Somitogenesis: Development of Paraxial Mesoderm

Formation of the Mesoderm

• During gastrulation, the epiblast cells that migrate through the primitive streak and occupy positions between the epiblast and endoderm will form mesoderm

Mesodermal Derivatives

The mesoderm with differentiate into the

• chordamesoderm,

• the paraxial mesoderm,

• the intermediate mesoderm,

• and lateral plate mesoderm.

Chordamesoderm

• Chordamesoderm generates the prechordal plate and the notochord

• Migrates through the node and is located at the midline

  • Also called axial mesoderm or dorsal mesoderm.

• Chordamesoderm is an important signaling center

Paraxial Mesoderm

• Is adjacent to the axial mesoderm and neural tube

• Paraxial mesoderm in the head, or head mesoderm is unsegmented and will form skeleton, muscle, and connective tissue of face and skull, along with contributions from the cranial neural crest

• Starting just posterior to the otic placode, the paraxial mesoderm is segmented into somites.

• Somties are epithelial "block like", clusters of cells adjacent to the neural tube.

• Somites will form muscle, bone, connective tissue, and dermis in the back of the embryo and muscles in the limbs.

Presomitic Mesoderm

• At the posterior end of the embryo, cells that have just migrated through the primitive groove are mesenchymal and unsegmented and are called premositic mesoderm

• Presomitic mesoderm will unto mesenchymal to epithelial transition (MET) to form the epithelial somites

Initial Somite Structure

Newly found somites have epithelial outer layer and a mesenchymal core, the somitocele.

Somite Organization

• As a somite matures the ventral part will undergo an EMT, generating the sclerotome

• The dorsal epithelial region is the dermomyotome

• The sclerotome will form the vertebrae and associated tendons and rib cartilage

• The dermomyotome will make skeletal muscle and dermis of the back

• The ventral portion is the myotome and will form musculature

• The dorsal surface is the dermatome and will form the dermis of the back.

Somite Differentiation

• Cranial somites differentiate from presomitic mesoderm earlier than caudal somites and are developmentally older than caudal somites.

Specifying Paraxial Mesoderm

• The lateral plate mesoderm expresses high level of BMP4, the notochord and the lateral portion of the somites expresses Noggin

• BMP4 signaling converts mesoderm to lateral plate mesoderm, so inhibiting the BMP signal is important for specifying somites. 

• Brachyury, Tbx6 and Mesogenin are pioneer transcription factors for presomitic mesoderm.

• The cells in the primitive streak region are bipotential, they can form either mesoderm or neural

• Tbx6 mice fail to form paraxial mesoderm and the bipotential progenitors produce three neural tubes.

• FGF8 and Wnt3a promote formation of the neuromesoderm progenitors (NMP) in the primitive streak region

• Retinoic acid is synthesized in the somites and neural tube and promotes the differentiation of presomitic mesoderm into somites

• The balance of counteracting morphogenetic gradients is critical for somite formation

Brachyury

• Means short tail and describes a spontaneous mice mutant.

• Heterozygotes have a short tail, and homozygotes are embryonic lethal will loss of trunk and tail mesoderm.

• Brachyury has a DNA binding domain (the T-box(, which is conserved in other related transcription factors, known as Tbx.

Sacral Agenesis with Vertebral Anomalies (SAVA)

• Homozygous mutations in the human T gene (TBXT)

• No sacrum

• Vertical clefting of all vertebral bodies.

Retinoic Acid as a Morphogen

• Retinoic acid is not a protein, but a small lipophilic molecule derived from vitamin A

• Receptors (RAR and RXR) are inside the cell, and when bound by retinoic acid act as transcription factors.

• We can't visualize where in an embryo RA is, so we look for the enzymes, like Raldh, which produce retinoic acid, and Cyp26b, which degrades retinoic acid.

Somite Identity

• Hox genes are expressed in overlapping domains in the paraxial mesoderm.

