- Email: [email protected]
Retinal neural progenitors express topographic map markers
Neurochemistry International 61 (2012) 859–865
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Retinal neural progenitors express topographic map markers James Goolsby a,c,⇑, Michael Atamas a, Sarah Rollor a, David Asanuma a, Rosemary Schuh a,c, Tapas Makar a,b,c, Paul S. Fishman a,c, Christopher T. Bever Jr. a,b,c, David Trisler a,b,c,⇑ a
Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Multiple Sclerosis Center of Excellence, East VAMHCS, Baltimore, MD 21201, USA c VAMHCS, Baltimore, MD 21201, USA b
a r t i c l e
i n f o
Article history: Available online 15 February 2012 Keywords: Retina Antigen Neural connection Synapse formation
a b s t r a c t Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-topoint map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems. Published by Elsevier Ltd.
1. Introduction Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Topographic order of projecting neurons is maintained in many of the neural circuits of the nervous system. Retinal ganglion cell axons, for example, project to visual centers of the brain forming circuits in topographic order (DeLong and Columbre, 1965; Sperry, 1963). Similarly, analogous projecting neurons of the auditory (Knudsen and Knudsen, 1983), somatosensory (Schlaggar and O’Leary, 1994) and motor (Lance-Jones and Landmesser, 1980) systems maintain topographic order when forming circuits. Topographically graded/distributed molecular markers of cell position have been shown to direct proper projection of axons to form appropriate neural circuits in these brain systems. We reported the first topographically graded molecule, TOPDV, in 1978 (Trisler et al., 1981) and we reported an orthogonal gradient of TOPAP molecules in 1990 (Trisler, 1990). Subsequently, a host of topo⇑ Corresponding authors. Address: Department of Neurology, University of Maryland School of Medicine, BRB 12-041, 655 W. Baltimore Street, Baltimore, MD 21201, USA. E-mail addresses: [email protected] (J. Goolsby), dtris001@ umaryland.edu (D. Trisler). 0197-0186/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.neuint.2012.02.010
graphical marker molecules have been reported in the visual system: retina, optic nerve, optic chiasm, optic tract and optic tectum (Fig. 1). In retina, topographically graded cell surface molecules TOPDV (Fig. 1 and Refs. Trisler, 1990; Trisler and Collins, 1987; Trisler et al., 1981), JONES (Constantine-Paton et al., 1986), Ephrin B1 and Ephrin B2 (Braisted et al., 1997) are more abundant in dorsal retina than in ventral retina as are transcription factors Tbx-5 (Koshiba-Takeuchi et al., 2000) and Xvent2 and diffusible morphogens BMP-4 (Hogan, 1996) and Radar (Rissi et al., 1995). Conversely, cell surface receptor EphB2 (Braisted and et al., 1997) as well as transcription factors Pax-2 (Mey and Thanos, 2000) and Vax2 (Barbieri and et al., 1999) and diffusible morphogens Sonic hedgehog (Ekker and et al., 1995) and retinoic acid (Wagner et al., 2000) are more abundant in ventral retina than in dorsal retina. Similarly, there are comparable graded molecules along the anterior–posterior axis of retina. Together, these orthogonally graded molecules can be used to identify cell position in the innate retinal map. In order to study the feasibility of using fetal neural stem cells or fetal neurons for replacing lost neuronal circuits in topographically ordered neural systems and the involvement of molecular maps in this order, we transplanted embryonic chick retinal cells derived from ventral retina into host retina. We found that when cells derived from ventral retina were implanted in dorsal host
860
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
Fig. 1. Topographic molecules can be used to identify cell position in the chick retina and optic tectum. (A) Orthogonal topographic gradients of TOPDV and TOPAP molecules are present in retina and complementary inverted gradients are present in the optic tectum. (B) Topographically distributed diffusible morphogens (boldface), transcription factors (italics) and cell surface effector molecules (normal type) have been reported in the visual system. The individual molecules are shown at their site of highest concentration in either dorsal (D), ventral (V), anterior (A) or posterior (P) retina and optic tectum and along the projection pathway from retina to tectum via the optic nerve, optic chiasm and optic tract (Goolsby, 2004).
retina, the cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors express topographic markers of cell position in retina. Indeed, we found that retinal neural progenitors express topographic markers: dorsal retinal stem cells expressed more Ephrin B2 than ventral stem cells and ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells.
2. Results 2.1. Topographic projection of implanted retinal ganglion cell axons to optic tectum Retinal cells derived from embryonic day 8 (E8) ventral retina were labeled with a fluorescent dye (Cell Tracker Orange) and injected into the vitreal space of host E6 chick eyes, 3 days after
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
861
ganglion cells appear in E3 chick retina. The labeled cells were found to migrate into both ventral and dorsal host retina from the vitreal space and implant in all retinal cell strata. Transplanted embryos were allowed to develop to E20. In order to determine the projection target of axons of ganglion cells derived from the transplanted cells, crystals of fluorescent dye (DiA) were injected into dorsal optic tectum, the normal target region for ventral retinal ganglion cell axon projection. DiA then was retrogradely transported to the cell bodies and dendritic arborizations of retinal ganglion cells that projected axons to the dorsal field of the optic tectum. Ganglion cells in dorsal host retina that developed from the cells transplanted from ventral retina were found to project their axons to dorsal optic tectum, the normal target for ventral retinal ganglion cells (Fig. 2B). Thus, these transplanted ventral cells projected to targets appropriate for the site of origin of the cells rather than the site of implant in the host retina. The ventral ganglion cells implanted in dorsal retina sent their axons passing over ventral tectum where their surrounding endogenous neighbor dorsal ganglion neurons projected and synapsed and projected their axons onto dorsal tectum where ventral retinal ganglion neurons normally project and synapse. The Fig. 2B shows the ganglion cell surface of en face images of whole mounted dorsal retina. Cell Tracker Orange labeled (red) ventral retinal progenitor cells that differentiated into ganglion cells are labeled with DiA (green) that was retrogradely transported along the axon to the cell soma and dendritic arbors from crystals placed in dorsal tectum. The field of each image is filled with the soma and dendrites of endogenous dorsal ganglion cells that are unlabeled because they project their axons to ventral tectum where there was no DiA to be transported. Ninety-three percent (196/211) of ventral cells transplanted into ventral retina were labeled with DiA transported from dorsal tectum (not shown). So, these ventral cells implanted in ventral retina projected to dorsal tectum like their endogenous neighbors. Significantly, 23% (49/164) of ventral cells transplanted into dorsal retina were labeled with DiA transported from dorsal tectum. Thus, these ventral cells, implanted in dorsal retina, projected axons beyond ventral tectum where their endogenous neighbors projected to selective targets in dorsal tectum – targets appropriate for ventral retinal ganglion cells. 2.2. Neural progenitors express topographic markers Two types of continuously dividing neural progenitors were cultured from embryonic retina. One was freely floating neurospheres and the others were monolayer flat cells that adhere to the tissue culture flask. Pure cultures of these adherent continuously dividing flat progenitor cells were obtained by repeatedly mechanically removing the neurons that adhere to the flat adherent progenitor cells in primary cultures by shaking the neurons off, then dissociating the flat cells and serially passaging the dividing flat cells. These cultured adherent flat cells express both neuronal NCAM and neurofilament as well as astroglial GFAP (Fig. 4B) and oligodendroglial galactocerebroside, however, oligodendroglial CNPase was not found (not shown). These adherent cells are neural because chick retina is not vascularized so the only cells in chick retina are neural cells. The chick retina contains photoreceptors, neurons, Müller glial cells and neural progenitor cells. We show below that both the floating neurosphere cells and the continuously dividing adherent flat cells are retinal progenitors that differentiate into both neurons and glia. Both floating neurospheres and adherent flat cells were examined by PCR and Western blot for expression of topographic molecules. Topographic molecules examined were Ephrin B2, which is more abundant in dorsal retina than ventral retina and Pax-2 and Ventroptin, which are more abundant in ventral retina than in dorsal retina.
