Somitogenesis Flashcards
Where does the somite originate from?
Paraxial mesoderm (which forms into sclerotome, myotome and dermatome)- produced by the presomitic mesoderm
What doe the intermediate and lateral mesoderm differentiate into?
Intermediate mesoderm- kidney and gonads
lateral mesoderm- splanchnic, somitic, extra-embryonic
What is the presomitic mesoderm (PSM) generated by?
The cranial (rostral) primitive streak & the tail bud, involving proliferation, specification and emigration
What pathways are invovled in somitogenesis and how do they interact?
Wnt, FGF, RA and BMP pathways are required, interacting with each other directly or indirectly, via positive & negative feedback loops
How does the embryo grow during somitogenesis?
Traditionally, the PSM is thought of as “regressing” that is moving backwards, leaving somites behind as it goes. In fact, the whole embryo is growing during this phase, and all tissues are moving, relative to one another
- somites laid down sequentially, cranial to caudal
In what manner are somites formed (e.g. spacially, temprorally)?
Formed regularly, with the time characteristic of the species (e.g. 1 pair each in 2h mouse, ~5h human, 1.5h chick)
What does the number of somite pairs developed depend on?
The total somites formed also characteristic of the species, independent of variations in embryonic size (e.g. zebrafish 33 pairs, human 44, mouse 65)
How do somite halves differ?
Cranial & caudal somite halves are different
What encodes somite fate?
Each somite has different fate, encoded in the PSM by Hox genes
How are somites initially paired and what do thety differentiate to?
Initially paired epithelial spheres each side of neural tube Differentiate to: Dermomyotome and sclerotome (vertebrae) Myotome: epimere, hypomere, limb muscle Dermatome: dorsal dermis
What is the fate of the most cranial 5 somites?
Occipital bone
What does the notochord develop into?
Nuclues pulposus
What happens if neural arches fail to fuse?
Spina bifida occulta
What abnormalities of the sclerotome exist?
Abnormal segmentation Hemivertebrae, or fused vertebrae Scoliosis (lateral curve of spine) Klippel-Feil syndrome (brevicollis) Short neck, reduced number of cervical vertebrae Failed fusion or non-union of arches Spina bifida occulta Non-union of sternum: Split xiphoid process
What is the Fate of the myotome?
Myotome into epimere, hypomere, limb muscle
Epimere: epaxial extensor muscles of spine
Dorsal ramus of spinal nerve
Hypomere: flexors of spine; outer, intermediate, inner layers in thorax & abdomen
Ventral ramus of spinal nerve
Nerve/muscle segmentation
Where do nerves grow in from?
Nerves grow in from spinal nerves of neural tube
What are Myoblasts attracted by?
Myoblasts attracted by HGF (scatter factor, only in limb field mesoderm; c-met receptor in myotome)
What are muscle patterns dicated by?
Connective tissue
Why do muscles produce trophic signals?
For nerves to follow
What does dermatome form?
Dermis, subdermal connective tissue
What is the fate of the dermatome?
Dermatome: area of skin supplied by a single spinal nerve & its ganglion
Dorsal dermis from dermomyotome
Adjacent spinal nerve - sensory innervation
Initial segmental pattern in limb buds converted to proximo-distal pattern
Rotation of limbs: opposite fore & hind
Outwards fore: elbows caudal
Inward hind: knees cranial
How is differentiation of the somite controlled?
LOL JOKE HAHAHAHA
The slide says:
Shh secreted from notochord, along with NT-2, Wnt1,3 from the dorsal neural tube and BMP4/FGF from the lateral plate mesoderm = contributes to somite differentiation
What do the signalling molecules involved in somitogenesis differentiate?
Notochord secretes Shh which induces Pax1 in ventral paraxial mesoderm to produce sclerotome.
Wnt1/3 released from dorsal (roof) plate of neural tube induces Myf5 in medial part which forms body wall muscles
NT-3 also secreted from neural tube acts on dorsal part of mesoderm which will form dermis
Wnt secreted from epidermis and BMP4/FGF from lateral plate mesoderm induces Pax3 and MyoD in lateral paraxial mesoderm which will form epaxial muscles
(read slide 20 of somitogenesis it’s lit)
What is the syndetome?
