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ANAT 381 > Ryan Lecture 4 > Flashcards

Ryan Lecture 4 Flashcards

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1
Q

What is important in forming a-p axis in chick embryos

A

Gravity is important
Egg spins in hen ~10-15 times/hr for 20 hrs

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2
Q

What does egg spinning do - a-p chick embryos

A

Organizes heavier to lighter particles within developing embryo
Lighter yolk components lie on one side of blastoderm = become POSTerior
Primitive streak begins at posterior and extends anteriorly

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3
Q

How many eggs do chicks lay

A

One egg per day
Only one oviduct develops, not bilateral

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4
Q

What happens during chick gastrulation

A

Primitive streak regresses
Ant side= hensens node

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5
Q

Describe primitive streak in chick embryos

A

Extends from post towards ant
Hensens node = organizer region at ant end

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6
Q

What happens when cut chick embryo like a pie

A

Each wedge will form primitive streak
Has dorsal ventral axis
Early chick embryo is radially symmetrical
Tissues around = competent to respond to signals coming from primitive streak

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7
Q

Describe the new ectopic streak - chick embryos

A

Forms in ant half, expresses similar molecular markers as endogenous primitive streak (brachyury)
If split into ant and post half =
Ant has new ectopic primitive streak, functions as normal, same gene expression
Post= endogenous primitive streak

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8
Q

What is important for primitive streak - a-p axis formation - exp gen

A

Vg1
Do exp = vg1 expressing cells placed in ant region, 180 to koller’s sickle (endogenous primitive streak)
Can use cell pellet= groups of cells
Could also use beads soaked in protein vg1

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9
Q

What is important for primitive streak - a-p axis formation - formally set up

A

Experiment:
1. Transplant cells expressing signalling molecule at the opposite side of the embryo from
Koller’s sickle.
2. Allow embryos to develop until primitive streak is formed.
3. Perform whole mount in situ hybridization experiment to monitor expression of cNodal that is
normally expressed in the primitive streak

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10
Q

What is important for primitive streak - a-p axis formation - results

A

Monitor nodal expression in primitive streak
If do vg1 + wnt = nodal expression induced by vg1
Multiple genes looked at - many diff results

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11
Q

What happens if place vg1 pellet 90 degrees to koller’s sickle

A

Can also induce primitive streak BUT no expression endogenous streak
Position important

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12
Q

Summarize effects of pellet vg1 for experiments

A

Vg1 important for primitive streak formation
Position of pellet matters
If placed at ant end = both ectopic and endogenous primitive streaks form
If placed on lateral margin = only ectopic primitive streak forms, formation of endogenous streak inhibited (by cells, bc only want one streak, if too close=would have to share tissues, think Siamese twins)

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13
Q

What happens if 2 vg1 pellets implanted - results

A

Implant one then wait and implant second 180 degrees from first
Implanted before endogenous streak formed
If both bead added at t=0 = 2 streaks
If 2nd bead added at t=4hr =1st streak more developed than second
If 2nd bead added at t=6h = only get one streak, no induction of other

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14
Q

What happens if 2 vg1 pellets implanted - conclusions

A

Depends on timing of second 2nd implant
Suggests that vg1 induces both primitive streak and expression of a molecule that inhibits induction other primitive streak

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15
Q

Describe a-p axis in mouse

A

1st definitive axis in mouse embryo
Primitive streak and node form on opposite side of embryo from AVE

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16
Q

What does a-p axis in mouse depend on

A

2 signaling centres are required to pattern anterior end of embryo =
Node (organizer region located at ant end primitive streak)
Anterior visceral endoderm = AVE

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17
Q

Describe AVE - mouse a-p

A

Derived by distal visceral endoderm (DVE)
Position of AVE determined in part by physical constraints of uterus - if remove physical constraints… embryos develop without a-p axis

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18
Q

Describe expression patterns in a-p polarity of mouse

A

Dve = shifts to become. AVE
High nodal expression in proximal epiblast
Future post side forms primitive streak
DVE expresses Cerberus and lefty, low nodal in distal epiblast