Fig. 2. Retinal ganglion cells that develop from transplanted retinal progenitor cells project axons to targets in visual centers of the brain that are topographically appropriate for the retinal topographic site of origin of the progenitor rather that the topographic site of implant of the progenitor cell. (A) The topographic map of the retinotectal projection was demonstrated by injecting crystals of FAST BLUE into dorsal optic tectum and Diamido Yellow into ventral optic tectum of E16 chick in windowed eggs. The embryos were incubated for 2 days to allow retrograde transport of the dyes to ganglion cell soma in the retina. FAST BLUE from dorsal tectum was found in the soma of ventral retinal ganglion cells of en face whole retinal mounts and conversely Diamido Yellow in ventral tectum was found in the soma of dorsal retinal ganglion cells. (B) Dissociated E8 ventral retinal cells that were labeled with the fluorescent dye Cell Tracker Orange (CTO, red) were injected into the vitreal space of eyes of E6 chicks. The embryos were incubated to E20 to allow differentiation of the implanted cells. Embryonic ventral retinal cells were found to implant throughout the host retina both ventral and dorsal. At E20 implanted embryos were suspended in 4% paraformaldehyde at 4 °C for 4 days. Crystals of 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) were injected into dorsal optic tectum and the embryos were kept in 4% paraformaldehyde at 4 °C for 3 months. Implanted retinas were disected, cut into flower-shaped flat whole mounts and examined en face by fluorescence microscopy. Implanted Cell Tracker Orange labeled cells were scored for the presence of DiA. Some CTOlabeled ganglion cells (red) in dorsal retina, that were formed from ventral retinal cells, were found to have retrogradely transported DiA (green) from dorsal tectum to their soma and dendritic arbors, unlike their surrounding unlabeled endogenous dorsal retinal ganglion cells. Scale bar = 75 lm.
862
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
Fig. 3. Retinal progenitor cells express topographic molecular markers of cell position at both the mRNA and protein levels. E8 chick retinal cells and floating neurosphere progenitors and adherent flat progenitors, all derived from both dorsal and ventral retina were examined by semi-quantitative PCR for expression of mRNA that codes for (A). Ephrin B2, that is more abundant in dorsal embryonic chick retina than in ventral retina as well as (B). Pax-2 and (C), Ventroptin both of which are more abundant in ventral embryonic chick retina than in dorsal retina. Conventional semi-quantitative PCR methodology was used with cDNA primers Ephrin B2 50 -ATAAAGACCAAGCAGATAGC-30 , 50 TGATGACGATGAAAATGATG-30 ; Pax-2 50 -CAACCTTTCCACCCAACACC-30 , 50 -TACTCCCCCTGCCCATACC-30 ; Ventroptin 50 -TTTGGTGTGCTCTGTCTCCT-30 , 50 -AGTGTCTGGCT TCCCTGTTG-30 ; GAPDH 50 -AGCGTGACCCCAGCAACAT[C?G]-30 , 50 -CAGCCTTAGCAGCCCCAGTG-30 . The bar graphs are the average of three determinations for each mRNA in dorsal and ventral E8, floater and flat progenitor cells. Brackets show the SEMs. Both floater neurosphere and flat adherent progenitor cells expressed more Ephrin B2 in cells from dorsal retina more than ventral retina like E8 retinal cells. Both progenitor cell types also expressed more Pax-2 and Ventroptin in ventral cells than dorsal cells like E8 cells. T test ((A) E8 p < 0.05, float p < 0.05, flat p < 0.05; (B) E8 p < 0.001, float p < 0.05, flat p < 0.05; (C) E8 p < 0.001, float p < 0.05, flat p > 0.05). (D) E8 retinas and floater neurosphere progenitors were examined for topographic marker protein expression by standard Western blot methodology. E8 retinal cells and floating neurospheres derived from E8 retina expressed more Ephrin B2 protein in cells from dorsal retina that ventral retina. These cells also expressed more Pax-2 in ventral retina than in dorsal retina.
