TAMU BIOL 414 Riley Developmental Bio Exam 3

Stem cell asymmetry

produces two daughter cells, one stem cell and one differentiating cell

Stem cell linage/differentiation stages

Multi/pluripotent stem cell → committed stem cell → progenitor (transit-amplifying cell) → differentiated cell
loses potency with each step

intestinal crypt stem cells molecules

Wnt2b - controls proliferation
Bmp4 - reduces differentiation

intestinal crypt stem cells

continuous renewal at crypt
crypt → differentiation → transit-amplifying cells → stromal pericryptic cells → into villus, anoikis then apoptosis

mesenchymal stem cells

different from adult stem cells in bone marrow
small numbers in organs/tissue
highly pluripotent, unknown function
differentiation from substrate stiffness

induced pluripotent stem cells (IPS)

adult stem cell into pluripotent stem cell
express Sox2, Oct4

imaginal discs in Drosophila

precursor tissue for external tissue (legs, wings, eyes, etc.)
Concentric rings of epithelial cells “telescope” out to produce proximo(dach)-distal(dxl) axis

AP patterning molecules in wing

Hh - posterior compartment
Dpp - anterior compartment
both high near boundary
Shh and Bmp act similarly in vertebrates

DV boundary wing bud Drosophila

Wg expression at margin, drives proliferation and extension of proximo-distal axis

Drosophila eye development

eye has 800 ommatidia, each with 8 photoreceptor neurons
Egfr needed for normal eye development
ato refined by notch into evenly spaced R8 cells
R8 uses Egf to signal other receptors and pattern rest of disc, sequential activation

eye imaginal disc Drosophila

hh needed for AP axis in morphogenetic furrow
activates dpp and ato in furrow for photoreceptor differentiation
represses hairy for differentiation

morphogenetic furrow

marks G1 arrest, onset of fate specification
signaling wave that transforms undifferentiated cells into photoreceptors

4 ectodermal fates

outer ectoderm (epidermis)
cranial placodes (thickening that give rise to sensory organs, eye lens, ears
neural crest (schwann cells)
neural tube (CNS, spinal chord)

neuroectoderm/neural plate signaling

requires fgf + anti-Bmps (noggin, chordin, follistatin)
forms neural plate (beginning of CNS) through Sox2 + Sox3 to establish neural potential and delay differentiation

neurulation

morphogenesis of neural plate into neural tube
1st and 2nd mechanisms
both involve convergence

1st neurulation

epithelial folding
occurs in neural plate
medial becomes ventral, lateral becomes dorsal
formation of spinal chord and brain
cell shape changes through contraction of actin, extension of microtubules
convergence (meet at midline and pull)
filopodia pull sides together, fusion of epithelia

1st neurulation/neural tube failure to close

must close at closure 2 (anencephaly), vitamin B12/follate needed
and closure 5 (spina bifida)

midline defects

can cause external symptoms (cleft lip, cyclops) or only internal
cholesterol and shh needed for proper development
cholesterol deficiency causes “non syndromic” mental retardation

1st neurulation molecules involved

E-cadherin for dorsal
N-cadherin for tube

2nd neurulation

posterior region of embryo
cavitation
convergence → neural keel (zebrafish ONLY) → cavitation

1st neurulation vs 2nd neurulation

1st occurs in anterior/upper region, in neural plate, forms brain/ upper spine
2nd in posterior region, in tailbud/primitive streak, form lower spine
both use convergence and form spine

neural tube adult derivatives (what it forms into)

telencephalon
diencephalon
mesencephalon
metencephalon
myelencephalon

telencephalon forms

olfactory lobes, cerebrum (association)

diencephalon forms

retina

mesencephalon forms

midbrain → tectum (sensory processing center)

metencephalon forms

cerebellum (muscle/motor coordination)

myelencephalon forms

medulla (reflex center)

AP patterning regulation

mesodermal signals
CNS signaling: organization centers, segmentation

signaling centers in brain

regulate growth and patterning in midbrain and anterior hindbrain

Hindbrain segmentation

even/odd rhombomeres signal each other via ephrins (ligand) and eph receptors (RTK)
stop different rhombomeres from mixing
in spinal chord, segmentation by somites

DV patterning in neural tube

BMP4 & 7 in epidermis (dorsal)
Shh in notochord (ventral)

Shh function in neural tube

used for DV patterning (ventral)
located in notochord
induces floor plate, gradient also induces different ventral cell fates

How are dorsal fates determined in neural tube?

