Spécification cellulaire: développement de la rétine. Michel Cayouette Neurobiologie Cellulaire IRCM

Transcription

1 Spécification cellulaire: développement de la rétine Michel Cayouette Neurobiologie Cellulaire IRCM

2 How is cell diversity generated during CNS development?? Intrinsic vs Environmental Fundamental importance Stem cell therapy Source : Alberts et al., MBOC 4th edition, Malatesta et al., 2003

3 The vertebrate retina: A model system to study cell fate determination mechanisms

4 The retina is an extension of the neural tube surface ectoderm neuroepithelium basal RPE: retinal pigment epithelium apical retina lens -> A series of inductive events (influence of of one region of the embryo on the development of a nearby region) guide eye development -> The only part of the CNS that can be directly visualized without the need of invasive methods!

5 The retina develops from the anterior part of the optic cup? lens E9.5 P10 developmental period in rodents

6 Progression of retinal development RPE e11 E18 retina NBL lens GCL P4 P7 ONL ONL NBL INL INL GCL GCL

7 Basic organization of the vertebrate retina Advantages: 1) Simple layered structure 2) Manageable number of cell types 3) Cell-specific markers for all cell types 4) Easily accessible 5) Two eyes

8 Basic organization of the vertebrate retina Advantages: 1) Simple layered structure 2) Manageable number of cell types 3) Cell-specific markers for all cell types 4) Easily accessible 5) Two eyes Photoreceptors Bipolars Amacrines

9 1) Glaucoma Major diseases of the retina Retinal ganglion cell degeneration (optic nerve compression?) 2) Retinitis pigmentosa Degeneration of rods followed by degeneration of cones (dominant, recessive, and X- linked forms) 3) Age-related macular degeneration (AMD) Degeneration of the cones in macula (unknown pathogenesis, some genetic contribution) 4) Stargardt macular dystrophy Degeneration of cones (autosomal recessive) 5) Leber congenital amaurosis Childhood blindness (genetic disease - abnormal development of photoreceptors or very early degeneration) 6) Usher syndrome Hearing and vision loss (retinitis pigmentosa) - autosomal recessive disease

10 Retinal progenitor cells proliferate and give rise to retinal cell types

11 Cell lineage and cell genesis Question: Are retinal progenitor cells unipotent or multipotent? Retinal Progenitor Cell Labeling? Progenitor Neuron or glia

12 Classic studies: Turner and Cepko, Nature, 1987 (retroviral vectors) Holt et al., Neuron, 1988 (cell tracer) Wetts and Fraser, Science, 1988 (cell tracer) Using retroviral vectors to study cell lineage: -Infect only dividing cells -Replication incompetent -Integrates host cell s genome -Reporter gene (PLAP, GFP, etc.) Early infection (e14): Large clones, many cell types Late infection (P2): Small clones, few cell types 4 Rods 1 Müller 1 Bipolar 2 Rods 1 Bipolar 12 Rods 1 Müller 1 Bipolar 2 Amacrines

13 General conclusions from these studies: - There are no restricted retinal progenitors - Retinal progenitor cells are multipotent (young progenitors have more potential than late progenitors) - Late progenitors have lost the capacity to generate early-born cells (i.e. no early-born cells in clones generated from progenitors labeled late) Questions: How does the fate of daughter cells produced by progenitor cells change over time? How do progenitors give rise to the large number of clonal composition?

14 The order of cell birth in the retina Early Late Adapted from Rapaport et al., J. Comp. Neurol., 2004 How is the temporal order established?

15 Cell fate determination Extrinsic signaling Feedback inhibition signals?

16 Ganglion cells are the first cell types to be generated Question: Is it possible that, as the retina matures, the cellular environment changes such that it is not conducive to ganglion cell determination? Waid and McLoon, Development, 1998

17 Waid and McLoon, 1998 Ganglion cell production is inhibited by late retinal cell population Depletion of ganglion cells from the late population abolishes this inhibitory effect, indicating that ganglion cells secrete signals to prevent generation of too many ganglion cells -> several studies later demonstrated that the signals secreted by RGCs is Shh, which promotes proliferation of progenitors and blocks differentiation into RGCs.

