vertebrate retinal histogenesis

Vertebrate Retinal Histogenesis

Ed LevineHCI 3343

elevine@hci.utah.edu7-9537

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