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ANAT 381 > Ryan Lecture 4 > Flashcards

Ryan Lecture 4 Flashcards

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
Q

What is patterning along a-p axis dependent on in mouse

A

hox genes

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26
Q

Describe rna expression patterns in mice of certain proteins

A

Cdx2
Ant vs post = diff expressions
Mix 1 = long axis
Foxa2 = long axis
Mesp1 = wing like, mesodermal derived, will form underlying layer mesoderm

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27
Q

Describe hox gene clusters - a-p

A

3’—>5’
3’ genes = expressed more ant and early
5’ genes expressed more post and later
Order evolutionary conserved

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28
Q

How many hox genes clusters do species have

A

Flies = 1 cluster
Humans = 4 clusters

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29
Q

What do mutations in hox genes do

A

Cause homeotic transformations = transform one region to another

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30
Q

Describe the types of homeotic transformations possible

A

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

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31
Q

Describe antennapedia mutation in drosophila

A

Gain of function = express antennapedia in more anterior segment
Bring segment forwards = expression in ant end

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32
Q

Describe ultrabithorax mutation drosophila

A

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

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33
Q

Describe hox genes in vertebrates - gen `

A

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
Q

Describe anterior boundary of expression of hox genes

A

Gets more anterior the lower the number of cluster it is
See boundary of hind lim bud = in situ hybridization

35
Q

What are paralogs - describe

A

Members of diff hox clusters, show similar anterior boundary in somites
So like C4 more ant than C6

36
Q

What happens when you extend boundary of Hoxa10 anteriorly

A

Shape of ribs patterned by hox genes
So makes ant look more like post
So lose ribs

37
Q

What happens if you delete hox A10, C10, D10

A

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
Q

Are vertebrates externally and internally asymmetric

A

NOOO
Externally bilaterally symmetrical
But internally there are many asymmetries

39
Q

Describe normal vertebrate internally presentation

A

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
Q

Describe situs Inversus

A

Switched - mirror image, higher incidence heart defect but properly packed and connected
Could also be just one cavity - abd or thoracic

41
Q

Describe other internal abnormal presentations of vertebrates

A

Left isomerism = polysplenia
Right isomerism = asplenia
= lungs have same number of lobes

Situs inversus thoracalis
Situs inversus abdmoninalis
= Spleens could be missing

42
Q

Describe why we get many diff presentations of internal organs of vertebrates

A

Each organ as forming on a-p axis of body receives L-R cues independently as they develop= patterned differently

43
Q

Describe antisymmetry

A

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
Q

Describe directional asymmetry

A

Humans
Most individuals are asymmetrical in the same direction
Typically inherited
Genetic mutations

45
Q

Describe initial morphological asymmetries

A

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
Q

Name the 4 steps of L-R patterning

A

Initiation
Stabilization of signal
Propagation of signal
Translation of signal

47
Q

Describe step 1 = initiation - L-P chick

A

Disruption of bilateral symmetry
Occurs prior to node formation and at level of the node
Very species specific

48
Q

What is needed for initiation - describe

A

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
Q

What is a critical early step in establishing l vs r

A

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
Q

What is needed at level of node

A

Asymmetric gene expression - early chick = starts during gastrulation, more during gastrulation/neurulation, dwindles before organogenesis
Node monocilia = mouse, chick, xenopus, zebrafish = during gastrulation

51
Q

Describe asymmetric gene expression at node - chick

A

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
Q

Describe cilia at the node

A

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
Q

Describe kif3b knockout embryos

A

Kinesin motors
LR patterning defects
Mutants have stunted cilia = D,H,G which are important in early embryos
Mutant cilia immotile
Stumper cilia

54
Q

Describe cilia at mouse node

A

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
Q

How is cilia moving important = evidence for nodal flow

A

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
Q

How is cilia moving important = evidence for nodal flow = Results

A

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
Q

Describe idea of cilia

A

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
Q

What is the role of cilia

A

Nodal flow
Particles moves and smash open and release

59
Q

Describe cilia in lr patterning

A

Motile cilia at the node have now been found in mice, xenopus (gastrocoel roof plate), zebrafish (kuppfer’s vesicle) and rabbits (node)

60
Q

What do cilia defects do

A

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
Q

What is important for positioning cilia

A

Planar cell polarity

62
Q

Describe xenopus l-r

A

Movement of particles across gastrocoel roof plate
If put glue = disrupt lr patterning

63
Q

DESCRribe chick and pig embryos

A

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
Q

What about humans - nodal flow and cilia

A

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
Q

Describe step 2 = stabilization of the signal - lp

A

Maintenance of the signal that broke bilateral symmetry - patterning event conserved
At node and/or peirnodal region
Asymmetric expression of nodal in chick

66
Q

Describe step 3 = propagation of signal - lp

A

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
Q

Describe chick - nodal

A

Expressed only on left side of perinodal region
Patterning on left mostly

68
Q

Describe asymmetric nodal and lefty-2 in lat plate mesoderm

A

Conserved in all species
Controls morphogens by inhibition
Lefty 1 = expressed in midline, inhibits ndoal
Lefty 2 = expressed in lateral plate mesoderm

69
Q

How does lefty act

A

Classical feedback inhibitor of nodal signaling

70
Q

Describe lefty - reaction diffusion model hypothesis

A

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
Q

Describe principles of the reaction diffusion model

A

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
Q

Describe nodal left feedback loop

A

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
Q

Describe pitx2 expression

A

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
Q

What does bilateral pitx2 expression do

A

Randomized direction of heart looping in chick
Endogenous or ectopic
Which side saw higher level = threshold and that side made loop

75
Q

Describe pitx2 in xenopus - bilateral pitx2 expression

A

Frogs
Reverse direction of heart and gut looping
Counter clockwise
Pitx2 = downstream, effectors of model, clockwise

76
Q

Describe step 4 = translation of signal

A

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
Q

Describe generation of anatomical asymmetries- morphogenesis

A

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
Q

Describe cellular basis of asymmetric organogenesis

A

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
Q

Describe LR patterning cascade in chick embryos

A

Asymmetric expression of Pitx2c in developing organs asymmetric organogenesis - nodal and lefty present in cell species

80
Q

Describe lr patterning cascade summary - species specific

A

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
Q

Describe lr patterning cascade summary - evolutionarily conserved

A

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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