18 Intrinsic signaling Question: Are retinal progenitor cells intrinsically biased to generate particular cell types at any one time? Experiment 1: Can the early environment force late progenitors to make early-born cells? (Belliveau et al., 2000) Experiment 2: Can the late environment force early progenitors to make late-born cells? (Watanabe and Raff, 1992) P0 progenitor (test population) E15 progenitor (test population) E14 retinal cells P0 retinal cells -No change in cell fate decisions in the test (labeled) population of progenitors -Retinal progenitors show intrinsic limitations in the production of cell types

19 How is a given differentiation program turned on at a specific time?

20 Temporal identity factors in Drosophila neuroblasts Hunchback is both necessary and sufficient for the production of earlyborn neurons Pearson and Doe., Ann Rev Cell Dev Biol 20:619

21 Ikaros: a Hunchback ortholog Zinc finger protein (DNA binding and protein-protein interactions) 4 other family members (Aiolos, Helios, Eos, Pegasus)

22 Hypothesis Ikaros confers early temporal competence to retinal progenitor cells Predictions: 1) Should be expressed in retinal progenitors at the time early temporal competence is specified. 2) Knockout mutants should show a decrease or absence of early born cell types 3) Misexpression at late stages should be sufficient to induce the production of early-born cell types

23 Ikaros expression is temporally regulated in retinal progenitor cells E12 E14 E17 P0 P2 Adult Present - Early progenitors - Early-born postmitotic neurons Absent - Late progenitors - Late-born postmitotic neurons

24 Working Model

25 Adult Ikaros KO retinas have fewer early-born cells WT 1- Retinal Ganglion Cells KO RGCs (Brn3B +) H/E Brn3B 2- Amacrine cells WT KO 3- Horizontal cells Pax6

26 Adult Ikaros KO retinas have normal numbers of lateborn cells WT IK -/- Also, no change in photoreceptor cell number

27 Cell death is not affected in IK KO retinas WT Embryonic Day 16 KO TUNEL (No difference at E13 and P1)

28 P Should be expressed in retinal progenitors at the time early temporal competence is specified. - Expressed in early, but not late, retinal progenitors - Expressed in Horizontal, Amacrine, Ganglion Cells (early-born cell types) P Knockout mutants should show a decrease or absence of early-born cell types - IK KO show a decrease in amacrine, horizontal, and retinal ganglion cell production - Possibility of compensation by other family members Misexpression at late stages should be sufficient to induce the production of early-born cell types

29 Ikaros misexpression in late retinal progenitors with retroviral vectors Subretinal injections PLAP activity in infected clones PLAP IKAROS-PLAP 20 days P1 mouse

30 Ikaros misexpression in late progenitors affects clonal composition Photoreceptor Photoreceptor Control PLAP Muller Bipolar Amacrine Muller Ikaros PLAP Bipolar Amacrine Horizontal Amacrine Horizontal Bipolar

31 Ikaros misexpression in late progenitors induces production of early-born neurons

32 Ikaros misexpression in late progenitors is sufficient to confer competence to generate early-born neurons

33 Ikaros misexpression in late progenitors induces generation of early-born ganglion cells P0 retinal progenitor cells Infected with GFP or Ikaros-GFP retrovirus 4 days in culture Analyze clones GFP+ / Brn3B+ 16 Percent of Total Brn3B (0) 0 GFP Ikaros-GFP

34 P Should be expressed in retinal progenitors at the time early temporal competence is specified. - Expressed in early, but not late, retinal progenitors - Expressed in Horizontal, Amacrine, Ganglion Cells (early-born cell types) P P Knockout mutants should show a decrease or absence of early-born cell types - IK KO show a decrease in amacrine, horizontal, and retinal ganglion cell production - Possibility of compensation by other family members Misexpression at late stages should be sufficient to induce the production of early-born cell types - Misexpression in late progenitors induces early-born neuron production

35 Conclusions Ikaros expression is temporally controlled in developing retinal progenitors (present early, absent late) Misexpression of Ikaros in late progenitors is sufficient to confer back the competence to generate early-born cell types Ikaros expression in developing retinal progenitor cells is required for the normal production of early-born cell types specifically Model

36 Conservation or coincidence? Chris Doe and colleagues

37 Vertebrate castor (Casz1) Single gene in mouse and human Zinc finger transcription factor Required for vascular/cardiac development in Xenopus Function in the nervous system remains unknown

