Vertebrate Retinal Histogenesis
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Transcript of Vertebrate Retinal Histogenesis
The neural retina contains seven major cell classes derived from the neuroepithelium
RodsCones
Ganglion Cells
Horizontal Cells
Bipolar Cells
Muller Glia
Amacrine Cells
Outer Nuclear Layer (ONL)
Inner Nuclear Layer (INL)
Ganglion Cell Layer (GCL)
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration
Progenitor cell
Post-mitotic differentiated cells
prototypical development of CNS tissue
Tissue formation (cell production, histogenesis) from the perspective of developmental time
1 2 3 4 5 6 7 8developmental time
Pulse-chase labeling paradigm
•Fix cells at selected timepoints, stain for BrdU
Birthdating of differentiated cells
Single exposure of 3H-thy (or BrdU)
3H-thy / BrdU
Birthdating of differentiated cells
Heavily labeled differentiated cells are considered to be born soon after time of nucleotide labeling
3H-thy / BrdU
Birthdating of differentiated cells
•Cells that exit cell cycle are heavily labeled•Cells that continue to proliferate dilute the label and are lightly labeled
Cells of each class are born in discrete intervals
2 phases of histogenesis in the rat retina
•RGCs, Cones, Horizontal, and Amacrine cells are generated in phase 1•Amacrine, Rods, Bipolar cells and Muller glia are generated in phase 2•Many more cells are generated in phase 2
Cell number production is highest from E17 through P5
•The order of temporal progression is conserved across vertebrates with
some exceptions, especially when comparing horizontal cells and cones.
•The extent of overlap can vary greatly. In fast developing organisms (frogs
and fish), overlap of cell class intervals is extensive. In birds and mammals,
overlap is less and is loosely correlated with gestation period.
•The relative ratios of cell classes varies between species, especially with
respect to rods and cones
•In amphibians and fish, histogenesis begins during embryogenesis, but
majority of growth occurs posthatch from ciliary margin zone
•In birds and mammals, histogenesis is restricted to embryonic and/or early
postnatal periods
Retinal histogenesis is more like the second model
1 2 3 4 5 6 7 8developmental time
Non-overlapping cell class generation
Overlapping cell class generation
How is timing of temporal progression maintained?
1 2 3cell cycle # 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8developmental time
Tissue formation (cell production, histogenesis) from the perspective of developmental time and CC
Blocking proliferation at early stage of development prior to cell fate commitment does not prevent formation of late cell
types in Xenopus embryos
Harris and Hartenstein (1991) Neuron 6:499-515
Pharmacological approach:
Hydroxyurea: blocks ribonucleoside diphosphate reductase which converts ribonucleotides to deoxynucleotides, which are necessary for DNA biosynthesis
Aphidicolin: inhibitor of DNA Polymerase-alpha
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1 2 3cell cycle # 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8developmental time
1cell cycle #
1 2 3 4 5 6 7 8developmental time
Cell fate is not dependent on the number of CCs a progenitor cell passes through in Xenopus
Normal development
Development during blocked cell division
Loss of Chx10 causes severe cell number deficits in the developing retina
The cell number defect of the Chx10 null retina is correlated with changes in retinal progenitor cell (RPC) proliferation and cell cycle dynamics, not
differentiation (or cell death)
Green et al (2003) Development 130:539-552
The cell number defect of the Chx10 null retina is correlated with changes in retinal progenitor cell (RPC) proliferation and cell cycle dynamics, not
differentiation (or cell death)
Model: Chx10 regulates CC time, not developmental time
1 2 3cell cycle # 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8developmental time
developmental time
1 2 3cell cycle # 4 5
1 2 3 4 5 6 7 8
Development in absence of Chx10
Normal development
Counting cell cycles does not appear to regulate the timing of temporal progression
Are RPCs multipotent or lineage restricted?
multipotent lineage restricted
retrovirusdextran dye
cell-specific Cre with floxed reporter
Birthdating of differentiated cells
3H-thy or BrdU
lineage-restricted progenitor
multipotent progenitor
Lineage analysis of progenitor cells
?
