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ANAT 381 > Ryan Lecture 5 > Flashcards

Ryan Lecture 5 Flashcards

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1
Q

Describe compartment

A

A module of embryo that consists of a group of adjacent cells that do not mix with cells from neighbouring compartments
Display same gene expression profile - same molecular/genetic address, express similar patterns of gene

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2
Q

Describe compartment boundary

A

Border/region between 2 compartments that cells do not cross - stay with adjacent cells
Boundaries may be invisible = defines compartments, molecular boundaries

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3
Q

Describe 1970s discovery

A

Devleopemnt of techniques to create genetic mosaics in flies
Revealed boundaries that did not correspond with morphological landmarks = expressing genes in diff sub domains

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4
Q

Describe clones of cells within drosophila wing

A

Clones of cells within a compartment have jagged borders = proliferate across
Clones of some cells have straight border - at a-p boundary =does not align with wing vein, cells do not cross line

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5
Q

What are the 2 critical functions that boundaries perform

A

Prevent intermingling of cells = maintain population fo cells, cannot migrate
Provide positional info to flanking cells

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6
Q

Describe how to visualize compartment boundaries

A

Lineage marker
Shows that cells do not mix across boundary

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7
Q

Describe cell adhesion properties at compartment boundaries

A

Extra cellular proteins secreted by ecm =
Qualitative = diff sides secrete diff molecules, cells recognize other cells as diff
Quantitative = express diff levels of molecules

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8
Q

Describe the effect of diff amounts of E-cadherin = compartments and boundaries

A

Qualitative, p cad =red, e cad = green
E-cadherin = tighter
When p cad > e cad = cels move towards inside
When p cad = e cad = more intermixed
When p cad < e cad = red on outside, excludes red, so green matches wth cells that have higher affinity (green)
LEVEL OF PROTEIN CASUES SEGREGATION

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9
Q

Forming compartments and boundaries = 1

A

FIELD Of cells becomes subdivided by their interpretation of a morphogenetic gradient
Cells on left see low level morphogenetic
Cells on right see high level morphogen, cells =turn on diff patterns of gene expression

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10
Q

Forming compartments and boundaries = 2

A

Morphogen gradient induces transcription factors, adhesion/affinities
Response causes repression of that signal in cells

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11
Q

Forming compartments and boundaries = 3

A

Subdivisions maintained and refined by local cell cell interactions - short range signalling
Gradual refinement by feedback
Expresses one or other
Interface between= where border forms

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12
Q

Forming compartments and boundaries = 4

A

2 distinct populations = leads to formation of specializes cells at borders = boundaries
Cells are diff= recognize cells that look like them and cells that do not, not some level or types of factors

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13
Q

Forming compartments and boundaries = 5

A

Boundary influences surrounding cells - long range signalling - to regulate growth and patterning
Forms organizer region sometimes
Emits signals
Cells on either side respond diff bc diff patterns (tfs)

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14
Q

Forming compartments and boundaries = Gen

A

A= gradient, with threshold levels
B = inhibition
C= represses other cells = creates distinct domains

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15
Q

Where do organizers form

A

At compartment boundaries

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16
Q

What do organizers do

A

Communicate info to neighbouring compartemts by releasing signalling molecules or their inhibitors
Long range signaling
Short range signalling
Initial = morphogen gradient across cells, differential responses, at interface = set up new organizer region, new long and short range signalling

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17
Q

Give 3 ex’s of developmental compartments

A
  1. Drosophila = stripes in embryo, hox genes and ap compartments, wing
  2. Somites
  3. Vertebrate cns and hind brain (fore and midbrain)
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18
Q

Describe drosophila pair rule genes - developmental compartments

A

Stripes of eve and ftz
Quickly defined info
14 stripes of expression

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19
Q

Describe drosophila pair rule genes - gen

A

Stripes of pair rule genes turn on segment polarity genes
Components of wnt and shh signaling
Segment = contains eve and ftz
Parasegemnt = eve or ftz + GAP
(Eve and ftz come on in response to earlier patterning)
Wg = turn on in gap, wingless, wnt fam
En = posterior to wg, turn on engrailled

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20
Q

Describe drosophila pair rule genes - specifics

A

Parasegment = anterior end of one stripe to anterior end of next stripe
Wingless expressed between stripes eve and ftz
Engrailed expressed in cells expression eve and ftz but only 1 cell/stripe; 14 rows
Cells expressing engrailed is the posterior end of each segment