• The cranial limit of expression for some Hax genes correspond to changes in vertebrate identity

  • Cranial limit of Hox10 expression is at the boundary of thoracic and lumbar vertebrae.

• Hox gene expression in the presomitic mesoderm establishes somite identity.

• Changes in expression for Hox genes in the presomitic mesoderm alters somite identity.

• Loss of Hox10 paralogues, which is expressed in the lumbar and sacral region, causes these vertebrae to become thoracic like with ribs

• Misexpression of Haxa10 causes cranial somites to produce lumbar vertebrae

Somite Identity Continued

• Hox genes at the 3' end of the cluster are expressed at more cranial locations

• Hox genes at the 5' end of the cluster are expressed at more caudal locations

• Temporal collinearity of Hox genes refers to the correspondence between when a Hox gene is turned on with its spatial expression in the embryo

  • Hox genes at the 3' end are turned on first and are expressed at more cranial positions

  • Hox genes at the 5' end are turned on later and are expressed at more caudal positions.

• The chromatin state of the Hox clusters change with developmental time

• The 3' end of the cluster is opened first, leading to early transcription

• The loosened chromatin conformation progress toward the 5' end of the cluster, leading to later transcription.

Cranial to Caudal Somite Identity

• As Hox genes are activated the progenitors expressing the Hox genes enter the primitive streak and migrate into the presomitic mesoderm

• More cranial somites result from progenitors that enter early

• Caudal somites result from progenitors that enter late.

• In chick embryos, when presomitic mesoderm is transplanted from an older embryo to a younger embryo, the transplanted tissues retains its identity

  • Thoracic vertebrae are produced at cervical levels

• The transplanted presomitic mesoderm is already determined prior to somite formation

Somitogenesis

• Somitogenesis is the process of forming somites.

• The anterior end of the presomitic mesoderm is organized into groups of somitomeres.

• The somitomeres are generating epithelial connections that lead to somite formation.

• Somites and somitomeres are numbered such that the newest somite is SI

Clock and Wavefront Model

• Somitogenesis results in the regular formation of somites, such as that a new pair of somites is generated at regular intervals

• This periodicity is generated by establishing a location for a boundary to form, using a wavefront, and generating a time for the boundary to form with an internal clock.

The Wavefront

• The wavefront is also called the determination front, and is where the presomitic mesoderm becomes determined

• Determination occurs in the presomitic mesoderm

• The location of the determination front was identified by inverting chick somitomeres so that they caudal compartment was now cranial

• If a somitomere is already determined, then the caudal cells will retain their caudal identity.

• If a somitomere is not determined, then the caudal cells will adopt a cranial identity, conforming to their new environment

• The determination front is established by counteracting gradients of FGF8 and retinoic acid

• FGF8 in the tailbud keeps presomitic progenitors in an undifferentiated state

• Retinoic acid promotes determination

The Wavefront Continued

• Once in a region of low FGF8 the presomitic progenitors become competent to repsond to a molecular clock.

• FGF8 gradient is established in part through mRNA decay

• Only progenitor cells in the tailbud transcribe FGF8 as evidenced by RNA in situ hybridization for FGF8 introns, which are only in the pre-mRNA found in the nucleus

• Progenitor cells in the presomitic mesoderm have the FGF8 mRNA but they are not making new FGF8 mRNA.

• As progenitor cells in the presomitic mesoderm get further away from the elongating tailbud the FGF8 mRNA is degraded, reducing the concentration of secreted FGF8, leading to determination.

• Retinoic acid inhibits FGF8 transcription so presomitic progenitors cannot transcribe more FGF8 mRNA.

The Clock

• A molecular clock is characterized by oscillations in gene expression

  • A gene is turned on and then it is turned off

• Communications between cells is needed to communicate the changes in gene expression between neighboring cells

• Presomitic progenitors that are past the determination front will respond to the oscillating clock and begin the mesenchymal to epithelial process to generate a new somite.

