Dev Neuro: Stem cells, neural induction, and neurulation pwpts

stem cells

• capable of self renewal

• immortal (propagate indefinitely)

• high developmental potential

How are ESCs cultured?

on feeder layers made with ECM components and peptides

Molecular reprogramming goal

Manipulate somatic cell’s potency

1 mature cell type to another

transdifferentiation

mature cell to stem cell state

reprogramming

mature cell to intermediate state

dedifferentiation

region required to drive expression in specific area

regulatory region

why generate pluripotent stem cell lines from adult somatic cells?

• ethical issues with using embryonic stem cells

• Patient-specific stem cell lines preventing immune rejection

organoids

1. multiple organ-specific cell types

2. capable of recapitulating specific function of organ

3. spatially organized like organ

Induction of pluripotent stem cells experiment

1. Identified 24 genes exclusively expressed in stem cells

2. no single gene capable of reprogramming fibroblast cell to ESC

3. Expression of all 24 genes reprogrammed cell to ESC

4. Removed 1/24 genes for each —> 4 genes allowed for reprogramming

5. Test for pluripotency via chimera + teratoma

Therapeutic potentials for organoids

• disease modeling

• drug safety/efficacy testing

• organ replacement therapy

Zika infection effects

decrease in cortical and progenitor cells

A specialized, transient group of cells in the early embryo that instructs neighboring cells to adopt specific fates, inducing and patterning the body axis and nervous system

Organizer

How would you determine whether the same protein is secreted by Hanson’s node as is secreted by Spemann’s organizer?

Transplant organizers across species to see if same result ensues —> if so, molecules conserved

How was neural tissue determined to be default?

Animal cap not exposed to mesoderm removed —> cells dispersed and formed neural tissue, cells kept together and formed epidermis

Factor that differentiates ectoderm into epidermis

BMP (bone morphogenic factor)

TGF-β signaling pathway

Paracrine factor BMP —> homodimerizes + autophosphorylates —> activates Smad proteins creating Smad complex —> acts as TF to create proteins differentiating ectoderm to epidermis

Noggin and Chordin

BMP inhibitor secreted by Spemann’s organizer allowing differentiation of ectoderm into neural tissue

necessary condition

is it required to do something?

sufficient condition

a factor that alone is enough to produce the effect

How to test if a protein is sufficient?

Gain-of-function assay

How was it tested that Noggin is sufficient to induce neural tissue?

• Noggin mRNA injected into ventral side (gain-of-function assay)—> 2-headed embryo ensued

• Noggin mRNA injected into dorsal side —> larger head and CNS

How to test if a protein is necessary?

loss-of-function assay

How was it tested if Noggin and Chordin are necessary for neural tissue formation?

Loss-of-function assay —> severe head defects when both removed, but barely any changes when only one or the other removed

Neurulation

formation and closing of neural tube

cell-shape change in development where the apical (top) surface of a cell contracts, turning columnar epithelial cells into a wedge or bottle shape

apical constriction

tissue narrows (converges) along one axis and simultaneously elongates (extends) in the perpendicular axis

convergent extension

hinge points

• formed by apical constriction

• make bending possible

• where cells change shape (to small triangular prisms rather than cylindrical)

ring-like bundle of actin filaments located just beneath the plasma membrane in epithelial cells at APICAL SIDE, usually associated with adherens junctions, structure maintains cell shape and stabilizes cell-cell junctions

Actin belt

contractile, ring-like structure composed of both actin filaments and myosin II motor proteins that actively constricts

Actomyosin belt

thin, broad, sheet-like protrusions at the leading edge of moving cells, acting as the "motor" for cell migration, rich with actin to help with movement

Lamellipodia

thin, spike-like, sensory projections for environmental navigation

Filipodia

What is polarization of cells dependent on?

PCP (planar cell polarity) pathway

PCP pathway

1. Wnt activates Frizzled transmembrane protein

2. Disheveled activated and activates Rho-Rac pathway

3. Changes cytoskeleton helping direct cell movement

essential for convergent extension

What happens to PCP mutants?

Cannot undergo convergent extension since cells not polarized

How is the neural tube closed?

1. Fusion of folds

2. Separation from ectoderm

What is cell adhesion necessary for?

closure and separation of tissues in neurulation

Morpholino knockdown

antisense oligonucleotide that binds to mRNA and prevents binding or ribosome + translation (can also be used at splice junction)

How was it shown N-cadherin is necessary for neural tube closure?

• N-cadherin deleted (loss-of-function) via MO

• Blocked neural fold closure and spina bifida ensued

Spina Bifida

issues with posterior NT closure

Anecephaly

issues with anterior NT closure

contractile protein complex composed of actin and myosin, acting as the fundamental molecular motor for muscle contraction and various cell motility processes

actomyosin

What drives invagination (apical constriction)?

actomyosin restriction

What directs convergence and extension

Wnt/PCP pathway

What is responsible for the fusion of the converging neural folds?

Cadherin and other adhesive interactions

short sequences fused onto a protein for antibody assays

Epitope tags

how to see where protein located on tissue

immunohistochemistry