Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

Eagleson, G., Ferreiro, B. and Harris, W. A. (1995). Fate of the anterior neural ridge and the morphogenesis of the Xenopus forebrain. J. Neurobiol, 28, 146–58.

Ekker, S. C., Ungar, A. R., Greenstein, P. et al. (1995). Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr. Biol., 5, 944–55.

Esteve, P., Lopez-Rios, J. and Bovolenta, P. (2004). SFRP1 is required for the proper establishment of the eye field in the medaka fish. Mech. Dev., 121, 687–701.

Feldman, B., Gates, M. A., Egan, E. S. et al. (1998). Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature, 395, 181–5.

Fernandez-Garre, P., Rodriguez-Gallardo, L., Gallego-Diaz, V., Alvarez, I. S. and Puelles, L. (2002). Fate map of the chicken neural plate at stage 4. Development, 129, 2807–22.

Gallagher, B. C., Hainski, A. M. and Moody, S. A. (1991). Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus.

Development, 112, 1103–14.

Goudreau, G., Petrou, P., Reneker, L. W. et al. (2002). Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.

Proc. Natl. Acad. Sci. U. S. A., 99, 8719–24.

Grinblat, Y., Gamse, J., Patel, M. and Sive, H. (1998). Determination of the zebrafish forebrain: induction and patterning. Development, 125, 4403–16.

Grindley, J. C., Davidson, D. R. and Hill, R. E. (1995). The role of Pax-6 in eye and nasal development. Development, 121, 1433–42.

Gritsman, K., Zhang, J., Cheng, S. et al. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell, 97, 121–32.

Halder, G., Callaerts, P. and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science, 267, 1788–92.

Halder, G., Callaerts, P., Flister, S. et al. (1998). Eyeless initiates the expression of both sine oculis and eyes absent during Drosophila compound eye development.

Development, 125, 2181–91.

Hammerschmidt, M., Pelegri, F., Mullins, M. C. et al. (1996). Mutations affecting morphogenesis during gastrulation and tail formation in the zebrafish, Danio rerio.

Development, 123, 143–51.

Harrelson, Z., Kelly, R. G., Goldin, S. N. et al. (2004). Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development, 131, 5041–52.

Hatta, K., Kimmel, C. B., Ho, R. K. and Walker, C. (1991). The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system. Nature, 350, 339–41.

Heisenberg, C. P. and Nusslein-Volhard, C. (1997). The function of silberblick in the positioning of the eye anlage in the zebrafish embryo. Dev. Biol., 184, 85–94.

Heisenberg, C. P., Brand, M., Jiang, Y. J. et al. (1996). Genes involved in forebrain development in the zebrafish, Danio rerio. Development, 123, 191–203.

Heisenberg, C. P., Houart, C., Take-Uchi, M. et al. (2001). A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. Genes Dev., 15, 1427–34.

Hill, R. E., Favor, J., Hogan, B. L. et al. (1991). Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature, 354, 522–5.

Hirsch, N. and Harris, W. A. (1997). Xenopus Pax-6 and retinal development. J. Neurobiol., 32, 45–61.

Hollemann, T., Bellefroid, E. and Pieler, T. (1998). The Xenopus homologue of the Drosophila gene tailless has a function in early eye development. Development, 125, 2425–32.

Houart, C., Westerfield, M. and Wilson, S. W. (1998). A small population of anterior cells patterns the forebrain during zebrafish gastrulation. Nature, 391, 788–92.

Houart, C., Caneparo, L., Heisenberg, C. et al. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron, 35, 255–65.

Hsu, D. R., Economides, A. N., Wang, X., Eimon, P. M. and Harland, R. M. (1998). The Xenopus dorsalizing factor Gremlin identifies a novel family of secreted proteins that antagonize BMP activities. Mol. Cell, 1, 673–83.

Huang, S. and Moody, S. A. (1993). The retinal fate of Xenopus cleavage stage progenitors is dependent upon blastomere position and competence: studies of normal and regulated clones. J. Neurosci., 13, 3193–210.

