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Flashcards in Part 2 Deck (96)
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1
Q
How are organiser cell induced?
A
From signals by neieuwkoop centre (nodal-a TGF-beta, Wnt)
show distinct transcription profile including expression of BMP inhibitors
2
Q
What do BMP inhibitors do?
A
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
3
Q
Where is neuronal fate acquired?
A
In ectoderm, somite/lateral plate mesoderm fate acquired in mesoderm.
4
Q
What happens to the organiser during gastrulation?
A
It is differentiating and undergoing involution/convergent extension
Results in A/P D/V axis
5
Q
What happens once the neural plate is induced?
A
1. Neuralation= formation of neural tube
2. Patterning= discrete cells induced in the neural tube
6
Q
Dorsal patterning by BMPs and BMP signalling
A
1. Bmps expressed by surface ectoderm next to edges of induced neural plate
2. neural plate border and roof plate induced
3. roof plate cells upregulate BMP
4. 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
7
Q
What does recent work about Bmp in roof plate show ?
A
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
8
Q
Ventralisation of Shh
A
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
9
Q
How is a cell defined as a particular progenitor?
A
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
10
Q
How do dorsalising and ventralising effects come together?
A
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
11
Q
How many distinct domains are there along d-v axis of the spinal cord?
A
13
12
Q
What are the subdivisions of the mesoderm?
A
1. Intermediate mesoderm- Kidney and Gonads
2. Axial mesoderm- Prechordal and notochord
3. Paraxial mesoderm- Head and somites (sclertome, syndotome, myotome, endothelial cells and dermatome)
4. Lateral plate mesoderm- splanchic, somatic and extra embryonic
13
Q
What are somites?
A
Segmented paraxial mesoderm
Pre somatic mesoderm= non segmented tissue
mesoderm segmentation conserved throughout evolution
14
Q
How does somatic number dictate the number of vertebrae?
A
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
15
Q
Somite formation from paraxial mesoderm
A
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
16
Q
How to explain the periodicity and reproducibilty of somite formation?
A
Cells within psm must respond to:
-positional info
-mechanism that coordinates left and right
-mechanism that generates anterior and posterior boundary
-formation of cleft
17
Q
How is periodicity of somites formation established?
A
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
18
Q
What is the oscillation of C-hairy expression in the pre-somatic mesoderm?
A
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
19
Q
Regression of Primitive streak and adding new somites
A
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)
20
Q
How do boundary cells induce somite boundary formation?
A
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
21
Q
What is lunatic fringe?
A
Overexpress to inhibit notch
LOOK AT DIAGRAM IN NOTES
22
Q
How is the determination front specified?
A
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
23
Q
How does notch work?
A
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
24
Q
Oscillating pathway
A
Molecular oscillations- C-Hairy1- lunatic fringe- delta +notch (Ephring- cell adhesion- somite formaton)
25
Q
What is myogenesis?
A
Formation of muscular tissue in embryonic development
26
Q
Roles of myogensis?
A
1. Movement and posture
2. communicarion- speech, expression
3. Maintain body temp- heat released in contraction
4. Respiration
27
Q
What are factors in muscle wasting diseases?
A
-Injuries
-Ageing
-Muscle degenerative diseases
28
Q
How is muscle made?
A
-Stem cell (specification)
-Muscle progenitor cells- myoblast (differentiation)
-Differentiated muscle cells- Myotubes (maturation)
-Myofibers
-Muscle fibre
-Muscle fasculus (slow or fast muscle)
-Muscle
29
Q
How is MyoD isolated?
A
Treated or untreated fibroblast- mRNA- cDNA-subtracted cDNA enriched in muscle specific genes- MyoD
30
Q
What is MyoD?
A
A master regulatory gene- simple intro into any cell type drives process of differentiation
Myogenic determination factor
STructure of BHLH protein
31
Q
What are the members of the MyoD family?
A
MyoD, Myf5, Myogenin, MRF4
Function= transcription activator and form heterodimers with E12 or E47
All have function of differentiation in muscles
32
Q
Where do skeletal muscles originate from?
A
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)
33
Q
Where are MRFs expressed during embryo genesis?
A
Myoblasts
Have to be expressed at right place and time
Myf5 expressed first
Do loss of function to test activity of genes
34
Q
Gene targeting in ES cells
A
Electroporate
Select
Analyze colonies
Make chimeras
Implant
Test offspring for chimerism
Test for germline expression
Cross heterzygote
Analyze offspring for penotype
35
Q
Targeted inactivation studies of MRFs
A
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
36
Q
Signalling pathway controlling muscle gene activation
A
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)
37
Q
signalling pathway in myogenesis
A
-Specifying the expaxial muscle linkage (Shh and wnt to induce Myf5 and MyoD)
- specifying the hypaxial muscle linkage
38
Q
What are satellite cells?
A
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
39
Q
How does muscle regenerate and grow by satellite cells?
A
Quiescent
activated by stormuli under basal ganglia
After activation:
1. induction of Myf5 and MyoD
2. expression of both ^
3. proliferation and self renewal
4. differentiation and fusion to existing fibres
40
Q
Which diffusible morphogens establish D-V axis?
A
BMPs from surface ectoderm
Shh from axial mesoderm
41
Q
What are the structures in the larva which are produced in an adult fly?
A
Antenna/eye
Leg
Haltere
Wing
Genitalia
Mouth parts
42
Q
What are the wing imaginal discs?
A
30 cells - 50,000 cells
A wing disc grows from columnar epithelial cells by the end of the third ventricular instar