• Notch, Delta, and Notch transcriptional targets have oscillating expression in the presomitic mesoderm

  • Transcriptional targets includes Hes1, Hes7, and LFNG

• Notch transcriptional targets, like Hes7, will inhibit Notch and Delta, providing a negative feedback loop.

  • Notch turns on its inhibitor, which shuts, Notch signaling off, leading to oscillations

The Wavefront and Clock Come Together

• High levels of FGF8 prevent NOTCH from activating Mesp2

• In the S-IV region at the determination front the presomitic mesoderm has less FGF8 and is competent to respond to the Notch clock

• Notch turns on Mesp2, in the cranial half of the somitomere, which results in boundary formation which the neighboring caudal compartment and a new somite is formed.

Defect in Somitogenesis

• Mutations in Notch signaling components like LFNG and Delts (DII3) cause defects in somitogenesis, resulting in vertebral malformations

• Known as spondylocostal dysostosis in humans

• MESP2 mutations cause spondylocostal dysostosis

• Severe vertebral segmentation defects.

Human Somite Congenital Malformations

• caudal dysgenesis is agenesis or incomplete development of the lumbar vertebrae, sacrum, and coccyx, hypoplastic lower limbs and anorectal and genitourinary dysgenesis

• Associated with diabetic mothers.

Sirenomelia

• Type of caudal dysgenesis where fusion of the lower extremities causes the fetus to resemble a mermaid

• Cause is unknown

Sclerotome and Dermomyotome

• Paracrine signals from neighboring tissues induce transcription factors that promote sclerotome and dermomyotome formation

• SHH from the notochord and floorplate cause Pax1 expression in the sclerotome.

• As a somite matures it undergoes an EMT on the ventral side to generate the sclerotome

• The migratory cells will move to positions around the neural tube to generate the vertebrae

• The epithelial cells on the dorsal side from the dermomyotome

Sclerotome Development

• The migrating sclerotome cells move to different locations, based on their location

• Ventromedial cells are attracted to signals coming from the notochord and will form the vertebral body

• Dorsomedial cells receive a different signal and will migrate over the neural tube to form the arch and spine of the vertebrae.

Notochord Contribution

• The notochord is broken up by sclerotome cells that will generate the vertebral body

• The remaining notochord cells will form the nuclei pulposi, which form the gel-like mass at the center of the invertebral discs.

Resegmentation

• The caudal segment of each sclerotome will separate from the cranial segment

• The caudal segment will fuse with the cranial segment behind it, in a process known as resegmentation

• One vertebrae is generated from parts of two different sclerotomes (somites)

• The neural crest, forming the dorsal root ganglia and sensory neurons, and motor neurons axons migrate through the cranial segments to innervate muscle from the myotome.

• The myotome does not perform resegmentation

• The process generates muscles that are offset from the vertebrae so that the muscles can attach to two adjacent vertebrae, allowing the spine to bend.

Dermomyotome Development

• The dorsal portion of the somite that becomes the dermomyotome expresses Pax6

• Two different myotome regions are formed, one close to the neural tube, which will form epaxial muscles (back muscles) and one close to the lateral plate mesoderm, which will form hypaxial muscles (body wall and limb muscles)

• The myotome regions form from the dorsomedial lip (epimere) and the ventrolateral lip (hypomere) of the dermomyotome

• Each region produces myoblast, or muscle precursors, which will migrate under the dermatome where they will differentiate into the myotome and form epaxial and hypaxial muscles.

• Central portion of the dermomyotome will form the dermotome, which generates dermal tissue of the skin in the back

  • Other dermal tissue is generated from lateral plate mesoderm.

Muscle Hypertrophy

• Myostatin is a TGF-B family member that functions as a negative regulator of muscle growth.

• Homozygous mutation in one patient resulted in a well muscled child

• Mother was a professional athlete whose father and grandfather were also exceptionally strong.