Inoue, T., Nakamura, S. and Osumi, N. (2000). Fate mapping of the mouse prosencephalic neural plate. Dev. Biol., 219, 373–83.

Isaacs, H. V., Andreazzoli, M. and Slack, J. M. (1999). Anteroposterior patterning by mutual repression of orthodenticle and caudal-type transcription factors. Evol. Dev., 1, 143–52.

Kimmel, C. B., Warga, R. M. and Schilling, T. F. (1990). Origin and organization of the zebrafish fate map. Development, 108, 581–94.

Klein, P. S. and Melton, D. A. (1996). A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. U. S. A., 93, 8455–9.

Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell, 75, 1431–44.

Kumar, J. P. and Moses, K. (2001). EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification. Cell, 104, 687–97.

Kuroda, H., Wessely, O. and Robertis, E. M. (2004). Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus. PLoS Biol., 2, E92.

Lagutin, O., Zhu, C. C., Furuta, Y. et al. (2001). Six3 promotes the formation of ectopic optic vesicle-like structures in mouse embryos. Dev. Dyn., 221, 342–9.

Lagutin, O. V., Zhu, C. C., Kobayashi, D. et al. (2003). Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. Genes Dev., 17, 368–79.

Lewis, W. H. (1907). Experiments on the origin and differentiation of the optic vesicle in amphibia. Am. J. Anat., 7, 259–76.

Li, H. S., Yang, J. M., Jacobson, R. D., Pasko, D. and Sundin, O. (1994). Pax-6 is first expressed in a region of ectoderm anterior to the early neural plate: implications for stepwise determination of the lens. Dev. Biol., 162, 181–94.

Li, H., Tierney, C., Wen, L., Wu, J. Y. and Rao, Y. (1997). A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate.

Development, 124, 603–15.

Li, X., Perissi, V., Liu, F., Rose, D. W. and Rosenfeld, M. G. (2002). Tissue-specific regulation of retinal and pituitary precursor cell proliferation. Science, 297, 1180–3.

Logan, C. Y. and Nusse, R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol., 20, 781–810.

Loosli, F., Winkler, S. and Wittbrodt, J. (1999). Six3 over-expression initiates the formation of ectopic retina. Genes Dev., 13, 649–54.

Loosli, F., Winkler, S., Burgtorf, C. et al. (2001). Medaka eyeless is the key factor linking retinal determination and eye growth. Development, 128, 4035–44.

Loosli, F., Staub, W., Finger-Baier, K. C. et al. (2003). Loss of eyes in zebrafish caused by mutation of chokh/rx3. EMBO Rep., 4, 894–9.

Lopashov, G. V. and Stroeva, O. G. (1964). Development of the Eye; Experimental Studies. New York, NY: D. Davey.

Macdonald, R., Barth, K. A., Xu, Q. et al. (1995). Midline signaling is required for Pax gene regulation and patterning of the eyes. Development, 121, 3267–78.

Mangold, O. (1931). Das Determinationsproblem. III. Das Wirbeltierauge in der Entwicklung und Regeneration. Ergeb Biol., 7, 196–403.

Mao, B., Wu, W., Li, Y. et al. (2001). LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature, 411, 321–5.

Marlow, F., Zwartkruis, F., Malicki, J. et al. (1998). Functional interactions of genes mediating convergent extension, knypek and trilobite, during the partitioning of the eye primordium in zebrafish. Dev. Biol., 203, 382–99.

Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx homeobox gene is essential for vertebrate eye development. Nature, 387, 603–7.

Maurus, D., Heligon, C., Burger-Schwarzler, A., Brandli, A. W. and Kuhl, M. (2005). Noncanonical Wnt-4 signaling and EAF2 are required for eye development in

Xenopus laevis. EMBO J., 24, 1181–91.

Mikkola, I., Bruun, J. A., Holm, T. and Johansen, T. (2001). Superactivation of Pax6-mediated transactivation from paired domain-binding sites by DNA-independent recruitment of different homeodomain proteins. J. Biol. Chem., 276, 4109–18.