TGFB/BMP signaling

outcome of DV patterning

sensory neurons
interneurons (transmits impulses between neurons)
motor neurons

Where does DV specification begin?

in neural plate

Steps in CNS cell fate

1. induction of neural plate (Sox2 & Sox3, Fgf + anti-BMPs)

2. specification of AP identity (Hox genes, rhombomeres, eph/ephrin signaling) and DV identity (BMP4 & 7 for dorsal, Shh for ventral)

3. induction of pro-neural genes

   1. encode bHLH TFs

   2. expressed in pro-neural clusters

4. lateral inhibition

pro-neural genes

express neurogenin-1
in pro-neural clusters
encode bHLH TF

diversifying cell fates in pro-neural clusters (lateral inhibition)

pro-neural cluster expresses pro-neural gene (neurogenin-1)
one then expresses DI (induced by upregulation of neurogenin-1)
lateral inhibition (neurogenin-1 repressed by notch, cell expressing D1 binds Notch receptor on adjacent cells, stopping them from undergoing same process)

oligodendrocyte vs. schwann cells

both produce myelin sheath for neurons
schwann cells aid in neuron regeneration and can only wrap one axon segment

astrocytes

most abundant cells in brain
used in axon guidance, neuronal maintenance, neurotransmitter propagation

neural stem cells in ventricular zone

ventricular zone - primary source of neural stem cells and glia cells, lines brain ventricles
neural cells undergo interkinetic nuclear migration to establish cell fate and growth, where nuclei move to ventricular surface for mitosis and basally for DNA synthesis

layering of CNS cell fates in neural tube

ventricular zone (glial cells)
intermediate zone (gray matter)
margin (myelinated axons)
external granule cell layer (neuroblasts)

cortical layers migration

later a neuron is born, further it migrates

axonal pathfinding steps

1. pathway selection (follow specific route)

2. target selection (recognize and bind)

3. address selection (each axon binds to small subset of possible cells)

pathfinding requires growth cones as well

what regulates microtubule growth in axonal pathfinding

APC
Rho-GTPases regulate F-actin dynamics for growth/retraction of microspikes
treadmilling - more Rho at leading edge to move actin forward

Growth cones

motile sensory structure at tip of axon that navigates nervous tissue to target
responds to guidance cues (ECM, transmembrane proteins, diffusible factors)

growth cones and ECM

require adhesive surface (laminin highway for adhesion and migration)
laminin coats glia
uses NCAM, N-cadherin and FGFR activation controlling cytoskeleton movement

Haptotaxis (axonal pathfinding)

migration along adhesion gradient (ECM)

contact-dependent repulsion in axonal pathfinding

causes growth cone collapse
axons repulsed by ephrins (eph receptors), semaphorins, and silt/robo
make sure axons avoid posterior half of somites

semaphorins

all repel axonal pathfinding to make sure axons migrate through anterior half of somites
have SEMA domain
membrane bound (SEM1) or diffusible (SEM2 & SEM3)

what causes axon extension

diffusible chemical cues or chemotaxis
netrins (chemoattractant)

netrins

used as chemoattractant in axon extension
guide commissural axons (axons that cross midline) to floor plate and across midline
helped by shh (mammals)

slit/robo

chemorepellents for axon pathfinding
slit is expressed by midline cells and bind to robo receptors on axons, causing growth cone collapse
commissural axons can downregulate robo receptors to cross midline

neurotrophins

NGF, BDNF, NT3, NT4/5
regulate axon growth and plasticity
chemotaxis to target (target selection)
survival when axons synapse with targets

address selection

form synapses at specific contact points (neuromuscular junctions)
agrin induces ACh receptors to cluster, helping axons form anchors
B2 laminin helps with this clustering
axons from other neurons join to reinforce synapse

axon competition/neural pruning

multiple axons bind to synapse to reinforce, most active axon wins, rest die (neural pruning)
ensures muscle controlled by one neuron
once neuron is mature, can control a broader section of muscles