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21
Q

Describe drosophila pair rule genes - interaction between engrailed and wingless

A

Wingless diffused = signal
Sees wingless signal
Cells competent to respond
Releases shh and binds to patched receptor =
Turned on another morphogen = now have repressive effects on each other

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22
Q

Describe drosophila pair rule genes - gradient

A

Level of signals = determines if turn n bristle or not
Signal = engrailed makes shh, shh and wingless repress activity of cells responding to ether
Shh = high ant, low post
Wg = low ant, high post

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23
Q

Describe vertebrate segments - allow for

A

Repetition to form
Makes them diff - if have ribs or nah

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24
Q

What does segmentation do

A

Provides a developmental mechanism for evolution of increasingly sophisticated structures, species specific, has diff patterns

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25
What are somites
Masses of mesoderm formed from presomitic mesodermal Repetitive structures along a-p axis Anterior somites older than posterior somites
26
Describe somite formation rate
1 pair of somites formed every 90 mins in chick, 55 pairs in total in chick
27
Describe somite differentiation - gen
Differentiate to give rise to dermis, skeletal muscle, and vertebrae Presomitic paraxial mesoderm proliferates, as ages differentiates = Hypaxial dermomyotome, layer outer Inner layer = myotome Sclerotome and myotome give rise to diff tissues
28
Describe transverse section through trunk of chick embryo on days 2-4
2 day = epithelialized, young 3 day = dermomyotome, sclerotome Late 4 day = epithelial layer undergoes EMT transition as they migrate away, Gives rise to diff structures
29
Describe somitogenesis in zebrafish embryo
Continuous process as embryo grows and extends in a-p axis
30
When is mesoderm formed
At end of grastrulation Paraxial endosperm, gives rise to somite Intermediate mesoderm = kidneys, and gland Lateral plate mesoderm = l and r patterning, asymmetric gene expression
31
Describe mesoderm derivatives
Paraxial mesoderm —> somite —> sclerotome (cartilage), syndetome (tendons), myotome (skeletal muscle), endotome (endothelial cells, dorsal aorta), dermatome (Dermis, skeletal muscle)
32
Describe mesoderm formation
At end of gastrulation BMP expressed bilaterally at lat plate mesoderm In Center = notochord, high chord in (bmp antagonist)
33
What is important for parasail mesoderm specification
BMP signalling Molecules = chordin - notochord, paraxis - somites, pax2 - intermediate mesoderm Exp = transplant noggin secreting cells (same effect as chordin) in to lat plate mesoderm Result = somites Why = need low level bmp for somites to form, restricts somites to paraxial mesoderm
34
What else is also important for generating presomitic mesoderm
Tailbud Cells here highly proliferative Psm = presomitic mesoderm Nmp = neuromesodermal progenitors (bipotential = cells contribute to neural tube and mesoderm - psm) DMZ = dorsal marginal zone, has nmp cells
35
What is important for paraxial mesoderm differentiation
Tbx6 Expressed in psm Get smaller as embryos grows Most posterior end dorsal mrgainal zone Wnt —> t —> tbx6 —> d2i Tbx6 downstream wnt and t Important for paraxial mesoderm patterning
36
Describe mouse tbx6 knockout
Paraxial mesoderm transformed into neural tube Neural tube strained w/ sox2, mutant = ectopic neural tubes express sox2, lose identity as mesoderm cels and somite progenitors Pax6 = tf expressed in DMZ of forming neural tube, mutant = also show dorsal ventral patterning with respect to pax6, ectopic neural tubes express px6 dorsally Conclusions = TBX6 IN NMPS (neuromesodermal progenitors ) AND PSM (presomitic mesoderm) REQUIRED FOR SOMITE IDENTITY
37
Describe somites in stage 6 chick embryo
Paired even and repeated 90 min cycle
38
How do somites know when and where to form
Embryological clock measures dev Rate of somite segmentation 1pr/90 min, must be coordinated with overall dev and occur in parallel on right an left sides If move to cooler temp, somites take longer to form?
39
Describe clock wavefront model of somite formation- when and where
Cooke and zeeman 1976 Intersection of system to regulate where a boundary will form (wavefornt) and when a boundary should form (clock) Refer to wave front as differentiation front
40
Describe role of retinoids acid in paraxial mesoderm development
High ant, low post
41
Describe role of fgf8 in paraxial mesoderm development