Moens, C. B., Yan, Y. L., Appel, B., Force, A. G. and Kimmel, C. B. (1996). valentino: a zebrafish gene required for normal hindbrain segmentation. Development, 122, 3981–90.

Moody, S. A. (1987). Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev. Biol., 122, 300–19.

Moore, K. B. and Moody, S. A. (1999). Animal-vegetal asymmetries influence the earliest steps in retina fate commitment in Xenopus. Dev. Biol., 212, 25–41.

Muller, F., Albert, S., Blader, P. et al. (2000). Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS.

Development, 127, 3889–97.

Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene ‘knockdown’ in zebrafish.

Nat. Genet., 26, 216–20.

Nieuwkoop, P. D. (1963). Pattern formation in artificially activated ectoderm (Rana pipiens and Ambystoma punctatum). Dev. Biol., 7, 255–79.

Nieuwkoop, P. D. and Nigtevecht, G. V. (1954). Neural activation and transformation in explants of competent ectoderm under the influence of fragments of anterior notochord in urodeles. J. Embryol. Exp. Morphol., 2, 175–93.

Nieuwkoop, P. D., Botterenbrood, E. C., Kremer, A. et al. (1952). Activation and organization of the central nervous system in Amphibians. J. Exp. Zool., 120, 1–108.

Nomura, M. and Li, E. (1998). Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature, 393, 786–90.

Odenthal, J. and Nusslein-Volhard, C. (1998). Fork head domain genes in zebrafish. Dev. Genes Evol., 208, 245–58.

Ohuchi, H., Tomonari, S., Itoh, H., Mikawa, T. and Noji, S. (1999). Identification of chick rax/rx genes with overlapping patterns of expression during early eye and brain development. Mech. Dev., 85, 193–5.

Oliver, G., Mailhos, A., Wehr, R. et al. (1995). Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development, 121, 4045–55.

Oliver, G., Loosli, F., Koster, R., Wittbrodt, J. and Gruss, P. (1996). Ectopic lens induction in fish in response to the murine homeobox gene Six3. Mech. Dev., 60, 233–9.

Papaioannou, V. E. (2001). T-box genes in development: from hydra to humans. Int. Rev. Cytol., 207, 1–70.

Pera, E. M. and Kessel, M. (1997). Patterning of the chick forebrain anlage by the prechordal plate. Development, 124, 4153–62.

Perron, M., Boy, S., Amato, M. A. et al. (2003). A novel function for Hedgehog signaling in retinal pigment epithelium differentiation. Development, 130, 1565–77.

Piccolo, S., Agius, E., Leyns, L. et al. (1999). The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature, 397, 707–10.

Pignoni, F., Hu, B., Zavitz, K. H. et al. (1997). The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell, 91, 881–91.

Pogoda, H. M., Solnica-Krezel, L., Driever, W. and Meyer, D. (2000). The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation. Curr. Biol., 10, 1041–9.

Porter, F. D., Drago, J., Xu, Y. et al. (1997). Lhx2, a LIM homeobox gene, is required for eye, forebrain, and definitive erythrocyte development. Development, 124, 2935–44.

Punzo, C., Plaza, S., Seimiya, M. et al. (2004). Functional divergence between eyeless and twin of eyeless in Drosophila melanogaster. Development, 131, 3943–53. Erratum in Development 2004 Sep, 131, 4635.

Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans. Science, 265, 785–9.

Rasmussen, J. T., Deardorff, M. A., Tan, C. et al. (2001). Regulation of eye development by frizzled signaling in Xenopus. Proc. Natl. Acad. Sci. U. S. A., 98, 3861–6.

Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B. (1998). Cyclops encodes a nodal-related factor involved in midline signaling. Proc. Natl. Acad. Sci. U. S. A., 95, 9932–7.

Richard-Parpaillon, L., Heligon, C., Chesnel, F., Boujard, D. and Philpott, A. (2002). The IGF pathway regulates head formation by inhibiting Wnt signaling in Xenopus. Dev. Biol., 244, 407–17.