The syndetome is the progenitor population for tendons
Name properties of the somite halves: cranio-caudal
Cranio-caudal halves of somites have different properties
Neural crest migration
Motor neurone migration
Formation of vertebrae
Specified in PSM, before somite boundary formation
Several genes expressed in half stripes in somitomeres S0 and S-1
Caudal half: HES1, Delta1 (Notch signalling)
Cranial half: MesP (bHLH, Mesp2 nulls lose cranial identity), EphA4 (EPH receptor)
What do Cranial & caudal halves make?
Centrum
Where do intersegmental arteries between somites come to lie?
Midway over bodies
Why do myotomes bridge intervertebral disks?
So they can move vertebrae
Where do spinal nerves pass through?
Spinal nerves pass through somites, but come to run through intervertebral foramina
Where is somite axial identitiy encoded?
Presomitic mesoderm
What TF is important in somitogenesis?
Hox genes-
Hox genes bind DNA in a sequence-specific fashion
They regulate expression of adjacent/nearby genes
Confer positional identity along the A-P (cranio-caudal) axis of the trunk
(highly conserved)
Desribe Hox gene evolution
Vertebrates have 4 Hox gene clusters, which have arisen from two duplication events of an ancestral chromosome
Gene order along the chromosome corresponds to the position of expression within the embryo
Gene transcription within each Hox cluster displays spatio-temporal co-linearity
Why are Hox genes important?
- they are involved in somite identity
- code their vertebral region (e.g. cervical/thoracic/lumbar/sacral/caudal
How is the regular formation of somites achieved?
The clock and wavefront model
In theory, what is needed is a clock mechanism to mark time, plus a wavefront or “trigger” to form a somite at each set period
Oscillations of the clock are generated by delayed negative feedback on gene expression
The trigger is provided by a gradient(s) threshold(s) of signalling proteins
Have ago at explaining the wave of gene expression in somitogenesis
(good luck xxx)
PSM cells have cyclic gene expression, with the same period as somite production: part of the somitogenesis clock. Typically, there is an apparent wave in PSM, and a persistent stripe cranially (rostrally). The “wave” is actually rapid switching on and off of expression, and suggests close coupling between cells, which has been shown in real time at the single cell level using a luciferase reporte
Describe the three-tier model of segmentation clock
bottom tier: single cell oscilators- so gene expression transcribes and translates protein which then inhibits further gene expression in a negative feedback loop
Middle tier: local synchronisation- involves 2 cells communicating with eachother (but that’s all i can decipher)
Upper tier: global control of slowing and arrest- results in elongation of A/P axis
What TFs cycle in PSM in all species?
The expression of Hes/Her transcription factors, effectors of Notch signalling, cycle in PSM in all species
Explain the single cell genetic oscillators: e.g. hes/her
Single cell genetic oscillators.
(A) Single-cell oscillator- gene products repress gene activity. This negative-feedback loop involves a sequence of steps. The Her/Hes gene is transcribed with a basal rate and mRNA is transported from the nucleus and translated. Proteins are transported back into the nucleus where they dimerize to form a transcriptional repressor. This repressor accumulates and binds the gene promoter to inhibit gene expression. Finally, when gene products (mRNA, protein, dimer) decay, the gene repression is released and the cycle can start over again.
Oscillations of mRNA and protein concentrations in the single cell oscillator.
What proteins modify Notch signalling?
FRINGE :) :) :) e.g. lunatic
What cyclic genes are involved in somitogenesis?
of-course, none of this is well understood
Cyclic genes belonging to the Notch and FGF pathways oscillate in opposite phase to cyclic genes of the Wnt pathway. A large number of the cyclic genes are involved in negative feedback loops. The basic circuitry of the three signalling pathways is represented.
All 3 pathways influence expression of Tbox genes e.g. Tbx6
Describe local synchronization through intercellular communication in the segmentation clock:
How neighboring oscillating cells send and receive signals via the Delta-Notch pathway:
The genes that encode the Delta ligands are repressed by the same factors that control the single-cell oscillator feedback loop.