38 Casz1 is expressed in mid/late stage progenitors in mouse retinogenesis Mattar et al., Neuron, 2015

39 Casz1 loss-of-function Mattar et al., Neuron, 2015

40 Casz1 inactivation increases early-born fates at the expense of mid/late fates Mattar et al., Neuron, 2015

41 Casz1 overexpression increases mid/late-born fates at the expense of early-born fates Mattar et al., Neuron, 2015

42 Model A conserved regulatory logic controls temporal patterning in the retina Mattar et al., Neuron, 2015

43 Asymmetric cell division: a conserved mechanism to generate cell diversity A B

44 Essential steps to generating an asymmetric cell division: lessons from invertebrates extrinsic intrinsic

45 Generating an asymmetric cell division 1. Setting-up the axis of polarity The Par polarity complex In mutants for either Par3, Par6, or apkc, the others mislocalize, spindles are randomly oriented and cell fate determinants are uniformly distributed

46 Generating an asymmetric cell division 2. Regulating asymmetric protein localization The apkc-lgl-myosin pathway - Par-3/6 locally inactivates Lgl by apkc-dependent phosphorylation - Unphosphorylated (active) Lgl inhibits Myosin II on the basal cell cortex, whereas apical Myosin II is active and prevents accumulation of determinants - Myosin VI (purple) promotes asymmetric localization of segregating determinants by vesicular transport.

47 Differential inheritance of cell fate determinants instructs asymmetric outcomes Numb is necessary and sufficient for sensory organ precursor asymmetric division Hair + Socket cell Neuron + Sheath cell Rhyu et al., Cell, 1994

48 Generating an asymmetric cell division 3. Regulating mitotic spindle orientation The Insc-Pins-Gαi-Mud pathway Bowman et al., Dev. Cell, 2006

49 Asymmetric cell divisions in vertebrates

50 The study that started it all Chenn and McConnell, Cell, 1995

51 Live imaging provides direct evidence for varying division plane in the developing cortex Chenn and McConnell, Cell, 1995

52 Asymmetric cell divisions in the neocortex a limited role for division orientation WT minsc O/E LGN KO Postiglione et al., Neuron, 2011

53 Asymmetric divisions in the retina: possible outcomes Diversify progenitor pool Difficult to study Steady increase in progenitors (self-renewal) Stem cell mode of division Increase diversity of postmitotic cell types

54 Variable division planes in the developing retina Horizontal Intermediate Vertical metaphase NE Plane anaphase NE Plane γ tubulin DNA Cayouette et al., J. Neurosci., 2001

55 The proportion of vertical divisions changes over time during development

56 Cell division orientation correlates with cell fate apical apical Cayouette and Raff, Development, 2003

57 Numb Homologs in Vertebrates PRD PTB: Phospho Tyrosine Binding Domain PRD: ProlineRich Domain Q: Poly-Glutamine Repeats PRD Notch signalling inhibitor Endocytosis Proteasome-mediated degradation PRD Zhong et al, Development, Studies of Numb/NbL function in cortical development report conflicting results -It remains unknown whether Numb actually regulates asymmetric cell divisions in situ

58 Numb localizes asymmetrically in retinal progenitors Kechad, Jolicoeur, et al., J. Neurosci., 2012

59 Numb is inherited asymmetrically in some retinal progenitor cell divisions Kechad, Jolicoeur, et al., J. Neurosci., 2012

60 Studying Numb function in asymmetric divisions: clonal inactivation - Study cell autonomous function - Allow temporal-specific inactivation - Lineage analysis

61 Numb is not required for self-renewing asymmetric cell divisions at early stages Kechad, Jolicoeur, et al., J. Neurosci., 2012

62 Numb is required for terminal asymmetric cell divisions at late stages CLE CLE-Cre asymmetric terminal symmetric terminal D % of 2-cell clones 100% 80% 60% 40% 20% 0% ** Pr + Pr 12% 8% 4% 0% * Pr + Amacrine *** Pr + Bipolar CLE CLE-Cre ** Pr + Müller Kechad, Jolicoeur, et al., J. Neurosci., 2012

63 Possible models of Numb function in terminal asymmetric cell divisions Prediction: Numb GOF should increase non-photoreceptor fate Prediction: Numb GOF should increase symmetric and reduce asymmetric terminal divisions

64 Numb gain-of-function increases symmetric terminal divisions at the expense of asymmetric terminal divisions (Ph + Ph) (Ph + X) àsame phenotype as Numb loss-of-function!! àhow do we reconcile these findings?

65 A suggested model for Numb function in retinal development Numb GOF N A N A + symmetric Notch (low) = symmetric fates

66 Notch signaling is increased in Numb/NbL cdko retinas control cdko Notch-ICD

67 Blocking Notch signaling phenocopies Numb LOF and GOF

68 Model