EAST 501 Lecture Notes - Lecture 13: Spina Bifida, Cilium, Torban
21 Jun 2018
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12- E. Torban
18- Planar polarity and primary cilium: implications for development and disease
1- Planar cell polarity pathway
●Drosophila planar cell polarity
○Cartoon of a fruit fly wing.
○Each fruit fly wing has a single hair positioned at the most distant vertex of the cell, and
they are all oriented in the same way.
■Drosophila wing hairs are normally highly organized = planar polarity.
■Certain mutants, however, exhibit disorganization of hair patterns.
●Planar cell polarity in animals
○See quite well in vertebrates: organized direction of scales in fish and skin.
○In mice: organized hair growth.
■Mutants: lose this polarity, see swirls of hair.
○Almost all cells have PCP.
○In addition to apico-basal polarity, also have PCP (from left to right, proximal to distal).
●Planar cell polarity (PCP)
○The PCP pathway regulates:
■Uniform orientation of the cellular structures within the epithelial plane.
■Directional cellular movements.
■Oriented cell division.
■Tissue elongation known as convergent extension (lengthening in one direction
and narrowing in the perpendicular direction).
■Generation, orientation, and function of primary cilium.
●Drosophila planar cell polarity
○Among genes involved is the Van Gogh (strabismus).
●Wing hair PCP phenotypes
○Different kinds of mutants.
●Planar cell polarity
○Three groups of genes:
■Core PCP proteins: Frizzled, Van Gogh Flamingo, Dishevelled, Prickle, Diego
■Global PCP proteins: Fat, Dachsous, and Four-jointed
●Mutations in any of these two groups of proteins will lead to
consequences on the whole body.
■PCP effectors: Fuzzy, Inturned, Fritz, mwh
○Final read out = activation of multiple actin regulators → actin is rearranged in response
to activation of the planar cell polarity pathway.
●PCP signalling leads to the asymmetric distributions of core PCP proteins
○Gradient of Wnt ligand produces small concentration changes among proximal and distal
sides.
■Fz incorporates into distal side, Vang incorporates into proximal side.
■Dgo and Dsh accumulate with Fz, and Pk binds to Vang.
○Have complex Pk-Vang on one side: Pk can never go to the other side because will be
destined for degradation.
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12- E. Torban
■Extracellular domain of Fz binds to extracellular domain of Vang of the
neighboring cell.
●Mutations in Vangl2 cause a severe NTD phenotype in Looptail
mouse
○Most research has been done in Drosophila, but some research has been done on mice.
○For many years, were trying to find a gene mutated in the Looptail
mouse.
■Discovered it was one of the two homologues of the Van Gogh gene.
■Mice have a very characteristic loop tail when are heterozygous.
■Homozygous mice die early in gestation (neural tube is completely open).
■Mutation causes misfolding of the protein → no PCP.
●Neural tube formation
○Neural tube originates from the flat neural plate. Originally, the neural plate is flat, thick,
and short.
■PCP regulates the cell to acquire projections so the cell can move directionally
towards the center, and the neural plate becomes longer and narrower.
■The neural folds generated bend, meet, and fuse.
○In the looptail mice, the folds are too distant and never meet → the neural tube stays
open.
■Typical PCP defect (completely open neural tube seen in Vangl2, Flamingo3,
Dishevelled1/2, Frizzled3/6, homozygous mice).
●NTD patients have increased bone fractures
○10-30% of patients with NTD (spina bifida
) have statistically more frequent fracture
events comparing to general population matched by age and gender.
■Second most common congenital defect in humans = spina bifida.
○The fractures occurs from minor stresses.
○Many SB patients have paralysis or less activity. Thus, fractures were considered
secondary to altered activity and reduced vertical load.
■What if the mutants that cause NTD also predisposed to osteoporosis?
●Overlap between genes involved in NTD and bone formation
○Looked at the overlap of NTD genes with genes involved in bone development → found
46 genes.