High post, low ant
42
Describe role of retinoids acid and fgf 8 in paraxial mesoderm development - gen
Antagonistic signals along the a-p axis pattern NMPS during paraxial mesoderm dev Antagonists important in positioning of somites Tbx6 represses sox2
43
Describe role of retinoids acid and fgf 8 in paraxial mesoderm development - specifics
Retinoic acid high at anterior - raldh2 involved in RA synthesis Fgf8 high at posterior Mesp = expressed at boundary of low RA/low FGF front = indicates where next somite will form Primarily expressed in psm
44
What regulates epithelialization during somite boundary formation
Eph-Ephrin signalling
45
Describe whole role of eph-ephrin signalling, steps 1-4
1. Mesp restricted to anterior half somitomere minus 1 2. Unregulated ephA4 in anterior half 3. EphA4 upregulates binding partner ephrinB2 in presumptive posterior SO (somite about to form), post half 4. Triggers epithelialization and formation of a boundary
46
Describe role of eph-ephrin signalling, steps generally
S-1 mesp —> ephA4 then goes and to next half ant somite, and turns on ephrinB2 ahead = ant s-1 and post s-0 somite epithelialization Cells of psm = mesenchymal But Turning on genes = epithelialization and detaches from psm and moves on
47
When is ephA4 EXPRESSED
AS NEW somites form -chick embryos
48
Describe where somites form
Opposing FGF/RA gradients —> mesp —> eph/ephrin
49
Describe when somites forms
Only one boundary formed at a time
50
What controls when somites form
Notch = time keeper Juxtacrine signalling molecule
51
Describe notch - time keeper of somites
Oscillates in segmentally define pattern All species have at least 1 notch target gene that oscillates Turns on and off v rapidly In cells Waves of notch expression
52
Describe notch - time keeper of somites NEGATIVE FEEDBACK
Notch target inhibits notch signaling Inhibitor - target is unstable When inhibitor gets degraded notch signalling comes back on Waves of notch signaling creates clock
53
How do waves of notch signalling connect to differentiation front
Targets of notch signalling = lunatic fringe, hairy (hairy enhancer of split) = genes that oscillate = important Ant out of sync with post Turns off and on in cells sequentially
54
What is wave of genes expression due to - notch time keeper
Sequential activation of gene expression not due to movements of cells
55
Describe whole somite formation process
1 cycle of notch/target takes ~90 minutes Mesp - needs low RA and fgf, NOTCH target in anterior half repressed notch activity in anterior half…eph/ephrin.. met (mesenchyme to epithelial transition determines boundary) High FGf at posterior prevents cells from being competent to respond to notch signal, so not form somites early, no mesp so no somites
56
Describe compartments - somites and A-P axis
Reiterated compartments resemble one another but give rise to different derivatives, e.g. somites that form cervical vertebrae do not form ribs; but somites that form thoracic vertebrae do (determined by position along AP axis before somitogenesis)
57
Recall = hox gene expression
Spatiotemporal colinearity of hox gene expression in psm Layered on, turned on a-p, 3’earlier than 5’, ant post, identity of somites IS CORRELATED WITH CHROMATIN REMODELING = genes come on at diff times, opens at 3’ end and then gradually progresses
58
What do chicks have compared to mouse - vertebral pattern along a-p axis
Chicks have more cervical vertebrae
59
What happens when you transplant presomitic mesoderm from region that would normally form thoracic vertebrae caudal (posterior) to the 1st somite in a younger embryo?
Transplantation exp Psm of thoracic vertebrae from post, older embryo Psm of cervical vertebra in chick embryo = Gain of function, ant to posterior transformation Has ribs - looks like thoracic, PSM ALREADY HAS PATTERNING INFO
60
WHAT happens when transplant thoracic somites into cervical region
Ribs from on neck vertebrae, derived from thoracic somites Carries a-p info already Due to notch gene expresssion
61
Transplant cervical somites into thoracic region
Ribs do not form on vertebrae derived from cervical somites Chick embryo = flat, so easy to do on one side
62
How do you stop making somites
Consider rate of oscillation vs rate of axis elongation (proliferation/wnt signal) If axis elongation sustained in balance with clock rate = infinite #, if sustain and balance with wavefront would make get infinite somites If axis elongation is less than clock rate = somite formation will use up PSM
63
Is there a 1:1 relationship between somite number and vertebrae #