Roessler, E., Belloni, E., Gaudenz, K. et al. (1996). Mutations in the human Sonic Hedgehog gene cause holoprosencephaly. Nat. Genet., 14, 357–60.

Saha, M. S. and Grainger, R. M. (1992). A labile period in the determination of the anterior-posterior axis during early neural development in Xenopus. Neuron, 8, 1003–14.

Sampath, K., Rubinstein, A. L., Cheng, A. M. et al. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signaling. Nature, 395, 185–9.

Seimiya, M. and Gehring, W. J. (2000). The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism. Development, 127, 1879–86.

Seo, H. C., Drivenes, O. Ellingsen, S. and Fjose, A. (1998). Expression of two zebrafish homologs of the murine Six3 gene demarcates the initial eye primordia. Mech. Dev., 73, 45–57.

Shimamura, K. and Rubenstein, J. L. (1997). Inductive interactions direct early regionalization of the mouse forebrain. Development, 124, 2709–18.

Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and Rubenstein, J. L. (1995). Longitudinal organization of the anterior neural plate and neural tube. Development, 121, 3923–33.

Shinya, M., Eschbach, C., Clark, M., Lehrach, H. and Furutani-Seiki, M. (2000). Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate. Mech. Dev., 98, 3–17.

Silva, A. C., Filipe, M., Kuerner, K. M., Steinbeisser, H. and Belo, J. A. (2003). Endogenous Cerberus activity is required for anterior head specification in Xenopus.

Development, 130, 4943–53.

Song, J., Oh, S. P., Schrewe, H. et al. (1999). The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice. Dev. Biol., 213, 157–169.

Spemann, H. (1901). Uber Correlationen in der Entwickelung des Auges. Verh. Anat. Ges., 15, 61–79.

Stenman, J., Yu, R. T., Evans, R. M. and Campbell, K. (2003). Tlx and Pax6 co-operate genetically to establish the pallio-subpallial boundary in the embryonic mouse telencephalon. Development, 130, 1113–22.

Stern, C. D. (2002). Induction and initial patterning of the nervous system – the chick embryo enters the scene. Curr. Opin. Genet. Dev., 12, 447–51.

Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A. and Stern, C. D. (2000). Initiation of neural induction by FGF signaling before gastrulation. Nature, 406, 74–8.

Takabatake, Y., Takabatake, T., Sasagawa, S. and Takeshima, K. (2002). Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development. Dev. Growth Differ., 44, 257–71.

Toy, J. and Sundin, O. H. (1999). Expression of the optx2 homeobox gene during mouse development. Mech. Dev., 83, 183–6.

Toy, J., Yang, J. M., Leppert, G. S. and Sundin, O. H. (1998). The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes. Proc. Natl. Acad. Sci. U. S. A., 95, 10643–8.

Tucker, P., Laemle, L., Munson, A. et al. (2001). The eyeless mouse mutation (ey1) removes an alternative start codon from the Rx/rax homeobox gene. Genesis, 31, 43–53.

Uren, A., Reichsman, F., Anest, V. et al. (2000). Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling. J. Biol. Chem., 275, 4374–82.

van de Water, S., van de Wetering, M. Joore, J. et al. (2001). Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant. Development, 128, 3877–88.

Varga, Z. M., Wegner, J. and Westerfield, M. (1999). Anterior movement of ventral diencephalic precursors separates the primordial eye field in the neural plate and requires cyclops. Development, 126, 5533–46.

Viczian, A. S., Bang, A. G., Harris, W. A. and Zuber, M. E. (2006). Expression of Xenopus laevis Lhx2 during eye development and evidence for divergent expression among vertebrates. Dev. Dyn., 235, 1133–41.

Voronina, V. A., Kozhemyakina, E. A., O’Kernick, C. M. et al. (2004). Mutations in the human RAX homeobox gene in a patient with anophthalmia and sclerocornea. Hum. Mol. Genet., 13, 315–22.

Walther, C. and Gruss, P. (1991). Pax-6, a murine paired box gene, is expressed in the developing CNS. Development, 113, 1435–49.