Both floating and flat stem cells express Ephrin B2, Pax-2 and Ventroptin at both the mRNA (Fig. 3A–C) and protein levels (Fig. 3D). Ephrin B2 was more abundant in dorsal floating and flat progenitor cells than in ventral cells like the E8 embryonic retina itself. Conversely, Pax-2 and Ventroptin were more abundant in ventral floating and flat progenitors than in dorsal progenitors, like E8 embryonic retina. 2.3. Implanted neural progenitors differentiate morphologically into the functional classes of retinal cells Chick neural retina has six major functional classes of cells. Photoreceptor rods and cones are the cell bodies in the outer cell stratum (outer nuclear layer). They synapse with bipolar neurons and horizontal interneurons in the outer synaptic layer. The inner cell stratum (inner nuclear layer) contains the cell bodies of horizontal, bipolar and amacrine neurons as well as Müller glial cells. Bipolar neurons synapse with ganglion neurons and amacrine interneurons in the inner synaptic layer. Ganglion neuron cell bodies are in the ganglion cell layer. They send their axons along the retinal surface to the optic nerve head and via the optic nerve to visual
centers in the brain. Müller cells are large glial cells that span the retina from the outer limiting membrane to the inner limiting membrane (Fig. 4). Acutely dissociated embryonic retinal cells containing neural progenitors were transplanted into host retina as were cultured retinal flat progenitor cells transplanted into other host retinas. Progenitors from the acutely dissociated cells were identified in retina by BrdU labeling of dividing transplanted progenitor cells in the host retina 2 days (E8) after transplantation. At E20 double-labeled (CTO + FITC-anti-BrdU) cells were found in all retinal cell strata. BrdU containing transplanted stem cells implanted in the photoreceptor and ganglion cell layers of retina are shown in Fig. 4C. The photoreceptor layer contained 12.3% (8/65) of CTOlabeled cells that were also labeled with BrdU; the inner nuclear layer contained 14.2% (9/65) that were double-labeled and the ganglion cell layer 13.4% (10/75). Transplanted cells differentiated morphologically into each of the functional classes of cells in retina (Fig. 4D). The cell functional class-specific morphology of the transplanted cells was assigned using images of Golgi-stained cells first reported by Ramón y Cajal (Thorpe and Glickstein, 1972). Acutely dissociated retinal
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
863
progenitors differentiated into photoreceptor-like (Fig. 4D: C–E) (54/306, 18%) and horizontal-like (Fig. 4D: F–H) (45/306, 15%), bipolar-like (Fig. 4D: I and J) (28/306, 9%), amacrine-like (Fig. 4D: K and L) (62/306, 20%) and ganglion-like (Fig. 4D: M–O) (90/306, 29%) neurons as well as Müller-like glial (Fig. 4D: A and B) (27/ 306, 9%) cells. Adherent flat progenitor cells differentiated into horizontal-like (946/247, 19%), amacrine-like (105/247, 43%) and ganglion-like (58/247, 23%) neurons as well as Müller-like glial (38/ 247, 15%) cells, but no photoreceptors and bipolar neurons were found to derive from the implanted flat progenitor cells. Thus, the acutely dissociated progenitor cells differentiated into all cell types, whereas the flat progenitor cells differentiated into both neurons and glia but not photoreceptor or bipolar cells. These results show that retinal progenitors can enter the retina from the vitreal space, migrate through the retina, implant in all retinal strata and appropriately differentiate morphologically for the class of cells in each stratum. 3. Discussion The major finding of this study is that neural progenitor cells express topographic marker molecules, and after transplantation into host retina, the progenitor cells that become projecting neurons project to targets appropriate for their site of origin rather than for their site of implant. 3.1. Retinal progenitor cell phenotypes
Fig. 4. Retinal progenitor cells implant in all retinal strata and express morphologies appropriate for the stratum of implant. (A) An image of E19 chick retina stained with Toluidin Blue that stains cell bodies of each stratum of retina. Drawn on this image are the representative morphologies of cells of each functional class of retina first shown by Cajal with Golgi silver stain (Thorpe and Glickstein, 1972). (B) Retinal flat stem cells that adhere to the plastic cell culture flask stained with (A) anti-NCAM antibody show cell surface staining and (B) anti-GFAP antibody showing intermediate filament GFAP staining. All flat stem cells expressed both neuronal NCAM and glial GFAP. Scale bar = 50 lm. (C) Images of retinas with implanted stem cells after S-phase BrdU incorporation labeled with antibody to BrdU. E8 retinal cells were labeled with Cell Tracker Orange (red) and injected into the vitreal space of host chick embryo eyes. Two days post-injection 10 lg of BrdU were injected into the amniotic space of the embryo. At E20 retinas were stained with anti-BrdU (green). CTO stained stem cells with nuclei containing incorporated BrdU (yellow) are shown implanted in the photoreceptor layer and the ganglion cell layers of host retina. The retinas were counterstained with DAPI (blue) to show the retinal cell nuclei and retinal cell strata. (D) Images of implanted cells that express morphologies appropriate for the stratum of implant. Transplanted CTO-labeled E8 cells differentiated morphologically into (A) Müller-like (small arrow), bipolar-like (solid arrowhead) and amacrine-like cells (open arrowhead); (B) Müller-like cell; (C) diagonal cone-like photoreceptor; (D) rod-like photoreceptor; (E) conventional cone-like photoreceptor; (F–H) horizontal-like neuron; (I and J) bipolar-like neuron; (K and L) amacrine-like neuron; (M–O) ganglion-like cell. The data show that transplanted cells migrate across the retina, implant in all strata of retina and differentiate morphologically into cells appropriate for the stratum of implant. Scale bar = 100 lm in panels (A–M) and 200 lm in panels (N and O).
Two types of neural stem cells were found in embryonic chick retina. One was freely floating neurospheres and the other was adherent flat stem cells that attach to the culture flask surface (Bernardos et al., 2007; Goolsby et al., 1999). These flat cells may be an actual second progenitor cell type or they may be Müller cells, the only previously known cell in chick retina that adheres to tissue culture flasks – Müller cells that can become progenitor cells. In culture, these flat cells express both neuronal NCAM and neurofilament genes and glial GFAP and galactocerebroside genes. If these flat cells are Müller cells or derived from Müller cells, it suggests that Müller cells behave as progenitor cells in embryonic retina like radioglial cells behave in embryonic brain. Both floating progenitors and flat progenitors were able to migrate to all cell strata of the host retina and differentiate morphologically into cell types appropriate for cells in each stratum. The flat cells had more limited potential for differentiation, but they did differentiate into both neurons and glia. 3.2. Topographic markers in retinal progenitor cells Both progenitor cell types in retina expressed topographic markers of cell position in a graded fashion. The progenitor cells expressed relative levels of the topographic molecule examined that were appropriate for the site of origin of the progenitor cells in retina. The progenitor cells examined for expression of topographic markers in this study were chick E8 cells. We do not know whether earlier progenitor cells express these markers and we do not know the embryonic age of appearance of these markers in progenitor cells. We have not found these topographic markers in embryonic stem cells or in adult bone marrow hematopoietic stem cells. It would be of interest to learn how stem cells, that do not express topographic markers, behave after transplantation when they differentiate into projection neurons in a topographically mapped field of the host. Do they project axons along with their neighbors
864
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
to targets appropriate for their site of implant? Are these unmarked stem cells potential universal donors for topographic projection neuron replacement?
3.3. Topographic order of projecting neurons Three homologous classic models of projecting axons maintaining topographic order are, first, the visual system of goldfish, frogs and chick embryos where retinal ganglion cell axons project to optic tectum and other visual centers in the thalamus (DeLong and Columbre, 1965; Sperry, 1963). Secondly, the somatosensory system of rodent whisker barrels where topographic order is maintained along the sensory pathway from whisker barrel to ganglion V to brainstem to thalamus to sensory cortex (Schlaggar and O’Leary, 1994). And thirdly, the motor system of chicks and quail/chick chimeras where segments of embryonic spinal cord is surgically transplanted in reversed order or placed in an ectopic position along the spinal tract – motor neurons derived from the misplaced stem cells in the early cord project to targets appropriate for the site of origin of the motor neurons rather than for the site of implant (Lance-Jones and Landmesser, 1980). These models predicted molecular maps of cell position. In the visual system misplaced transplanted retinal ganglion cells maintained topographic order when projecting to the tectum, but the order was based on site of cell origin not site of cell implant. Some of the ventral cells transplanted into dorsal retina were labeled with DiA from dorsal tectum and other ventral cells were not labeled with DiA. Ventral cells in dorsal retina that were not labeled with DiA may be unlabeled because they did not reach the dorsal tectum due to their growth cones being misrouted, migrating over other retinal ganglion cell axons instead of on the tectal surface where the growth cones could read the topographical molecular map. On the other hand, some ventral cells in dorsal retina may not have had sufficient time (E6 to E20) to differentiate, to project their axons from the retina along the optic nerve, the optic tract and over ventral tectum to reach targets in dorsal tectum. In the present study, visual images detected in dorsal retina and transmitted to visual centers in the brain by ganglion cells derived from stem cells of ventral retinal origin would be interpreted by the brain as being images in the ventral visual field leading to confusion and perhaps, as a consequence, a misstep. Thus, a progenitor cell that expresses topographic markers cannot be used to functionally replace missing projection neurons at all positions in a topographically mapped field.