NAHHHH Humans = 52 somites, 33 vertebrae Mouse = 65 somites, 29 + 25 (tail) vertebrae Snake = <500 somites, 100-300 vertebrae
64
Describe snake vs mice
Snakes = long coil of somites
65
What mechanisms/pathways can we alter to get more segments - snake case study
Tail boys elongation - more proliferation Fgf, wnt, RA gradients Notch delta signalling -rate of on/off gene expression Eph-ephrin B2 epithelialization Extra layers of patterning? Species specific things that overlay basic
66
What questions can we ask- snake case study
1. How could alterations in one of these mechanisms lead to changes in segment number to make the axis of a snake as compared to that of a mouse? 2.What would be the ramifications to the other three mechanisms based upon your proposed change?
67
What does data from corn snakes show - snake case study
They have 3 fold more oscillations in lunatic fringe expression in PSM compared with similar stage mouse embryos More rapid oscillations
68
What is fore vs hind limb
Fore = arms Hind = legs
69
Name all 3 limb axes
Proximal-distal Dorsal-ventral Anterior-posterior
70
Describe proximal distal limb axis
Proximal = closest to the body - shoulder/pelvic girdle Distal = tips of digits
71
Describe dorsal ventral limb axis
Dorsal = back of limb/hand - top of foot Ventral = front of limb/hand - bottom of foot NAILS on dorsal side
72
Describe anterior posterior limb axis
Mostly looked at relative to hand/foot Anterior = thumb/big toe, digit 1 Posterior = pinky, little toe, digit 5
73
Describe limb bud morphogenesis
Begins as outpouching of cells - limb buds Limb bud = posterior lat plate mesoderm - skeletal precursors Somite cells - muscles precursors Overly ectoderm - outer layer, inside =mesoderms (lpm + somites)
74
Do both hind and fore limb buds form at same time
Forelimb bud forms 1/2 days before hindlimb bud
75
Describe transverse scanning electron micrograph through limb bud
Mesenchymal mesoderm cells Thin ectoderm
76
Describe limb bud days 9-11
Mouse Limb buds continue to grow, get bigger Day 33 in humans
77
Describe limb bud E11
Apical ectodermal ridge - AER is now clearly visible = thicker ectoderm Forms at dorsal ventral boundary
78
Describe limb bud day 12
Limb bud becomes paddle shaped, flattens Indentations that will become inter digit region are visible Programmed cell death = apoptosis in inter digit region important to prevent webbing (some species need webbing tho like ducks) Human = 36 days
79
Describe digits growth in humans
Between days 48 and 56 Web at 48 has By 56 days most webbing gone
80
What is limb bud formed by
Out pouching of lateral plate mesoderm at the correct position along a-p axis in parallel Cells from dorsal region lat plate mesoderm =undergo epithelial to mesenchymal transition
81
How is position of outpouching determine of limb bud - exp 1 1918
Detwiler and Harrison = showed removing groups of cells blocked limb bud growth
82
How is position of outpouching determine of limb bud - exp 2 1925 / 1933
1925 = hertwig transplanted groups of cells to new places and go a limb, depends on stage and patterning info that comes with it 1925/1933 = balinsky - showed that optic vesicle (ear), pituitary (thickened ectoderm that will become pituitary) and/or olfactory sac (lining of nose) could induce limb bud in inter limb region (note = all derived from ectodermal placode = thickened region)
83
How is position of outpouching determine of limb bud - exp 3 1971
Rosenquist = marked groups of cells and watched where they go = fate mapping
84
Describe role of RA and FGF8 along a-p axis - limb bud
Hox genes expressed along a-p axis regulate expression of RA and FGF RA and FGF8 act as antagonistic signals to induce Tbx5 expression (homeobox gene) FGF8 high in ant region RA high in forelimb field HOXC6 gene Opposing gradients
85
What determines position of forelimb bud
Tbx5
86
Describe RA and FGF8 knockout exp in limb bud
Looking at rare = RA response element cloned upstream LacZ gene - turns blue Rdh10 knockout = block RA expression, get extension of FGF ant expression = moves boundary Also lose expression Tbx5 In situ hybridization for FGF8 expression - can see it is more expressed in heart
87
What signals position of Lat plate mesoderm outgrowth
Fgf10 = 4 blocks expression, where outgrowth, turned on after Tbx5 If transplant fgf beads or cells = can induce limbs If plant close to forelimb = look like wing If plant in middle = chimera, both features If plant closed to hind = looks like leg
88
What can FGF do - limb buds
Induce ectopic limb buds Can induce normally patterned bud, with digits Chick embryo

ANAT 381 (24 decks)

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