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19
Q

What does Cerberus do

A

Inhibits wnt

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20
Q

What does lefty do

A

Inhibits nodal

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21
Q

What exactly happens - a-p in mouse

A

DVE tilts and migrates over epiblast in one direction
Which determines future anterior end of the embryo = AVE

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22
Q

WHAT IS AVE NEEDED FOR IN MOUSE

A

PATTERNINg the anterior end of embryo
Inhibits primitive streak formation (via inhibition of nodal, expression in primitive streak)
At E6.5 = plays a role in patterning anterior primitive streak

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23
Q

Describe mouse gastrulation - a-p

A

Starts to form at future post end = primitive streak
Endoderm leaves epiblast layer
Then next = mesoderm
On inside = ectoderm
Definitive endoderm on outside

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24
Q

Describe role of node - a-p mouse

A

Node = responsible for creation of body plan - patterning
Will form at most distal end embryo
Both express many genes also found in chick and frog organizer tissues = Hesens node and spemann mangold organizer

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25
What is patterning along a-p axis dependent on in mouse
hox genes
26
Describe rna expression patterns in mice of certain proteins
Cdx2 Ant vs post = diff expressions Mix 1 = long axis Foxa2 = long axis Mesp1 = wing like, mesodermal derived, will form underlying layer mesoderm
27
Describe hox gene clusters - a-p
3’—>5’ 3’ genes = expressed more ant and early 5’ genes expressed more post and later Order evolutionary conserved
28
How many hox genes clusters do species have
Flies = 1 cluster Humans = 4 clusters
29
What do mutations in hox genes do
Cause homeotic transformations = transform one region to another
30
Describe the types of homeotic transformations possible
Loss of function = cause post —> anterior transformations, to make more ant phenotype Gain of function mutations = cause anterior to posterior transformations, bring more posterior gene forward
31
Describe antennapedia mutation in drosophila
Gain of function = express antennapedia in more anterior segment Bring segment forwards = expression in ant end
32
Describe ultrabithorax mutation drosophila
Lose of function = lose ultrabithorax expression in T3 segment Hox gene expression pattern in T3 looks like the segment that is anterior to it Post transformed to ant So get 2 pairs of wings=extra segment with wings
33
Describe hox genes in vertebrates - gen `
3’ cluster = more ant expression than genes at 5’ end cluster Not all clusters have same fam members Some have additional Have to knockout multiple members
34
Describe anterior boundary of expression of hox genes
Gets more anterior the lower the number of cluster it is See boundary of hind lim bud = in situ hybridization
35
What are paralogs - describe
Members of diff hox clusters, show similar anterior boundary in somites So like C4 more ant than C6
36
What happens when you extend boundary of Hoxa10 anteriorly
Shape of ribs patterned by hox genes So makes ant look more like post So lose ribs
37
What happens if you delete hox A10, C10, D10
Normally extend to pattern lumbar region So now look more anterior Have to knockout all to get pattern = A,C, & D Lumbar vertebrae now have ribs = look like thoracic vertebrae Posterior segment looks more like anterior segment = posterior to anterior transformation
38
Are vertebrates externally and internally asymmetric
NOOO Externally bilaterally symmetrical But internally there are many asymmetries
39
Describe normal vertebrate internally presentation
Situs solitus Heart on left Left lung bilolobed, right lung trilobed Liver mostly on right Stomach on left Kidneys sit on diff a-p axis, not even across Happens early in embryo Evolutionarily conserved pattern Allows for efficient packing of organs within body cavity
40
Describe situs Inversus
Switched - mirror image, higher incidence heart defect but properly packed and connected Could also be just one cavity - abd or thoracic
41
Describe other internal abnormal presentations of vertebrates
Left isomerism = polysplenia Right isomerism = asplenia = lungs have same number of lobes Situs inversus thoracalis Situs inversus abdmoninalis = Spleens could be missing
42
Describe why we get many diff presentations of internal organs of vertebrates