The Delta gene is transcribed and the mRNA exported to the cytoplasm. The mRNA is translated, and the protein undergoes post-translational modifications before becoming an active ligand for Notch receptors on the neighboring cell.
Upon ligand/receptor binding, the Notch intracellular domain (NICD) is proteolytically cleaved from the Notch receptor of the neighboring cell and translocates to the nucleus, where it positively regulates the transcription of cyclic genes, such as the Hes/Her gene.
Notch signaling is not required for basal Hes/Her gene transcription. The details of the feedback loop of single-cell oscillator are dimmed to highlight coupling in this figure. Pre-somitic mesoderm cells communicate with multiple neighbors, effectively sitting in a 3D lattice of mobile coupled oscillators.
What is expressed in a pulsatile manner in the causal PSM and how do they act?
Notch and DLL
Notch protein precedes DLL
Notch translated and translocates to cell membrane
Notch & DLL at cell membrane = activation
Activation = NICD for Notch effectors, while DLL endocytosed
What holds cells in the presomitic state? (part of the wavefront trigger)
A gradient of FGF8 protein, formed by mRNA instability.
Describe the “wavefront” or gradient threshold
FGF & Wnt gradients similar, but produced by RNA and protein decay respectively
Cells leaving the tail bud stop de novo production of Wnt3a protein, so levels drop with time
When a threshold level is reached, cycling stops, and a cell is ready to become part of a somite
FGF may enhance Wnt activity, through Akt & Gsk3beta (sexy)
A Retinoic Acid gradient, formed by RALDH2, is in the opposite direction and antagonizes FGF
What positions the determination front?
Antagonistic gradients of FGF/Wnt signalling and RA signalling
What happens as the embryo extends posteriorly?
The determination front moves caudally
What happens to cells that reach the determination front ?
They are exposed to the periodic clock signal, initiating the segmentation program by repressing Tbox genes and activating simultaneously expression of genes such as Mesp2 in a stripe domain that prefigures the future segment. This establishes the segmental pattern of the presumptive somites.
Describe global gradients and then teach me xx
Global gradients control slowing and arrest of oscillating cells.
An elongating vertebrate axis showing cyclic gene expression patterns in blue/white and arrested segments and somites (solid boundaries).
Gradients of fibroblast growth factor (FGF) and Wnt (brown) span the tissue from the posterior. An opposing shorter-range retinoic acid (RA) gradient expands from the recently formed somites. The actual shape of these gradients is not known.
Sustained high-frequency oscillations are observed in the posterior region of the tissue. Oscillators gradually slow down as they approach the wavefront of arrest.
The panel on the left shows the different stages that single-cell oscillators undergo as they traverse the tissue. The right panel highlights the maturation program of pre-somitic mesoderm cells, running in parallel to segment length specification controlled by the clock.
What happens when a group of cells at the right phase of the oscillating cycle hits the wavefront of FGF/Wnt/RA?
Cycling stops and the cells are induced to express Mesp2, which requires Tbx6 expression.
It is Mesp2, interacting with Tbx6 and residual Notch NICD, that sets the boundary for somite border formation
What does Notch coordinate?
cyclic expression among PSM cells, synchronizing the clock and the spread of expression
What does the Wnt, FGF and RA threshold represent?
Represents the wavefront with Wnt3a
High levels (above threshold): clock on Low levels (below threshold): clock off
What happens to cells at the correct phase of cycling?
Cells at the correct phase of cycling are competent to respond to the wavefront, switching on Mesp2 and stopping cycling
What do Mesp2, Tbx6 and Notch do?
Mesp2, Tbx6 and Notch interact to promote somite border formation and cranio-caudal somite compartments
In the WT, what does RA do?
Prevents PSM lateralization
What happens in raldh2 -/-?
lateralization and asymmetric segmentation
What does Mutation of Human Orthologs of the Segmentation Clock Genes Leads to ?
Congential scoliosis
Name some dull, annoying Human mutations
Spondylocostal dysostoses
Multiple vertebral segmentation defects
Often with rib defects
DLL3 (Bulman et al, 2003)
Notch ligand
LFNG (Sparrow et al, 2006)
Notch modulator
MESP2 (Whittock et al 2004)
No1cares (Khadija et al 2017)