Clones of the mutant cells can grow along with the disc to make mosaic structure
43
Q
What was Zeidler's research on Pas3?
A
WT marked with GFP, mutant not GFP
Mosaic disc- some cells wt and some mutant and mixing is random
44
Q
What are the imaginal discs?
A
- Set aside in embryo
- Grow in lava
- Evert to make the adult during the pupal stages
45
Q
What is Pax6/ eyeless?
A
Eyeless drosophila mutant with no eyes
Eyeless is a homeobox TF homologous to vertebrate Pax6
Mutations in Pax6 responsible for human aniridia (Loss of iris)
Loss of mouse Pax6 gives rise to small eye mutants
46
Q
What happens if human pax6 is missexpressed in flies?
A
Extra eyes
Necessary to make eye
Sufficient to make eye (even inter-species)
47
Q
What is the network eyeless is part of?
A
Eyeless gives rise to either eyes absent or sine occulis which go to dachshund and eye specification
48
Q
Where are drosophila eyes?
A
2 either side of head- most vision happens here
3 on top- oceli- for orientation

Each large eye consists of 800 amatidia (hexagonal and curved)
Each large eye has 8 photoreceptors, 4 cone cells, 2 primary, secondary and tertiary pigment cells
Most have bristle, socket and neuron and sheath cell
49
Q
What do the cone and pigment and granule cells do?
A
Cone- secrete overlying lens
Pigment- lattice optically insulates each eye unit
Granules- red pigment= eyes look red- insulating stray light that shines not direct to the eyes so see where they are
50
Q
What is a neurocyrstalline lattice?
A
Rhobdmeres= light sensitive structures
Equator= divides D and V eyes

R1-6= photoreceptors project axons into the lamina
R7-8 photoreceptors project into the medulla crossing left to right and vice versa
51
Q
Neural superposition eye
A
-Angling tubes means any single object is seen by multiple rhapdomeres because they overlap slightly but they innervate different photoreceptors
- single image projected onto photoreceptors
52
Q
The growth of the eye/antennal imaginal disc during larva life
A
No receptors in first or second instar but lots in third

Where eye develops there is a morphogenic furrow rich in actin, few photoreceptors in furrow

- R8 cells recruited first
- each row represents approx 2hrs pf development time
- Ammatidal clusters- neurons cross optic stalk, innervate specific places in brain
53
Q
How do imaginal discs grow during life?
A
- Asynchronous proliferation
- First mitotic wave= before furrow
- G1 arrest in morphogenetic furrow
- S/G2/m- behind furrow (2nd mitotic wave)
- G1/0 arrest