Each organ as forming on a-p axis of body receives L-R cues independently as they develop= patterned differently
43
Describe antisymmetry
Snails Dextral (clockwise) and sinistral (counterclockwise) are equally common Almost never inherited = random, dextral and sinistral phenotypes in offspring are equally common regardless of parental phenotype 29 cases in plants and animals = mostly
44
Describe directional asymmetry
Humans Most individuals are asymmetrical in the same direction Typically inherited Genetic mutations
45
Describe initial morphological asymmetries
Early chick embryos Bilaterally symmetric = ~24hrs —> heart looping = ~ 39hrs, rightwardly looping, first l-p we see patterning —> axial rotation = ~50hrs Mouse has to contort extra = since endoderm outside, = flips inside out, axial rotation
46
Name the 4 steps of L-R patterning
Initiation Stabilization of signal Propagation of signal Translation of signal
47
Describe step 1 = initiation - L-P chick
Disruption of bilateral symmetry Occurs prior to node formation and at level of the node Very species specific
48
What is needed for initiation - describe
H+/K+ - atpase = Xenopus and chick = pump protons, move ions, gives l-r patterning, active during cleavage and dwindles at gastrulation Gap junctions = xenopus, chick, some cleavage but mostly gastrulation, cells at midline do not connect by gap junctions, know they are on diff sides, just not which side
49
What is a critical early step in establishing l vs r
Asymmetric cell movement at hensen’s node Cell move diff = from right to left side If put glue in embryo and block celll movements = will change l and r patterning
50
What is needed at level of node
Asymmetric gene expression - early chick = starts during gastrulation, more during gastrulation/neurulation, dwindles before organogenesis Node monocilia = mouse, chick, xenopus, zebrafish = during gastrulation
51
Describe asymmetric gene expression at node - chick
Shh on left side of hensens node Cldn10 on right side hensens node = similar at first then gradually increased, so only on right Very important for l and r patterning and early Tjs = block paracellualr movements of ions, so will block ability of ions to move from surface between cells to signal to underlying tissue
52
Describe cilia at the node
Mouse rabbit zebrafish, xenopus Bit diff in mouse Node = teardrop shaped Planar cell polarity Ant and post Each of cells have a cilia that sits at post end cell and at particular angle
53
Describe kif3b knockout embryos
Kinesin motors LR patterning defects Mutants have stunted cilia = D,H,G which are important in early embryos Mutant cilia immotile Stumper cilia
54
Describe cilia at mouse node
Actually move and turn 12 hr cycles in mouse = have standard times At 2-6 am on days of dev cilia rotate on node
55
How is cilia moving important = evidence for nodal flow
Artificial flow can affect situs Cilia at post end and move in v coordinated fashion Exp = positioned chambers, mutant embryos w/o and did experiment so flow would move l—>r or r—>l Peristaltic pump experiment Usually flows from r—>l but can switch to l —> r
56
How is cilia moving important = evidence for nodal flow = Results
Reverse of situs of wild type embryos at presomite stage by fast rightward flow = override endogenous flow Artificial flow can correct situs inversus defects in embryos with immotile cilia
57
Describe idea of cilia
Do not know what molecule is that is being asymmetrically distributed Immotile cilia = crown cells Motile cilia = pit cells Create flow/movement bc surrounding node = crown cells, If abandon immotile cilia = get calcium flux released = important
58
What is the role of cilia
Nodal flow Particles moves and smash open and release
59
Describe cilia in lr patterning
Motile cilia at the node have now been found in mice, xenopus (gastrocoel roof plate), zebrafish (kuppfer’s vesicle) and rabbits (node)
60
What do cilia defects do
Defects that affect cilia motility, structural proteins and other comments affect laterality Experiments indicate that monociliary movement is important but do not indicate that nodal flow initiates asymmetry
61
What is important for positioning cilia
Planar cell polarity
62
Describe xenopus l-r
Movement of particles across gastrocoel roof plate If put glue = disrupt lr patterning
63
DESCRribe chick and pig embryos
Chick have cilia on cells of hensens node but no flow, rounded shape, due to #?, shape of node? = asymmetric gene expression at hensens node in chick Similarly = no nodal flow at node in pig embryos