Wargelius, A., Seo, H. C., Austbo, L. and Fjose, A. (2003). Retinal expression of zebrafish six3.1 and its regulation by Pax6. Biochem. Biophys. Res. Commun., 309, 475–81.

Wawersik, S. and Maas, R. L. (2000). Vertebrate eye development as modeled in Drosophila. Hum. Mol. Genet., 9, 917–25.

Wilson, S. I. and Edlund, T. (2001). Neural induction: toward a unifying mechanism. Nat. Neurosci., 4 (Suppl.), 1161–8.

Wilson, S. I., Graziano, E., Harland, R., Jessell, T. M. and Edlund, T. (2000). An early requirement for FGF signaling in the acquisition of neural cell fate in the chick embryo. Curr. Biol., 10, 421–9.

Winkler, S., Loosli, F., Henrich, T., Wakamatsu, Y. and Wittbrodt, J. (2000). The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye. Development, 127, 1911–19.

Woo, K. and Fraser, S. E. (1995). Order and coherence in the fate map of the zebrafish nervous system. Development, 121, 2595–609.

Yu, R. T., Chiang, M. Y., Tanabe, T. et al. (2000). The orphan nuclear receptor Tlx regulates Pax2 and is essential for vision. Proc. Natl. Acad. Sci. U. S. A., 97, 2621–5.

Zhang, L., Mathers, P. H. and Jamrich, M. (2000). Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice. Genesis, 28, 135–42.

Zhou, X., Hollemann, T., Pieler, T. and Gruss, P. (2000). Cloning and expression of xSix3, the Xenopus homologue of murine Six3. Mech. Dev., 91, 327–30.

Zuber, M. E., Perron, M., Philpott, A., Bang, A. and Harris, W. A. (1999). Giant eyes in Xenopus laevis by over-expression of XOptx2. Cell, 98, 341–52.

Zuber, M. E., Gestri, G., Viczian, A. S., Barsacchi, G. and Harris, W. A. (2003). Specification of the vertebrate eye by a network of eye field transcription factors.

Development, 130, 5155–67.

better understanding of normal retinal development requires working out the mechanism(s) promoting progression through the cell cycle and maintenance and departure from the cell cycle. This chapter focuses on these issues – how retinal progenitors proceed through the cell cycle and how and when they produce postmitotic daughters. Much of this occurs relatively early in development, primarily from late optic vesicle and optic cup stages. Finally, the significance of sequentially ordered cell production will be explored in the context of the mechanism(s) of cell fate determination.

3.2The cell cycle in the retina

The neuroepithelium of the optic vesicle and cup that generates the retina proceeds through the various stages of the cell cycle as in any other tissue in the body (Figure 3.1a). Chromosomes are duplicated during a DNA synthesis period called ‘S-phase’, and the cells become tetraploid. Subsequently they undergo a division whereby the chromosome pairs are sorted, aligned, segregated and separated to two daughters during mitotic or ‘M-phase’ of the cell cycle. In M-phase the chromosome complement is reduced to the normal two of a diploid cell. Between M- and S-phase are two temporal gaps, of varying length, called interphase (Figure 3.1). The first gap, G1 interphase, is between M- and S-phases, and G2 interphase is between S- and M-phases. Therefore cells are diploid during G1 and tetraploid during G2. Finally, a cell leaves the cell cycle after M-phase, sometime during G1. It may or may not retain the potential to re-enter the cycle and is said to be in a stage referred to as G0 (Figure 3.1b).

Early investigators made two seminal observations about cell division in the CNS. First, dyes that bind nucleic acids readily demonstrated the chromatin that aggregates during M- phase of the cell cycle. Such profiles are called mitotic figures, and the great majority line the lumen of the neural tube (Figure 3.1b, for review see Sidman, 1970; Jacobson, 1978; Rakic, 1981). In the retina this corresponds to the outermost (or scleral) surface, which, upon formation of the optic cup, is adjacent to the retinal pigment epithelium (Figures 3.1b, 3.2A– C, H–K). Second, using spectrophotometry to measure DNA content, it was early noted that profiles farthest away from the mitotic figures have approximately double the concentration of DNA, and are tetraploid (Figure 3.2D–G) (Sauer and Chittenden, 1959). Among the hypotheses advanced to account for these observations was that DNA replication, which occurs during S-phase, is spatially separate from cytokinesis, occurring in M-phase, with the nuclei migrating between the inner and outer surfaces of the neuroepithelium during interphase periods (Figure 3.1b).