Determination of the lineage of a neuroblast in the rat retina
(A) A virus containing a functional -galactosidase gene is injected and after 4-6 weeks the retina is stained for the presence of the enzyme. (B) Stained cells forming a strip across the neural retina, including five rods, a rod terminal (t), and a Muller glia cell (mg).
Turner and Cepko (1987) Nature 328: 131
xenopus
mouse
Individual progenitors can give rise to multiple cell classes
Rat: Turner and Cepko (1987) Nature 328: 131Mouse:Xenopus:
retrovirus
lineage-restricted progenitor
multipotent progenitor
?
Are postnatal rat RPCs multipotent or lineage restricted?
retrovirus
lineage-restricted progenitor
multipotent progenitor
Result: Both lineages were observed
retrovirus
lineage-restricted progenitor
multipotent progenitors
As were 2 cell clones, each cell with a different fate
AND
How is multipotential character maintained?
Pax6 is required for the multipotent state of mouse RPCs
Conditional ablation of Pax6 in the peripheral retina at the optic cup stage causes severe defects in the generation of major retinal cell types
Marquardt et al. (2001) Cell 105: 43
Marquardt et al. (2001) Cell 105: 43
Model of requirement for Pax6 in regulating multipotential state
Early cell fate in the vertebrate retina is flexible and influenced by extrinsic factors
E15 retina cells labeled with thymidine and mixed with other E15 cells (isochronic) are not as likely to differentiate as rods. Instead, they primarily make retinal ganglion cells (RGC). If the same cells are mixed with P1 cells (heterochronic), they tend to differentiate as rods.
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•Reh shows similar results using heterochronic co-cultures of dissociated mouse and rat cells J.Neurobiology (1992) 23:1067-1083
•In contrast, Morrow et al observe no influence of P0 cells on E16 cells with respect to rod differentiation J. Neurosci (1998) 18:3738-3748
Watanabe and Raff (1990) Neuron 4:461Early retinal environment is inhibitory to rod differentiation (fewer late retinal cells expressed opsin if cultured in excess of early retinal cells (not specific to retinal cells - observed with other cells as well) (we don’t know what the late cells become e.g. other late cell type, blocked from maturation, early cell type
Adler and Hatlee (1989) Science 243:391-393Repka and Adler (1992) Dev Biol 153:242-2549Cell type produced by dissociated mitotic cell is not specified intrinsically but rather dictated by environment - isolate chick retinal cells give rise to photoreceptors regardless of age (tested up to E8) - argues for model that photoreceptor is default phenotype
Other data supporting the model that cell fate determination is dependent on extracellular environment
James et al (2003) J.Neurosci 23:8193-8203Used RGC markers to show that late rat RPCs can differentiate into RGCs if exposed to early cellular environment (E3 chick retinal cells/CM) (EXAMPLE OF RPCs HAVING POTENTIAL TO GO BACK IN LINEAGE)Also found that early environment (E3 chick retinal cells/CM) inhibits opsin expression in late RPCs
Belliveau and Cepko (1998) Development 124:1119-1131E16 rat RPCs cultured in excess of P0 cells inhibited amacrine cell production (NEGATIVE FEEDBACK MODEL)
Belliveau et al (2000) J Neurosci 20:2247-2254Confirms that early retinal environment is inhibitory to rod differentiation, and also observed an increase in bipolar cells
Sonic hedgehog promotes rod differentiation and progenitor proliferation in retinal cells
rhodopsin antibody:
Control +shh
Levine et al., 1997, J.Neuroscience 17: 6277
Other extrinsic factors that regulate cell fate determination in the vertebrate retina:retinoic acid
notch/delta FGFactivinTGF, lamininneurotrophins (NGF, BDNF, NT-3)CNTF
Differentiated cells feedback to limit cell fate/differentiation
mitotic progenitors
differentiating neurons & glia
Reh and Kljavin (1989) J. Neurosci 9:4179-4189Cell type produced by dissociated mitotic cell is reflective of age that cell is initially isolated - Argues for intrinsic information (used E14 and P1 rat cells)
Cayouette et al (2003) Neuron 40:897-904Clonal analysis of proliferation and differentiation in E16 rat explants and dissociated cellsResults: The 2 culture conditions generate very similar clones (in size and composition) even though the environments are very different
Data supporting the model that cell fate determination is programmed cell intrinsically
Alexaides and Cepko (1997) Development 124:1119-1131Early VC1.1+ rat RPCs generate amacrine and horizontal cells and VC1.1- RPCs generate conesLate VC1.1+ rat RPCs generate amacrine and rod cellsThese results argue that intrinsic differences correlate with bias of cell output
Model of temporal progression
Timing appears to be regulated by changes in both cell-extrinsic and cell-intrinsic factors and responses and by
negative feedback mechanisms
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration
Histogenesis occurs as a wave of differentiation from thecentral to peripheral retina
Histogenesis occurs as a wave of differentiation from thecentral to peripheral retina
mitotic progenitors
differentiating neurons & glia
Histogenesis occurs as a wave of differentiation from thecentral to peripheral retina