Retino-tectal pathfinding

fish/amphibians: retinal neurons pass midline
birds/mammals: retinal ganglia cells (RGCs) project to both ipsilateral & contralateral using expression of slit, netrin, semaphorin, and ephrins
axons form reversed pattern in tectum (posterior to anterior)
ephrin signaling creates “map” in tectum
competition still, neural activity required to maintain

Hubel & Wiesel experiments

sewed one eye of kitten shut for 3 months, eye permanently blind
shut both eyes, no blindness (no neural activity so no binding)
showed a critical period for validation

Neural crest cell fates

depends on origin
cartilage, schwann cells, neurons, melanocytes (pigment)
cranial, cardiac, vagal, sacral, and trunk

neural crest fate specification molecules after neural plate

Wnt8 + bmp gradient + Wnt1

Trunk neural crest steps + fate

1. specification

2. delamination (form PNS)

3. migration

schwann cells, sympathetic neurons, pigment cells

Trunk neural crest specification

induced by Bmp + Wnt
sox10 specifies pre-migratory crest
reduced sox10 = less pigment due to impaired melanocyte

Trunk neural crest delamination

snail = tf used in EMT
loss of adherin junctions as detach from neural tube
Rac1 & RhoA = GTPases regulating actin polymerization

Trunk neural crest migration (2 pathways)

Ventral pathway - PNS
-uses Foxd3
-preferred by early migratory cells

Dorsolateral pathway - melanocytes/MITF
-MITF

regulated by ephrins

Stem Cell factor (STF)

chemotaxis target (attractant)
misexpression redirects migration
also promotes surival/proliferation
SCF = ligand, Kit = RTK

Vagal and sacral neural crest

-express RET
-found in ganglia of gut

cranial neural crest

nerves and facial structure (bone, muscle)

Cardiac neural crest

-heart forms near rhombomere 7 (posterior hindbrain)
-cardiac crest from r7 and anterior spinal chord
-cardiac NC migrate to form aortic arches
-this requires Pax3

Hox genes and neural crest cell behavior

-expresses Hoxa1 & Hoxb1
-NC origin in hindbrain
-hox genes needed for proper dev. of pharyngeal arch

where do placodes arise from

preplacodal ectoderm (PPE) from anterior neural plate
dlx3 expression defines this area

preplacodal specification steps

1. BMP establishes competence in ventral ectoderm

2. Fgf + anti-BMP from dorsal tissue induce PPE fate

Anterior placodes + pax6

olfactory & lens placode
induction of pax6 with DK1, cerberus, and frzb
pax6 suppressed by shh

lens placode

sequential activation for lens compentence
combinatorial signals to specify lens placode

lens→ retina
BMP induces Sox2
Fgf induces L-Maf

Nasal placode molecules

also requires Pax6
induced by Fgf from forebrain
-later shh

nasal placode physical development

nasal pit → olfactory epithelium with neurons → olfactory bulb
combinatorial activation

otic placode molecules

induced by fgf in hindbrain & mesoderm
-later maintained by wnt

otic placode physical development

invagination to form optic vesicle → neuroblast formation → delaminate and migrate to hindbrain → extend and innervate sensory hair cells

otic vesicle hair cells

arise from equivalence group expressing Atoh1

Trigeminal placode molecules

sensory neurons
wnt + fgf from midbrain/hindbrain induce

epibranchial placodes/vagus nerve

sensory ganglia
fgf from hindbrain and mesoderm

trigeminal & epibranchial placodes arise from

placodes (sensory)
neural tube (motor)
neural crest (schwann)