Cells can never divide again after that
54
Q
Analysis of the mosaic mutant ammatidia
A
Sev and Boss can be removed from outer photoreceptors without losing R7
- Sev absolutely required for R7 (autonomous)
- Boss is absolutely required for R8 (non-autonomous)
55
Q
What is Sev?
A
Sevenless
Expressed R8,1,6,4,3,7
Transmembrane receptor tyrosine kinase
Activator Ras/Raf/tRK pathway
56
Q
What is boss?
A
Transmembrane tethered ligand specific sev
Expressed exclusively in R8
57
Q
What is oncogenesis?
A
Forming organs, derive multiple cell types and organise them
58
Q
What is pattern formation?
A
Organisation of cells within the body
Coordinate to know where they are positioned and how they relate to neighbouring cells
During development axis are made to provide regional coordinates
59
Q
What are the three axes of the vertebrae limb?
A
Humerus= stylopod
Ulna= zeugcopod
Carpals= Autopod
60
Q
How do limb buds first appear?
A
First appear as protrusions from the flank at the precise positions along the AP axis of the embryo
(not visible but already defined- put cells in area where limb should not form and you get a limb bud)
61
Q
What do the 2 T-box TF specify?
A
1. Tbx5- Forelimb
2. Tbx4- Hind limb
Restrict each other through negative feedback loop
Precisely expressed in the forming limb and no where else
If you put Tbx5 in hind limb if will form leg instead
62
Q
Where is the forelimb?
A
The most anterior part of the limb boundary
63
Q
What does Hox do?
A
Provides an permissive environment that allows for synthesis of RA in the lateral mesoderm that is the place where the limb bud forms
RA induces tbx genes
Hox - RA - tbx TF
64
Q
How is limb formation initiated?
A
Use FGF beads- observed if placed bead early in development then you see an ectopic limb forming in flank of embryo where bead was planted

Identity depends on where the bead is implanted
65
Q
What is the model of initiation of limb bud formation?
A
Hox- RA- tbx- fgf10 (mesoderm) - Fgf8 (ectoderm)

Wnt causes Fgf10 in mesoderm to express wnt3a and form the apical ectoderm ridge`
66
Q
What is the morphology of the early limb bud?
A
Defined limb field
Thickening of the limb bud at ridge- start producing more Fgf8+4
Progress zone-proximal
apical ectodermal ridge (AER)
Polarising ridge (zpa)
67
Q
How do the different regions work together to form the limb bud?
A
-Positional info along the proximal distal axis
- John saunders took embryos at different stages of development, removed thickening (AER), let grow and examined skeletal element
- AER removed at day 3, chick embryo developed but proximal not distal
68
Q
What happened to John Saunders embryo if AER was removed at day 3 1/2 and day 4?
A
3 1/2= no sign of digits
4= full development of skeletal but truncated
69
Q
What does John Saunders experiment show about the limb bud formation?
A
AER plays role in proximal role in maintaining progress zone (limb growth)
more time cells spend in the progress zone, more distal elements of the limb bud formed
70
Q
What is the progress zone model of proxima-distal patterning?
A
As progress zone extends it becomes more distal
Map didnt match model as you won't be able to see the cells there later on in time- cells already present at start that will contribute to all proximal- distal
71
Q
What is the 2 signal model of proximo-distal patterning?
A
RA expressing distal direction and FGF opposing it
Differentiate according to the signal they've been exposed to
Interpretation of positional info is conserved in flies and vertebrates
RA= proximal
Fgfs wnt = distal
72
Q
What are the major limb segment markers?
A
Styloid- Meis
Zengopod- Hox1.1
Autopod- Hox1.3

Drosophila homologes
Distaless= Hoxd1.3
Homathorax = Meis
73
Q
What controls antero-posterior patterning?
A
ZPA- polarising ridge
Anterior graft of ZPA creates a major image duplication of autopods (graft performed early also loss of ulna)
Graft added distal then there are double digits
74
Q
Wolpert french flag model
A
Highest conc gradient you would expect to specify most posterior digit
Middle= Intermediate
Lowest= Most anterior digit

A-P patterning controlled by morphogen gradient
75
Q
What is the phenotype of shh mutant limb?
A
Consistent with the morphogen gradient hypothesis
-Absence of shh there is a complete loss of distal most skeletal elements
- Also, loss of identity of zengopods (Ulna and radius)
76
Q
What are the 3 genes essential for dorso-ventral patterning?
A
Wnt7a, En1 and Lmx1b (drosophila homologue= antererous and controls dorsal fate)

