Part 2 Flashcards
How are organiser cell induced?
From signals by neieuwkoop centre (nodal-a TGF-beta, Wnt)
show distinct transcription profile including expression of BMP inhibitors
What do BMP inhibitors do?
BMP inhibitors are secreted from the organsier and act on cells close to the organiser, to prevent the BMP signalling pathway being activated in them
Express particular TF to acquire cell fates
Where is neuronal fate acquired?
In ectoderm, somite/lateral plate mesoderm fate acquired in mesoderm.
What happens to the organiser during gastrulation?
It is differentiating and undergoing involution/convergent extension
Results in A/P D/V axis
What happens once the neural plate is induced?
- Neuralation= formation of neural tube
2. Patterning= discrete cells induced in the neural tube
Dorsal patterning by BMPs and BMP signalling
- Bmps expressed by surface ectoderm next to edges of induced neural plate
- neural plate border and roof plate induced
- roof plate cells upregulate BMP
- Secreted BMP diffuse into the dorsal neural tube, induce expression of TF (PAX 6,7,3) that cause cells to acquire dorsal identity and form dorsal progenitors
What does recent work about Bmp in roof plate show ?
Was thought bmp act as a morphogen to induce different types of dorsal cells
Recent work shows that the roof plate expresses many different bmps which induce particular dorsal cell type
Ventralisation of Shh
Soon after it differentiates into axial mesoderm cells start to make Shh morphogen
-Expressed in notochord and then floor plate
-diffuses out of nc/fp to neural tube
Genes encoding different TF transcribed and translated down d-v axis leaving:
-overlapping regions
-shh morphogen gradient
Mature neurons on outer (mantle zone) as cells differentiate into neurons the differentiating progeny move laterally
How is a cell defined as a particular progenitor?
The array of TF that expressed by that cell
Shh acts early stage to confer D-V pattern of TF on progenitor cells
Distinct progenitors differentiate into distinct neuronal subsets
How do dorsalising and ventralising effects come together?
Shh acts as morphogen to regulate TF expression
much involves De-repression
Example
- GliR represses NKX6.1, 2.1, 6.2
- SHH present- GliR to GliA means that Nkx can be transcribed
Together Shh and BMP signalling pathway pattern D-V axis
How many distinct domains are there along d-v axis of the spinal cord?
13
What are the subdivisions of the mesoderm?
- Intermediate mesoderm- Kidney and Gonads
- Axial mesoderm- Prechordal and notochord
- Paraxial mesoderm- Head and somites (sclertome, syndotome, myotome, endothelial cells and dermatome)
- Lateral plate mesoderm- splanchic, somatic and extra embryonic
What are somites?
Segmented paraxial mesoderm
Pre somatic mesoderm= non segmented tissue
mesoderm segmentation conserved throughout evolution
How does somatic number dictate the number of vertebrae?
Humans are born with 33 vertebrae by adulthood the have 24 and 9 fused ones Embryos somite number human=38-44 chick=55 mouse=65 zebrafish= 33
Somite formation from paraxial mesoderm
Somites form in pairs from paraxial mesoderm
Paraxial mesoderm forms in a continuous manner until proper somite number has been reached
number is fixed for given species
primitive streak present until somite no longer forms
presomtic mesoderm- non segmented but pre figuresthe future of segmentation of somites
How to explain the periodicity and reproducibilty of somite formation?
Cells within psm must respond to:
- positional info
- mechanism that coordinates left and right
- mechanism that generates anterior and posterior boundary
- formation of cleft
How is periodicity of somites formation established?
The clock and wavefront model (cooke and zeeman 1976)
predict ‘clock’ that ticks in posterior psm and drives molecular oscilation that predicts periodicity of somites
Where cells hit the travelling wavefront an abrupt change of property occurs leading to the decision to form somites
What is the oscillation of C-hairy expression in the pre-somatic mesoderm?
take c-hairy gene, synthesise probe and do in-situ hybridisation
look at expression of pattern
(tried experiemtn again and counted embryo somites)
-bisected embryo in centre, did in-situ hybridisation of one half
-both sides had same number even tho hald was fixed
Oscillation =90 mins n
Time for pair of somites to form
Regression of Primitive streak and adding new somites
Each block is 30mins
12 oscillations close to end of somites- 12 somites =18h
Rest when cell fate decision takes place
The position s-2 (somite minus 2)
How do boundary cells induce somite boundary formation?