4. Materials and methods 4.1. Progenitor cell culture White leghorn chick (Gallus gallus domesticus) retinas from E8 embryos were dissociated and cultured in DMEM containing 10% FBS as reported (Trisler, 1990). From primary cultures, floating neurospheres were cultured separately. Neurons were physically removed from the monolayer of flat continuously dividing adherent progenitor cells by repeatedly shaking off the neurons and passaging the flat cells until there were pure cultures of flat cells.
4.2. Retinal cell labeling Retinal cells were labeled with Cell Tracker Orange (CTO) (Molecular Probes) as previously reported (Trisler et al., 1996).
4.3. Retinal cell transplantation CTO-labeled retinal cells were injected into the vitreal space of E6 chick embryos in windowed eggs as previously reported (Trisler et al., 1986, 1996). Transplanted embryos were incubated to E16 or E20. 4.4. Topographic labeling of optic tectum The topographic map of the retinotectal projection was demonstrated by injecting crystals of FAST BLUE into dorsal tectum and Diamido Yellow into ventral tectum. The embryos were incubated for 2 days to allow retrograde transport of the dyes to the ganglion cell bodies in retina. For transplanted retinas the embryos were incubated to E20 to allow as much time as possible for differentiation of the implanted progenitor cells. At E20 the embryos were killed and suspended in 4% paraformaldehyde at 4 °C for 4 days then crystals of 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) (Molecular Probes) were injected into dorsal tectum. The embryos were suspended in 4% paraformaldehyde at 4 °C for 3 months to allow retrograde diffusion of DiA from tectum to retina. 4.5. Semi-quantitative PCR Expression of topographic molecule mRNA in retinal progenitor cells was determined by standard semi-quantitative PCR as previously reported (Pessac et al., 2011). 4.6. Western blot Topographic marker protein expression by retinal progenitor cells was determined by standard Western blot analysis as previously reported (Savitt et al., 1995). Conflicts of interest J.G., C.T.B. and D.T. hold USA (10/982,381) and International (PTC/US04/37122) Patents Pending. Acknowledgements The authors thank Deborah Yarnell and David Ford for technical assistance in this project. This work was supported by R.S. (VA REAP), T.M. (VA Merit), P.S.F. (VA REAP), C.T.B. (VA Merit) and D.T. (VA Merit and Abraxis Biosci.). References Barbieri, A.M. et al., 1999. A homeobox gene, vax2 controls the patterning of the eye dorsoventral axis. Proc. Natl. Acad. Sci. USA 96, 10729–10734. Bernardos, R.L., Barthel, L.K., Meyers, J.R., Raymond, P.A., 2007. Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells. J. Neurosci. 27, 7028–7040. Braisted, J.E. et al., 1997. Graded and laminin-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system. Dev. Biol. 191, 14–28. Constantine-Paton, M. et al., 1986. A cell surface molecule distributed in a dorsoventral gradient in perinatal rat retina. Nature 324, 459–462. DeLong, G.R., Columbre, A.J., 1965. Development of the retinotectal topographic projection in the chick embryo. Exp. Neurol. 14, 351–363. Ekker, S.C. et al., 1995. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr. Biol. 5, 944–955. Goolsby, J., 2004. Transplantation of stem cells into the central nervous system. Ph.D. Dissertation. Goolsby, J., Rosenthal, C., Bever, C., Trisler, D., 1999. Retinal engineering: engrafted retinal cells of each functional class migrate and implant in the proper retinal stratum. Soc. Neurosci. Abst. 25, 1310. Hogan, B.L., 1996. Bone morphogenic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580–1594.
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865 Knudsen, E.I., Knudsen, P.F., 1983. Space-mapped auditory projections from the inferior colliculus to the optic tectum in the barn owl (Tyto alba). J. Comp. Neurol. 218, 187–196. Koshiba-Takeuchi, K. et al., 2000. Tbx5 and the retinotectum projection. Science 287, 134–137. Lance-Jones, C., Landmesser, L., 1980. Motorneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord. J. Physiol. 302, 581–602. Mey, J., Thanos, S., 2000. Development of the visual system of the chick. I. Cell differentiation and histogenesis. Brain Res. Brain Res. Rev. 32, 343–379. Pessac, B., Ait Bara, M., Ford, D., Patibandla, G., Trisler, D., 2011. Hematopoietic progenitors express embryonic stem cell and germ layer genes. C. R. Biol. 334, 300–306. Ramón y Cajal, S., 1972. The Structure of the Retina (Thorpe, S. A., Glicustein, M., Compilers and Translators). Charles C. Thomas, Springfield. Rissi, M. et al., 1995. Zebrafish Radar: a new member of the TGF-beta superfamily defines dorsal regions of the neural plate and the embryonic retina. Mech. Dev. 49, 223–234. Savitt, J., Trisler, D., Hilt, D., 1995. Molecular cloning of TOPAP: a topographically graded molecule in the developing chick visual system. Neuron 14, 253–261.
865
Schlaggar, B.L., O’Leary, D.M., 1994. Early development of the somatotropic map and barrel patterning in rat somatosensory cortex. J. Comp. Neurol. 346, 80–96. Sperry, R.W., 1963. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. USA 50, 703–710. Trisler, D., 1990. Cell recognition and pattern formation in the developing nervous system. J. Exp. Biol. 153, 11–27. Trisler, D., Collins, F., 1987. Corresponding spatial gradients of TOP molecules in the developing retina and optic tectum. Science 237, 1208–1209. Trisler, G.D., Schneider, M.D., Nirenberg, M., 1981. A topographic gradient of molecules in retina can be used to identify neuron position. Proc. Natl. Acad. Sci. USA 78, 2145–2149. Trisler, D., Bekenstein, J., Daniels, M.P., 1986. Antibody to a molecular marker of cell position inhibits synapse formation in retina. Proc. Natl. Acad. Sci. USA 83, 4194–4198. Trisler, D., Rutin, J., Pessac, B., 1996. Retinal engineering: engrafted neural cell lines locate in appropriate layers. Proc. Natl. Acad. Sci. USA 93, 6269– 6274. Wagner, E., McCaffery, P., Drager, U., 2000. Retinoic acid in the formation of the dorsoventral retina and its central projections. Dev. Biol. 222, 460–470.