mitotic progenitors
differentiating neurons & glia
Major Questions
I. How is neurogenesis initiated?
II. How is wave propogated?
III. Is each wave of differentiation coordinated?
Sonic hedgehog drives a wave of neurogenesis across the retina (similar to the Drosophila retina)
Neumann and Nuesslein-Volhard, Science 289: 2137
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration
The CMZ recapitulates the cellular and molecularThe CMZ recapitulates the cellular and molecularmechanisms of embryonic developmentmechanisms of embryonic development
Perron et al., 1998
The juvenile chick retina contains a CMZThe juvenile chick retina contains a CMZ
Reh and Fischer, 2001
mammalian pigmented Ciliary Epithelium:mammalian pigmented Ciliary Epithelium:a source of in-vitro derived retinal stem cellsa source of in-vitro derived retinal stem cells
Tropepe et al., 2000
Adult fish retina:Adult fish retina:3 intrinsic neurogenic cell populations3 intrinsic neurogenic cell populations
CMZ stem cells are multipotentCMZ stem cells are multipotentStem cells in mature retina are lineage restrictedStem cells in mature retina are lineage restricted
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration
Retinal regeneration in fish:Retinal regeneration in fish:stem cell source intrinsic to retinastem cell source intrinsic to retina
physicalphysicallesionlesion
neurotoxicneurotoxiclesionlesion photocoagulationphotocoagulation
What is the source of the regenerated cells?What is the source of the regenerated cells?
The rod precursor lineage in the mature retina isThe rod precursor lineage in the mature retina isthe source of regenerated cellsthe source of regenerated cells
Adult amphibian retina:Adult amphibian retina:1 intrinsic neurogenic cell population1 intrinsic neurogenic cell population
Retinal regeneration in amphibians:Retinal regeneration in amphibians:RPE transdifferentiationRPE transdifferentiation
Chicken Müller glia:Chicken Müller glia:Cells with regenerative potentialCells with regenerative potential
Reh and Fischer, 2001
Comparison of retinal development and stem cells in vertebratesComparison of retinal development and stem cells in vertebrates
PrimarilyPrimarilyembryonicembryonic
YesYes(juvenile)(juvenile)
AmacrineAmacrineBipolarBipolar
LimitedLimited
Muller gliaMuller glia
BirdsBirds
ThroughoutThroughoutlifelife
YesYes
HighHigh
All typesAll types
RPERPE
AmphibiansAmphibians
ThroughoutThroughoutlifelife
YesYes
HighHigh
All typesAll types
Rod progenitorsRod progenitorsIntrinsic stem cellsIntrinsic stem cells
FishFish
DevelopmentalDevelopmentalperiodperiod
CMZCMZ
CMZ outputCMZ output
RegenerativeRegenerativepotentialpotential
Regenerated cellRegenerated cellsourcesource
PropertyProperty
EmbryonicEmbryonic&&
PostnatalPostnatal
NoNo
NoneNone
NoneNone
MammalsMammals
NoneNone
Overview of Histogenesis
I. Temporal progression
II. Spatial progression
III. Adult stem cells
IV. Regeneration