KO of Lmx1b causes ventralization of the limb
77
Q
DV patterning pathway
A
Bmp - En1 - Wnt7a - Lmx1b
78
Q
Steps for Neural plate to early neural tube in verebrates
A
Step 1- Under the influence of signals that define the ant-pos axis an early teritory is established that express distinct TF:
-Rx
-Six3
-Pax6
3 TF on both sides

Step 2- Slightly later, shh from the prechordal mesoderm induces shh and floor plate like structures in the middle- splits eye field, Shh suppresses Pax6/Rx
79
Q
What can splitting the eye field lead to?
A
Cyclopia - single eye due to mutant -/- shh mouse wt and shh-null
80
Q
What is holoprosencephaly?
A
Failure of ventral forebrain to form- (and failure of pituituary, cyclopia, general problems withi midline of face) - due to mutations in Shh and Shh signalling
81
Q
In early development where is the eye field set up?
A
In anterior neural plate
Anterior neural plate and eye field borders ectoderm
82
Q
How does the eye form between 3-7 weeks in humans?
A
1. Eye field grows sideways and contact ectoderm
2. Ectoderm thickens (placode)
3. Eye field form double- layered cup
83
Q
Why does the part of the eye, eg. the retina, contain neurons?
A
Eyes grow sideways, forming the optic vesicle- this comes from neural tissue therefore part of the eye will contain neurons
84
Q
Where will the thickened placode form from?
A
The lens
85
Q
What happens to the cell behaviour controlled by six3 and Rx TF?
A
Migration and proliferation
86
Q
What is the series of reciprocal inductive interactions ?
A
1. Induction of the optic placode
2. Once induced the lens placode signals back to the optic vesicle causing it to undergoa morphological transformation invaginating and forming a 2 layered cup-like structure
3. Signals back to the lens placode to induce it to become the lens
87
Q
How do you form a transparent lens?
A
The lens placode invaginates and pinches off to form a hollow ball of cells
These cells are stem like- capable of self-renewing to give themselves or differentiating to lens fibre cells
88
Q
What are lens fibre cells?
A
Long, fibre-like, highly specialised cells that fill the inside of the lens and make lots of protein cyrstallin (transparent protein) before throwing out their nucleus
89
Q
Retinal and optic stalk development
A
The 2 layered cup retina consists of the outer retinal pigment epithelium and inner neural epithelium

Retinal progenitor cells give rise to:
Inner layer- ganglion cells, bipolar cells, amacrine cells, photorecepetors and muller glia
Outer layer- Pigmented cells (giving rise to colour)

Optic stalk give rise to astroyctes of optic nerve
90
Q
Retinal differentiation- what do the 2 layers of optic cup differentiate into?
A
Different directions
Outer layer cells- Retinal pigment epithelium= produce melanin
Inner layer cells- Stem cell like poplation= self renew or differentiate via progenitors to produce diverse ganglion, interneurons and light sensitve photoreceptor cells in the neural retina
91
Q
What gives rise to the different tissue types in the eye?
A
Neural ectoderm - retina and retinal pigment epithelium (RPE)
Surface ectoderm- Lens
Migrating cells- sclera and anterior chamber migrating cells.
92
Q
The early neural tube is only 1 cell wide
A
Neuroepithelium
93
Q
Neuroepithelium are proliferating and, nuclei undergoing interkinetic migration
A
G1 + S- Nucleus away from lumen
M + cytokinesis - nucleus close to lumen

Early divide symmetrically
Later divide assymetically- radial glia or second daughter
94
Q
What happens in weeks 5 onwards?
A
Week 5 Waves of differentiated cells form
Week 7-8:
1. Outer neuroblastic layer- neuroblasts in the region form rods and cones
2. Inner neuroblastic layer- neuroblasrs in this region form bipolar neurons
3. Innermost neuroblastic layer- neuroblasts in this region form ganglion cells
95
Q
How do we know that neuroblasts are stem/progenitor cells?
A
Many intrinsic signalling factors (shh, notch bmps) govern dfferentiation of the different neuronal cell types found in the retina
Induce expression specific TF that determine cell fate (intrinsically)(autonomously)

Rat pup- inject at 4-6 weeks, remove retina fix and stain section then analyze
96
Q
What is the problem for the eye with stem cells?
A
Stem cells decline over age and neurons in the eye can degenerate
Age related macular degeneration
SC in epithelium supported by retinal epithelial cells- made from different types of sc and are being transplanted into patients to overcome macular degeneration