Contents lists available at SciVerse ScienceDirect
Neurochemistry International journal homepage: www.elsevier.com/locate/nci
Retinal neural progenitors express topographic map markers James Goolsby a,c,⇑, Michael Atamas a, Sarah Rollor a, David Asanuma a, Rosemary Schuh a,c, Tapas Makar a,b,c, Paul S. Fishman a,c, Christopher T. Bever Jr. a,b,c, David Trisler a,b,c,⇑ a
Department of Neurology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Multiple Sclerosis Center of Excellence, East VAMHCS, Baltimore, MD 21201, USA c VAMHCS, Baltimore, MD 21201, USA b
a r t i c l e
i n f o
Article history: Available online 15 February 2012 Keywords: Retina Antigen Neural connection Synapse formation
a b s t r a c t Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-topoint map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems. Published by Elsevier Ltd.
1. Introduction Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Topographic order of projecting neurons is maintained in many of the neural circuits of the nervous system. Retinal ganglion cell axons, for example, project to visual centers of the brain forming circuits in topographic order (DeLong and Columbre, 1965; Sperry, 1963). Similarly, analogous projecting neurons of the auditory (Knudsen and Knudsen, 1983), somatosensory (Schlaggar and O’Leary, 1994) and motor (Lance-Jones and Landmesser, 1980) systems maintain topographic order when forming circuits. Topographically graded/distributed molecular markers of cell position have been shown to direct proper projection of axons to form appropriate neural circuits in these brain systems. We reported the first topographically graded molecule, TOPDV, in 1978 (Trisler et al., 1981) and we reported an orthogonal gradient of TOPAP molecules in 1990 (Trisler, 1990). Subsequently, a host of topo⇑ Corresponding authors. Address: Department of Neurology, University of Maryland School of Medicine, BRB 12-041, 655 W. Baltimore Street, Baltimore, MD 21201, USA. E-mail addresses: [email protected] (J. Goolsby), dtris001@ umaryland.edu (D. Trisler). 0197-0186/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.neuint.2012.02.010
graphical marker molecules have been reported in the visual system: retina, optic nerve, optic chiasm, optic tract and optic tectum (Fig. 1). In retina, topographically graded cell surface molecules TOPDV (Fig. 1 and Refs. Trisler, 1990; Trisler and Collins, 1987; Trisler et al., 1981), JONES (Constantine-Paton et al., 1986), Ephrin B1 and Ephrin B2 (Braisted et al., 1997) are more abundant in dorsal retina than in ventral retina as are transcription factors Tbx-5 (Koshiba-Takeuchi et al., 2000) and Xvent2 and diffusible morphogens BMP-4 (Hogan, 1996) and Radar (Rissi et al., 1995). Conversely, cell surface receptor EphB2 (Braisted and et al., 1997) as well as transcription factors Pax-2 (Mey and Thanos, 2000) and Vax2 (Barbieri and et al., 1999) and diffusible morphogens Sonic hedgehog (Ekker and et al., 1995) and retinoic acid (Wagner et al., 2000) are more abundant in ventral retina than in dorsal retina. Similarly, there are comparable graded molecules along the anterior–posterior axis of retina. Together, these orthogonally graded molecules can be used to identify cell position in the innate retinal map. In order to study the feasibility of using fetal neural stem cells or fetal neurons for replacing lost neuronal circuits in topographically ordered neural systems and the involvement of molecular maps in this order, we transplanted embryonic chick retinal cells derived from ventral retina into host retina. We found that when cells derived from ventral retina were implanted in dorsal host
860
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
Fig. 1. Topographic molecules can be used to identify cell position in the chick retina and optic tectum. (A) Orthogonal topographic gradients of TOPDV and TOPAP molecules are present in retina and complementary inverted gradients are present in the optic tectum. (B) Topographically distributed diffusible morphogens (boldface), transcription factors (italics) and cell surface effector molecules (normal type) have been reported in the visual system. The individual molecules are shown at their site of highest concentration in either dorsal (D), ventral (V), anterior (A) or posterior (P) retina and optic tectum and along the projection pathway from retina to tectum via the optic nerve, optic chiasm and optic tract (Goolsby, 2004).
retina, the cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors express topographic markers of cell position in retina. Indeed, we found that retinal neural progenitors express topographic markers: dorsal retinal stem cells expressed more Ephrin B2 than ventral stem cells and ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells.
2. Results 2.1. Topographic projection of implanted retinal ganglion cell axons to optic tectum Retinal cells derived from embryonic day 8 (E8) ventral retina were labeled with a fluorescent dye (Cell Tracker Orange) and injected into the vitreal space of host E6 chick eyes, 3 days after
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
861
ganglion cells appear in E3 chick retina. The labeled cells were found to migrate into both ventral and dorsal host retina from the vitreal space and implant in all retinal cell strata. Transplanted embryos were allowed to develop to E20. In order to determine the projection target of axons of ganglion cells derived from the transplanted cells, crystals of fluorescent dye (DiA) were injected into dorsal optic tectum, the normal target region for ventral retinal ganglion cell axon projection. DiA then was retrogradely transported to the cell bodies and dendritic arborizations of retinal ganglion cells that projected axons to the dorsal field of the optic tectum. Ganglion cells in dorsal host retina that developed from the cells transplanted from ventral retina were found to project their axons to dorsal optic tectum, the normal target for ventral retinal ganglion cells (Fig. 2B). Thus, these transplanted ventral cells projected to targets appropriate for the site of origin of the cells rather than the site of implant in the host retina. The ventral ganglion cells implanted in dorsal retina sent their axons passing over ventral tectum where their surrounding endogenous neighbor dorsal ganglion neurons projected and synapsed and projected their axons onto dorsal tectum where ventral retinal ganglion neurons normally project and synapse. The Fig. 2B shows the ganglion cell surface of en face images of whole mounted dorsal retina. Cell Tracker Orange labeled (red) ventral retinal progenitor cells that differentiated into ganglion cells are labeled with DiA (green) that was retrogradely transported along the axon to the cell soma and dendritic arbors from crystals placed in dorsal tectum. The field of each image is filled with the soma and dendrites of endogenous dorsal ganglion cells that are unlabeled because they project their axons to ventral tectum where there was no DiA to be transported. Ninety-three percent (196/211) of ventral cells transplanted into ventral retina were labeled with DiA transported from dorsal tectum (not shown). So, these ventral cells implanted in ventral retina projected to dorsal tectum like their endogenous neighbors. Significantly, 23% (49/164) of ventral cells transplanted into dorsal retina were labeled with DiA transported from dorsal tectum. Thus, these ventral cells, implanted in dorsal retina, projected axons beyond ventral tectum where their endogenous neighbors projected to selective targets in dorsal tectum – targets appropriate for ventral retinal ganglion cells. 2.2. Neural progenitors express topographic markers Two types of continuously dividing neural progenitors were cultured from embryonic retina. One was freely floating neurospheres and the others were monolayer flat cells that adhere to the tissue culture flask. Pure cultures of these adherent continuously dividing flat progenitor cells were obtained by repeatedly mechanically removing the neurons that adhere to the flat adherent progenitor cells in primary cultures by shaking the neurons off, then dissociating the flat cells and serially passaging the dividing flat cells. These cultured adherent flat cells express both neuronal NCAM and neurofilament as well as astroglial GFAP (Fig. 4B) and oligodendroglial galactocerebroside, however, oligodendroglial CNPase was not found (not shown). These adherent cells are neural because chick retina is not vascularized so the only cells in chick retina are neural cells. The chick retina contains photoreceptors, neurons, Müller glial cells and neural progenitor cells. We show below that both the floating neurosphere cells and the continuously dividing adherent flat cells are retinal progenitors that differentiate into both neurons and glia. Both floating neurospheres and adherent flat cells were examined by PCR and Western blot for expression of topographic molecules. Topographic molecules examined were Ephrin B2, which is more abundant in dorsal retina than ventral retina and Pax-2 and Ventroptin, which are more abundant in ventral retina than in dorsal retina.