64
What about humans - nodal flow and cilia
Humans with ciliary defects have associated laterality defects but nothing is known about if there is flow Kartagener syndrome = many cilia defects, infertile, cannot smell, have lr patterning defects = situs inversus Know cilia important but not specifics
65
Describe step 2 = stabilization of the signal - lp
Maintenance of the signal that broke bilateral symmetry - patterning event conserved At node and/or peirnodal region Asymmetric expression of nodal in chick
66
Describe step 3 = propagation of signal - lp
Transfer and maintenance of info on left and right sides embryo Midline acts as barrier = molecular and physical, midline = notochord, super strong solid bar, do not want lr signals to mix Asymmetric gene expression in lateral plate mesoderm Conserved in species
67
Describe chick - nodal
Expressed only on left side of perinodal region Patterning on left mostly
68
Describe asymmetric nodal and lefty-2 in lat plate mesoderm
Conserved in all species Controls morphogens by inhibition Lefty 1 = expressed in midline, inhibits ndoal Lefty 2 = expressed in lateral plate mesoderm
69
How does lefty act
Classical feedback inhibitor of nodal signaling
70
Describe lefty - reaction diffusion model hypothesis
Interactions between an activator and an inhibitor control signalling gradients within a field of cells and subsequently pattern them Activators turns on its own expression and activates its own inhibitor, also turns on other genes
71
Describe principles of the reaction diffusion model
Activator activates its own production Activator activates its inhibitor Inhibitor blocks auto activation of activator Inhibitor acts at a long range to restrict the effective range of the activator, can work at longer range
72
Describe nodal left feedback loop
Relative expression levels of nodal and lefty determine intensity of signal Nodal turns own expression but also turns on lefty Diff doses nodal signalling
73
Describe pitx2 expression
Asymmetric in left lateral plate mesoderm Pitx2 produces multiple transcripts Induced by nodal signalling Conserved in all species Stays in asymmetrically position tissues = left gonad - recall oviduct, one egg Pitx2c expressed in lat plate mesoderm Pitx2 isoforms = same homeodomain and transactivation domain Pitx2c = produced from internal promoter = starts only at exon 4
74
What does bilateral pitx2 expression do
Randomized direction of heart looping in chick Endogenous or ectopic Which side saw higher level = threshold and that side made loop
75
Describe pitx2 in xenopus - bilateral pitx2 expression
Frogs Reverse direction of heart and gut looping Counter clockwise Pitx2 = downstream, effectors of model, clockwise
76
Describe step 4 = translation of signal
Conversion of info into asymmetric morphogenesis Occurs along entire AP axis and at level of individual organs Pitx2c asymmetrically expressed in organs that exhibit asymmetry = independent interpretation of Pitx2c info, organs respond different to decreasing doses of Pitx2c, need diff doses
77
Describe generation of anatomical asymmetries- morphogenesis
Directional looping- diff loop = heart, stomach, intestine Asymmetric development = lung, liver Asymmetric regression = blood vessels,right oviduct = if overexpress pitx2 = can retain gonads on both sides
78
Describe cellular basis of asymmetric organogenesis
Differential Cell death - regression Differential Proliferation = smp, spleen Differential Cell elongation = chick gut (one side = cells stay columnar or other = get flat and elongated, important for tissue to shape and band) Differential Migration = zebrafish gut Rearrangement of actin-cytoskeleton Intrinsic/extrinsic forces = heart tube
79
Describe LR patterning cascade in chick embryos
Asymmetric expression of Pitx2c in developing organs asymmetric organogenesis - nodal and lefty present in cell species
80
Describe lr patterning cascade summary - species specific
Axis of LR symmetric oriented and info spread throughout multicellular fields, H+/K+ atpase and membrane potential, polycystin-2 and calcium signalling, gap junctions
81
Describe lr patterning cascade summary - evolutionarily conserved
Asymmetric pattern amplified and propagated by cascades of gene espreccion = lefty1, lefty2, nodal Proteins that influence morphogenesis are induced in target tissues = Pitx2c, morphogenetic proteins

ANAT 381 (24 decks)

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