Understanding of cell cycle and cell production was significantly advanced by the development of a new tool that relied on the incorporation of the radiolabelled nucleotide precursor of thymine, thymidine. It was important that the nucleotide be thymine since it is the one unique to DNA. Tritiated-thymidine (3H-TdR) is taken up by dividing cells and incorporated into DNA during S-phase. Radiolabelled cells are demonstrated by autoradiography of tissue sections (Figure 3.2D/E, H/I). Any cells postmitotic before the 3H-TdR was administered are not labelled. A larger cohort (eventually all) of the dividing cells is

labelled as the interval following administration increases, limited only by the availability of 3H-TdR. Eventually the 3H-TdR is dispersed, incorporated or metabolized, and the cells that continue to divide lose their signal (by approximately 50% each round). Recently other thymine precursors such as 5 -Bromo-2 -deoxy-uridine (BrdU) have been administered, ones for which antibodies are available and allow immunohistochemistry to demonstrate their presence (Figure 3.2F/G, J/K). Regardless of the label, use of short post-injection intervals allows the localization of S-phase nuclei and tracking their position through the cell cycle (Figure 3.1b, 3.2D–G).

One of the first tissues to be studied with the new techniques for tracking cell cycle and cell genesis was the vertebrate retina (Sidman, 1961). Confirming the earlier hypothesis, S-phase occurs at a site distant from the luminal surface of the neural tube (Figure 3.1b), as this is where radioor immunolabelled profiles are seen one hour following 3H-TdR (Figure 3.2D/E) or BrdU administration (Figure 3.2F/G). A thin cell-sparse marginal zone separates the labelled cells from the innermost (vitreal) surface of the retina. Several hours later the radiolabelled profiles will have migrated to the outer retinal surface where they can be observed as mitotic figures (Figure 3.2H/I). Following mitosis the progeny either return to the inner zone to again replicate DNA, or leave the cell cycle, migrate away from the neural tube lumen and take up adult laminar positions (Figure 3.1b). The transit of nuclei during the cell cycle has come to be known as ‘interkinetic nuclear migration’, and the neuroepithelium spanning the migratory pathway forms the ‘ventricular zone’.

3.3 The rate of progression through the cell cycle

The cell cycle is, by definition, a dynamic process studied by static neuroanatomical techniques only through time-consuming and resource-intensive experiments. However, a number of ways have evolved to derive data on cell cycle timing. The most straightforward is to simply count the number of cells in the retina at two time-points during development. More widely applied techniques for determining cell cycle timing involve short survivals following injection of a DNA synthesis marker (Figure 3.3). One can measure the proportion of labelled mitotic figures (Figure 3.3a) or count the labelled progenitors at successive intervals until the maximum number is reached (Figure 3.3b).

Zebrafish retinal progenitors rapidly increase in absolute numbers at stages prior to 16 hours post-fertilization (hpf) and after 24 hpf. However, between 16 hpf and 24 hpf the increase slows considerably. Computing cell cycle timing from these data gave

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Figure 3.1 (cont.) epithelium but remains separated by the potential space of the obliterated neural tube lumen (optic vesicle). The nuclei of progenitors undergo S-phase distal to the neural tube lumen but enter M-phase at the luminal surface. A vitreally extending cell process retracts as cells round up for M-phase and again extends outward. During the interphase periods the cell nuclei migrate out (G2) and in (G1) within this process, in what is known as interkinetic nuclear migration. Following M-phase daughter cells are faced with the decision of exiting the cell cycle, migrating to the proper laminar position, extending axons and dendrites and differentiating, or re-entering S-phase to once again replicate DNA.