Fig. 2. Retinal ganglion cells that develop from transplanted retinal progenitor cells project axons to targets in visual centers of the brain that are topographically appropriate for the retinal topographic site of origin of the progenitor rather that the topographic site of implant of the progenitor cell. (A) The topographic map of the retinotectal projection was demonstrated by injecting crystals of FAST BLUE into dorsal optic tectum and Diamido Yellow into ventral optic tectum of E16 chick in windowed eggs. The embryos were incubated for 2 days to allow retrograde transport of the dyes to ganglion cell soma in the retina. FAST BLUE from dorsal tectum was found in the soma of ventral retinal ganglion cells of en face whole retinal mounts and conversely Diamido Yellow in ventral tectum was found in the soma of dorsal retinal ganglion cells. (B) Dissociated E8 ventral retinal cells that were labeled with the fluorescent dye Cell Tracker Orange (CTO, red) were injected into the vitreal space of eyes of E6 chicks. The embryos were incubated to E20 to allow differentiation of the implanted cells. Embryonic ventral retinal cells were found to implant throughout the host retina both ventral and dorsal. At E20 implanted embryos were suspended in 4% paraformaldehyde at 4 °C for 4 days. Crystals of 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) were injected into dorsal optic tectum and the embryos were kept in 4% paraformaldehyde at 4 °C for 3 months. Implanted retinas were disected, cut into flower-shaped flat whole mounts and examined en face by fluorescence microscopy. Implanted Cell Tracker Orange labeled cells were scored for the presence of DiA. Some CTOlabeled ganglion cells (red) in dorsal retina, that were formed from ventral retinal cells, were found to have retrogradely transported DiA (green) from dorsal tectum to their soma and dendritic arbors, unlike their surrounding unlabeled endogenous dorsal retinal ganglion cells. Scale bar = 75 lm.
862
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
Fig. 3. Retinal progenitor cells express topographic molecular markers of cell position at both the mRNA and protein levels. E8 chick retinal cells and floating neurosphere progenitors and adherent flat progenitors, all derived from both dorsal and ventral retina were examined by semi-quantitative PCR for expression of mRNA that codes for (A). Ephrin B2, that is more abundant in dorsal embryonic chick retina than in ventral retina as well as (B). Pax-2 and (C), Ventroptin both of which are more abundant in ventral embryonic chick retina than in dorsal retina. Conventional semi-quantitative PCR methodology was used with cDNA primers Ephrin B2 50 -ATAAAGACCAAGCAGATAGC-30 , 50 TGATGACGATGAAAATGATG-30 ; Pax-2 50 -CAACCTTTCCACCCAACACC-30 , 50 -TACTCCCCCTGCCCATACC-30 ; Ventroptin 50 -TTTGGTGTGCTCTGTCTCCT-30 , 50 -AGTGTCTGGCT TCCCTGTTG-30 ; GAPDH 50 -AGCGTGACCCCAGCAACAT[C?G]-30 , 50 -CAGCCTTAGCAGCCCCAGTG-30 . The bar graphs are the average of three determinations for each mRNA in dorsal and ventral E8, floater and flat progenitor cells. Brackets show the SEMs. Both floater neurosphere and flat adherent progenitor cells expressed more Ephrin B2 in cells from dorsal retina more than ventral retina like E8 retinal cells. Both progenitor cell types also expressed more Pax-2 and Ventroptin in ventral cells than dorsal cells like E8 cells. T test ((A) E8 p < 0.05, float p < 0.05, flat p < 0.05; (B) E8 p < 0.001, float p < 0.05, flat p < 0.05; (C) E8 p < 0.001, float p < 0.05, flat p > 0.05). (D) E8 retinas and floater neurosphere progenitors were examined for topographic marker protein expression by standard Western blot methodology. E8 retinal cells and floating neurospheres derived from E8 retina expressed more Ephrin B2 protein in cells from dorsal retina that ventral retina. These cells also expressed more Pax-2 in ventral retina than in dorsal retina.
Both floating and flat stem cells express Ephrin B2, Pax-2 and Ventroptin at both the mRNA (Fig. 3A–C) and protein levels (Fig. 3D). Ephrin B2 was more abundant in dorsal floating and flat progenitor cells than in ventral cells like the E8 embryonic retina itself. Conversely, Pax-2 and Ventroptin were more abundant in ventral floating and flat progenitors than in dorsal progenitors, like E8 embryonic retina. 2.3. Implanted neural progenitors differentiate morphologically into the functional classes of retinal cells Chick neural retina has six major functional classes of cells. Photoreceptor rods and cones are the cell bodies in the outer cell stratum (outer nuclear layer). They synapse with bipolar neurons and horizontal interneurons in the outer synaptic layer. The inner cell stratum (inner nuclear layer) contains the cell bodies of horizontal, bipolar and amacrine neurons as well as Müller glial cells. Bipolar neurons synapse with ganglion neurons and amacrine interneurons in the inner synaptic layer. Ganglion neuron cell bodies are in the ganglion cell layer. They send their axons along the retinal surface to the optic nerve head and via the optic nerve to visual
centers in the brain. Müller cells are large glial cells that span the retina from the outer limiting membrane to the inner limiting membrane (Fig. 4). Acutely dissociated embryonic retinal cells containing neural progenitors were transplanted into host retina as were cultured retinal flat progenitor cells transplanted into other host retinas. Progenitors from the acutely dissociated cells were identified in retina by BrdU labeling of dividing transplanted progenitor cells in the host retina 2 days (E8) after transplantation. At E20 double-labeled (CTO + FITC-anti-BrdU) cells were found in all retinal cell strata. BrdU containing transplanted stem cells implanted in the photoreceptor and ganglion cell layers of retina are shown in Fig. 4C. The photoreceptor layer contained 12.3% (8/65) of CTOlabeled cells that were also labeled with BrdU; the inner nuclear layer contained 14.2% (9/65) that were double-labeled and the ganglion cell layer 13.4% (10/75). Transplanted cells differentiated morphologically into each of the functional classes of cells in retina (Fig. 4D). The cell functional class-specific morphology of the transplanted cells was assigned using images of Golgi-stained cells first reported by Ramón y Cajal (Thorpe and Glickstein, 1972). Acutely dissociated retinal
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
863
progenitors differentiated into photoreceptor-like (Fig. 4D: C–E) (54/306, 18%) and horizontal-like (Fig. 4D: F–H) (45/306, 15%), bipolar-like (Fig. 4D: I and J) (28/306, 9%), amacrine-like (Fig. 4D: K and L) (62/306, 20%) and ganglion-like (Fig. 4D: M–O) (90/306, 29%) neurons as well as Müller-like glial (Fig. 4D: A and B) (27/ 306, 9%) cells. Adherent flat progenitor cells differentiated into horizontal-like (946/247, 19%), amacrine-like (105/247, 43%) and ganglion-like (58/247, 23%) neurons as well as Müller-like glial (38/ 247, 15%) cells, but no photoreceptors and bipolar neurons were found to derive from the implanted flat progenitor cells. Thus, the acutely dissociated progenitor cells differentiated into all cell types, whereas the flat progenitor cells differentiated into both neurons and glia but not photoreceptor or bipolar cells. These results show that retinal progenitors can enter the retina from the vitreal space, migrate through the retina, implant in all retinal strata and appropriately differentiate morphologically for the class of cells in each stratum. 3. Discussion The major finding of this study is that neural progenitor cells express topographic marker molecules, and after transplantation into host retina, the progenitor cells that become projecting neurons project to targets appropriate for their site of origin rather than for their site of implant. 3.1. Retinal progenitor cell phenotypes
