, middle portion of nephric duct induces kidney tubes (Mesonephros) differentiate

In Females - undergoes apoptosis - source of hematopoietic stem cells.

In Males - vas deferens

Stage 3: Metanephros (functional kidney in reptiles, birds and mammals) – generated by interactions of epithelial and mesenchymal cells of intermediate mesoderm

Metanephric mesenchyme --> induces formation of a branch from each nephric duct, ureteric buds

• Ureterine buds enter metanephric mesenchyme --> mesenchyme induces buds
• to branch

-At tips, the bud epithelium induces mesenchyme to condense -->differentiate into nephrons.

- Which induces ureterine bud to branch and grow