●Osteoblast differentiation is accompanied by increase in PCP gene expression
○Prepared osteoblast cultures and differentiate them for 21 days.
○Can see that for Vangl2 and Celsr2 (vertebrate Flamingo)
■Increased PCP gene expression.
●Trabecular bone structure in Vangl2+/- mice
○Bone volume compared to tissue volume is decreased.
●Cortical bone thickness and cell orientation are abnormal in Vangl2+/- mice
○Thickness is slightly decreased, and more round than oval.
○Biggest difference is seen via EM: mineralized cells are much more organized in WT (in
parallel) compared to in the mutant (disorganized).
○SO in NTD patients with mutations in PCP → intrinsic properties of the gene may cause
these bone defects.
●Summary I
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12- E. Torban
○PCP is a fundamental property of most cells.
○PCP signalling controls asymmetric arrangement of cellular CSK. This enables cell shape
changes and collective cell movements required for tissue morphogenesis.
○Loss of PCP affects morphogenesis of many tissues including the neural tube and
skeleton.
2- Primary cilium
●Types of eukaryotic cilia and flagella
○In algae or yeast, have flagella.
○Cilia and flagella: either sensory/ perception functions, or motility functions.
■In lower eukaryotes: required for cell motility, mating.
■In multiciliated cells: lots of different roles for cilia.
●Photoreceptors: have three parts (outer segment where rhodopsin resides,
the inner segment, and in the middle, the connecting cilium → part
where traffic between OS and IS occurs.
●If proteins in this segment are mutated, patients progress to retinal
degeneration and blindness.
●Primary cilia in renal cells
○Primary cilium was first described in the studies of ultrastructure of kidney epithelial
cells in 1898.
■Small hair that protrudes into the lumen.
■Can be detected by EM or fluorescence microscopy.
●Structure of the primary cilium
○The structure of cilia is very specific: consists of a microtubular core (= axoneme) which
resides on the basal body.
○The basal body is stabilized by fibers and attached to the PM.
○The axoneme is covered by the ciliary membrane.
○Various molecules allow the cilia to be a sensing hub for the cell: sticks out into the
lumen and relays information about chemicals, growth factors, mechanosensation, and
osmolarity to the inside of the cell.
●Ciliogenic cues
○Ciliogenesis is tightly linked to the cell cycle and occurs in G0/G1.
■Centrioles= absolutely necessary for mitosis (serve as the MTOC).
○As renal epithelial cells differentiate, they become taller and are more crowded exerting
tensile tension on each other.
■Appears to provide an additional cue to initiate ciliogenesis.
●Motile vs. nonmotile (primary) cilium
○Both have nine pairs of microtubular duplets, but motile cilia also have a pair of singlets.
■That's why motile cilia is often described as 9+2, and sensory cilia as 9+0.
○Motile cilia: sperm, respiratory tract (trachea, bronchi, alveoli), fallopian tubes, lining of
the ventricles → important to generate a directional movement of CSF.
○Sensory cilia: renal epithelia, photoreceptors, olfactory neurons, kinocilium of the
cochlea hair cells.
●Classification of cilia based on axoneme shape
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Document Summary
18- planar polarity and primary cilium: implications for development and disease. Each fruit fly wing has a single hair positioned at the most distant vertex of the cell, and they are all oriented in the same way. Drosophila wing hairs are normally highly organized = planar polarity. Certain mutants, however, exhibit disorganization of hair patterns. See quite well in vertebrates: organized direction of scales in fish and skin. Mutants: lose this polarity, see swirls of hair. In addition to apico-basal polarity, also have pcp (from left to right, proximal to distal). Uniform orientation of the cellular structures within the epithelial plane. Tissue elongation known as convergent extension (lengthening in one direction and narrowing in the perpendicular direction). Generation, orientation, and function of primary cilium. Among genes involved is the van gogh (strabismus). Core pcp proteins : frizzled, van gogh flamingo, dishevelled, prickle, diego. Global pcp proteins : fat, dachsous, and four-jointed lead to these two groups of proteins will.