Fig. 4. Retinal progenitor cells implant in all retinal strata and express morphologies appropriate for the stratum of implant. (A) An image of E19 chick retina stained with Toluidin Blue that stains cell bodies of each stratum of retina. Drawn on this image are the representative morphologies of cells of each functional class of retina first shown by Cajal with Golgi silver stain (Thorpe and Glickstein, 1972). (B) Retinal flat stem cells that adhere to the plastic cell culture flask stained with (A) anti-NCAM antibody show cell surface staining and (B) anti-GFAP antibody showing intermediate filament GFAP staining. All flat stem cells expressed both neuronal NCAM and glial GFAP. Scale bar = 50 lm. (C) Images of retinas with implanted stem cells after S-phase BrdU incorporation labeled with antibody to BrdU. E8 retinal cells were labeled with Cell Tracker Orange (red) and injected into the vitreal space of host chick embryo eyes. Two days post-injection 10 lg of BrdU were injected into the amniotic space of the embryo. At E20 retinas were stained with anti-BrdU (green). CTO stained stem cells with nuclei containing incorporated BrdU (yellow) are shown implanted in the photoreceptor layer and the ganglion cell layers of host retina. The retinas were counterstained with DAPI (blue) to show the retinal cell nuclei and retinal cell strata. (D) Images of implanted cells that express morphologies appropriate for the stratum of implant. Transplanted CTO-labeled E8 cells differentiated morphologically into (A) Müller-like (small arrow), bipolar-like (solid arrowhead) and amacrine-like cells (open arrowhead); (B) Müller-like cell; (C) diagonal cone-like photoreceptor; (D) rod-like photoreceptor; (E) conventional cone-like photoreceptor; (F–H) horizontal-like neuron; (I and J) bipolar-like neuron; (K and L) amacrine-like neuron; (M–O) ganglion-like cell. The data show that transplanted cells migrate across the retina, implant in all strata of retina and differentiate morphologically into cells appropriate for the stratum of implant. Scale bar = 100 lm in panels (A–M) and 200 lm in panels (N and O).
Two types of neural stem cells were found in embryonic chick retina. One was freely floating neurospheres and the other was adherent flat stem cells that attach to the culture flask surface (Bernardos et al., 2007; Goolsby et al., 1999). These flat cells may be an actual second progenitor cell type or they may be Müller cells, the only previously known cell in chick retina that adheres to tissue culture flasks – Müller cells that can become progenitor cells. In culture, these flat cells express both neuronal NCAM and neurofilament genes and glial GFAP and galactocerebroside genes. If these flat cells are Müller cells or derived from Müller cells, it suggests that Müller cells behave as progenitor cells in embryonic retina like radioglial cells behave in embryonic brain. Both floating progenitors and flat progenitors were able to migrate to all cell strata of the host retina and differentiate morphologically into cell types appropriate for cells in each stratum. The flat cells had more limited potential for differentiation, but they did differentiate into both neurons and glia. 3.2. Topographic markers in retinal progenitor cells Both progenitor cell types in retina expressed topographic markers of cell position in a graded fashion. The progenitor cells expressed relative levels of the topographic molecule examined that were appropriate for the site of origin of the progenitor cells in retina. The progenitor cells examined for expression of topographic markers in this study were chick E8 cells. We do not know whether earlier progenitor cells express these markers and we do not know the embryonic age of appearance of these markers in progenitor cells. We have not found these topographic markers in embryonic stem cells or in adult bone marrow hematopoietic stem cells. It would be of interest to learn how stem cells, that do not express topographic markers, behave after transplantation when they differentiate into projection neurons in a topographically mapped field of the host. Do they project axons along with their neighbors
864
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865
to targets appropriate for their site of implant? Are these unmarked stem cells potential universal donors for topographic projection neuron replacement?
3.3. Topographic order of projecting neurons Three homologous classic models of projecting axons maintaining topographic order are, first, the visual system of goldfish, frogs and chick embryos where retinal ganglion cell axons project to optic tectum and other visual centers in the thalamus (DeLong and Columbre, 1965; Sperry, 1963). Secondly, the somatosensory system of rodent whisker barrels where topographic order is maintained along the sensory pathway from whisker barrel to ganglion V to brainstem to thalamus to sensory cortex (Schlaggar and O’Leary, 1994). And thirdly, the motor system of chicks and quail/chick chimeras where segments of embryonic spinal cord is surgically transplanted in reversed order or placed in an ectopic position along the spinal tract – motor neurons derived from the misplaced stem cells in the early cord project to targets appropriate for the site of origin of the motor neurons rather than for the site of implant (Lance-Jones and Landmesser, 1980). These models predicted molecular maps of cell position. In the visual system misplaced transplanted retinal ganglion cells maintained topographic order when projecting to the tectum, but the order was based on site of cell origin not site of cell implant. Some of the ventral cells transplanted into dorsal retina were labeled with DiA from dorsal tectum and other ventral cells were not labeled with DiA. Ventral cells in dorsal retina that were not labeled with DiA may be unlabeled because they did not reach the dorsal tectum due to their growth cones being misrouted, migrating over other retinal ganglion cell axons instead of on the tectal surface where the growth cones could read the topographical molecular map. On the other hand, some ventral cells in dorsal retina may not have had sufficient time (E6 to E20) to differentiate, to project their axons from the retina along the optic nerve, the optic tract and over ventral tectum to reach targets in dorsal tectum. In the present study, visual images detected in dorsal retina and transmitted to visual centers in the brain by ganglion cells derived from stem cells of ventral retinal origin would be interpreted by the brain as being images in the ventral visual field leading to confusion and perhaps, as a consequence, a misstep. Thus, a progenitor cell that expresses topographic markers cannot be used to functionally replace missing projection neurons at all positions in a topographically mapped field.