Transplant cells at different A/P positions into centre part now have 2 somites form which are smaller
Tells us that the cells originally located at prospective AP boundary already have required respective info and instruction
-somite boundary region into non-boundary= new boundary
-boundary cells= instruct cells that are anterior to form boundary
What is lunatic fringe?
Overexpress to inhibit notch
LOOK AT DIAGRAM IN NOTES
How is the determination front specified?
Positioned at the interface of two opposing gradients
eg. RA and FGFb
FGF drives expression of Cyp26 which inhibits RA
so if you increase FGF you decrease RA
restrict Mesp2 by negative feedback loop
Tbxb + notch = Mesp2
How does notch work?
Works with high and low levels next to each other
clock results in oscillations of number of genes- 12 genes
ligand presented in the cell membrane drive cell-cell adhesion
Signal in 2 ways bidirectional
Oscillating pathway
Molecular oscillations- C-Hairy1- lunatic fringe- delta +notch (Ephring- cell adhesion- somite formaton)
What is myogenesis?
Formation of muscular tissue in embryonic development
Roles of myogensis?
- Movement and posture
- communicarion- speech, expression
- Maintain body temp- heat released in contraction
- Respiration
What are factors in muscle wasting diseases?
- Injuries
- Ageing
- Muscle degenerative diseases
How is muscle made?
- Stem cell (specification)
- Muscle progenitor cells- myoblast (differentiation)
- Differentiated muscle cells- Myotubes (maturation)
- Myofibers
- Muscle fibre
- Muscle fasculus (slow or fast muscle)
- Muscle
How is MyoD isolated?
Treated or untreated fibroblast- mRNA- cDNA-subtracted cDNA enriched in muscle specific genes- MyoD
What is MyoD?
A master regulatory gene- simple intro into any cell type drives process of differentiation
Myogenic determination factor
STructure of BHLH protein
What are the members of the MyoD family?
MyoD, Myf5, Myogenin, MRF4
Function= transcription activator and form heterodimers with E12 or E47
All have function of differentiation in muscles
Where do skeletal muscles originate from?
Dermomytome
contains progenitor for these muscles- trunk and limb
Express TF Pax3
In the trunk Pax3 positive cells contribute to the myotome with 2 domains:
1. Expaxial (medial)
2. hypoxial (lateral)
Where are MRFs expressed during embryo genesis?
Myoblasts
Have to be expressed at right place and time
Myf5 expressed first
Do loss of function to test activity of genes
Gene targeting in ES cells
Electroporate Select Analyze colonies Make chimeras Implant Test offspring for chimerism Test for germline expression Cross heterzygote Analyze offspring for penotype
Targeted inactivation studies of MRFs
Myf5 KO
- mice viable, no obvious muscle defects at birth, delay in myotome formation until the onse of MyoD expression
MyoD KO
- mice viable
-no obvious defects at birth
-increased Myf5 expression in somites compensates for lack of MyoD
MyF5/MyoD KO
-complete absence of skeletal muscle
-one of the 2 required to generate myoblasts
Myogenin KO
- mice die shortly after birth
-diaphragm defect , reduced myoblast density
- required for muscle differentiation
Signalling pathway controlling muscle gene activation
provide wnt and Shh signal to medial position close to notochord and neural tube- there myogenin and MRF4 are expressed due to Pax3 (wnt but low Shh)
signalling pathway in myogenesis
- Specifying the expaxial muscle linkage (Shh and wnt to induce Myf5 and MyoD)
- specifying the hypaxial muscle linkage
What are satellite cells?
Adult muscle specific cells
- Originate from somites
- Express marker unique to them- Pax7
- 32% of mouse muscle nuclei at birth
- 5% of mouse muscle nuclei at adult stage