4. Materials and methods 4.1. Progenitor cell culture White leghorn chick (Gallus gallus domesticus) retinas from E8 embryos were dissociated and cultured in DMEM containing 10% FBS as reported (Trisler, 1990). From primary cultures, floating neurospheres were cultured separately. Neurons were physically removed from the monolayer of flat continuously dividing adherent progenitor cells by repeatedly shaking off the neurons and passaging the flat cells until there were pure cultures of flat cells.
4.2. Retinal cell labeling Retinal cells were labeled with Cell Tracker Orange (CTO) (Molecular Probes) as previously reported (Trisler et al., 1996).
4.3. Retinal cell transplantation CTO-labeled retinal cells were injected into the vitreal space of E6 chick embryos in windowed eggs as previously reported (Trisler et al., 1986, 1996). Transplanted embryos were incubated to E16 or E20. 4.4. Topographic labeling of optic tectum The topographic map of the retinotectal projection was demonstrated by injecting crystals of FAST BLUE into dorsal tectum and Diamido Yellow into ventral tectum. The embryos were incubated for 2 days to allow retrograde transport of the dyes to the ganglion cell bodies in retina. For transplanted retinas the embryos were incubated to E20 to allow as much time as possible for differentiation of the implanted progenitor cells. At E20 the embryos were killed and suspended in 4% paraformaldehyde at 4 °C for 4 days then crystals of 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) (Molecular Probes) were injected into dorsal tectum. The embryos were suspended in 4% paraformaldehyde at 4 °C for 3 months to allow retrograde diffusion of DiA from tectum to retina. 4.5. Semi-quantitative PCR Expression of topographic molecule mRNA in retinal progenitor cells was determined by standard semi-quantitative PCR as previously reported (Pessac et al., 2011). 4.6. Western blot Topographic marker protein expression by retinal progenitor cells was determined by standard Western blot analysis as previously reported (Savitt et al., 1995). Conflicts of interest J.G., C.T.B. and D.T. hold USA (10/982,381) and International (PTC/US04/37122) Patents Pending. Acknowledgements The authors thank Deborah Yarnell and David Ford for technical assistance in this project. This work was supported by R.S. (VA REAP), T.M. (VA Merit), P.S.F. (VA REAP), C.T.B. (VA Merit) and D.T. (VA Merit and Abraxis Biosci.). References Barbieri, A.M. et al., 1999. A homeobox gene, vax2 controls the patterning of the eye dorsoventral axis. Proc. Natl. Acad. Sci. USA 96, 10729–10734. Bernardos, R.L., Barthel, L.K., Meyers, J.R., Raymond, P.A., 2007. Late-stage neuronal progenitors in the retina are radial Müller glia that function as retinal stem cells. J. Neurosci. 27, 7028–7040. Braisted, J.E. et al., 1997. Graded and laminin-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system. Dev. Biol. 191, 14–28. Constantine-Paton, M. et al., 1986. A cell surface molecule distributed in a dorsoventral gradient in perinatal rat retina. Nature 324, 459–462. DeLong, G.R., Columbre, A.J., 1965. Development of the retinotectal topographic projection in the chick embryo. Exp. Neurol. 14, 351–363. Ekker, S.C. et al., 1995. Patterning activities of vertebrate hedgehog proteins in the developing eye and brain. Curr. Biol. 5, 944–955. Goolsby, J., 2004. Transplantation of stem cells into the central nervous system. Ph.D. Dissertation. Goolsby, J., Rosenthal, C., Bever, C., Trisler, D., 1999. Retinal engineering: engrafted retinal cells of each functional class migrate and implant in the proper retinal stratum. Soc. Neurosci. Abst. 25, 1310. Hogan, B.L., 1996. Bone morphogenic proteins: multifunctional regulators of vertebrate development. Genes Dev. 10, 1580–1594.
J. Goolsby et al. / Neurochemistry International 61 (2012) 859–865 Knudsen, E.I., Knudsen, P.F., 1983. Space-mapped auditory projections from the inferior colliculus to the optic tectum in the barn owl (Tyto alba). J. Comp. Neurol. 218, 187–196. Koshiba-Takeuchi, K. et al., 2000. Tbx5 and the retinotectum projection. Science 287, 134–137. Lance-Jones, C., Landmesser, L., 1980. Motorneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord. J. Physiol. 302, 581–602. Mey, J., Thanos, S., 2000. Development of the visual system of the chick. I. Cell differentiation and histogenesis. Brain Res. Brain Res. Rev. 32, 343–379. Pessac, B., Ait Bara, M., Ford, D., Patibandla, G., Trisler, D., 2011. Hematopoietic progenitors express embryonic stem cell and germ layer genes. C. R. Biol. 334, 300–306. Ramón y Cajal, S., 1972. The Structure of the Retina (Thorpe, S. A., Glicustein, M., Compilers and Translators). Charles C. Thomas, Springfield. Rissi, M. et al., 1995. Zebrafish Radar: a new member of the TGF-beta superfamily defines dorsal regions of the neural plate and the embryonic retina. Mech. Dev. 49, 223–234. Savitt, J., Trisler, D., Hilt, D., 1995. Molecular cloning of TOPAP: a topographically graded molecule in the developing chick visual system. Neuron 14, 253–261.
865
Schlaggar, B.L., O’Leary, D.M., 1994. Early development of the somatotropic map and barrel patterning in rat somatosensory cortex. J. Comp. Neurol. 346, 80–96. Sperry, R.W., 1963. Chemoaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl. Acad. Sci. USA 50, 703–710. Trisler, D., 1990. Cell recognition and pattern formation in the developing nervous system. J. Exp. Biol. 153, 11–27. Trisler, D., Collins, F., 1987. Corresponding spatial gradients of TOP molecules in the developing retina and optic tectum. Science 237, 1208–1209. Trisler, G.D., Schneider, M.D., Nirenberg, M., 1981. A topographic gradient of molecules in retina can be used to identify neuron position. Proc. Natl. Acad. Sci. USA 78, 2145–2149. Trisler, D., Bekenstein, J., Daniels, M.P., 1986. Antibody to a molecular marker of cell position inhibits synapse formation in retina. Proc. Natl. Acad. Sci. USA 83, 4194–4198. Trisler, D., Rutin, J., Pessac, B., 1996. Retinal engineering: engrafted neural cell lines locate in appropriate layers. Proc. Natl. Acad. Sci. USA 93, 6269– 6274. Wagner, E., McCaffery, P., Drager, U., 2000. Retinoic acid in the formation of the dorsoventral retina and its central projections. Dev. Biol. 222, 460–470.