Spatial and Temporal Patterns of Neurogenesis in the Chick Retina

European Journal of Neuroscience, Vol. 3 , pp . 559-569 @ European Neuroscience Association 0953-81 &/91 $3.00

Spatial and Temporal Patterns of Neurogenesis in the Chick Retina

Carmen Prada’**, Jose Puga’, Luisa Perez-Mendez’, Rosario Lopez’ and Galo Ramirez2 ’ Departmento de Fisiologia, Facultad de Medicina, Universidad Cornplutense, 28040 Madrid, Spain 2Centro de Biologia Molecular (CSIC-UAM), Universidad Autdnonia, Canto Blanco, 28049 Madrid, Spain

Key words: chick retina, [3H]thymidine labelling, autoradiography, cell dissociation, cell birth, neurogenetic gradients

Abstract

Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal-to-ventral and temporal-to-nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Muller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Muller and bipolar cells. Finally, 1000’0 levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Muller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.

Introduction The accumulated evidence from studies dealing with the time and sequence of generation of the different cell types (neuronal and glial) in the central nervous system has contributed significantly to our understanding of the mechanisms by which cellular diversity is created from an apparently homogeneous population of neuroepithelial cells (Jacobson, 1978). In the case of the vertebrate retina, autoradiographic studies in different species (Xenopus, Jacobson, 1968; Holt et al., 1988; frog, Hollyfield, 1968; chick, Fujita and Horii, 1963; Kahn, 1973, 1974; mouse, Sidman, 1961; Carter-Dawson and La Vail, 1979; Young 1985; and cat, Zimmerman et al., 1988) have revealed (i) the existence of a central-to-peripheral gradient of neurogenesis, and (ii) that, in any portion of the retina, ganglion cells (the largest neurons in the retina, with axons projecting to diverse areas in the brain) start leaving the cycle before the different intemeuron (bipolar, horizontal and amacrine) and photoreceptor cell types. Cell generation patterns are not however identical in all vertebrate species: thus, while in Xenopus the full cell type complexity is attained in retina after only -25 h (between stages

24 and 37, Holt et al., 1988), retinal neurogenesis in the chick and mouse, extends for several days, with different cell types appearing in overlapping succession (Kahn, 1974; Young, 1985; Spence and Robson, 1989). Neurogenesis in the cat retina seems to occur essentially as in the two latter species (Zimmerman et al., 1988), although detailed studies are not available.

Although the avian retina would appear to be a very convenient experimental model for neurogenetic studies, the only overall analysis of chick retinal neurogenesis (Kahn, 1974) is not complete, and some of its conclusions, notably on photoreceptor birth dates, do not agree, either with previous evidence (Fujita and Horii, 1963; Morris, 1973), or with the more recent results of Spence and Robson (1989). This relatively scarce and somewhat contradictory information on the spatial and temporal patterns of chick retinal neurogenesis has prompted us to undertake this investigation, trying to improve our understanding of the early developmental events which condition the progressive build- up and final structure of the mature chick retina.

Correspondence to: Professor G . Ramirez, as above

Received 18 October 1990, revised 13 December 1990, accepted 6 March 1991

560 Neurogenesis in the chick retina

Materials and methods Rationale for PHlthymidine labelling: analysis of unlabelled cells It is possible to give enough [3H]thymidine to developing chick embryos so that every neuroepithelial cell entering the cycle will incorporate the precursor into DNA. The progeny of these cells will then appear labelled in autoradiographs prepared at later stages of development. Any cells having left the cycle up to the time of injection will, however, appear unlabelled. This technique of cumulative labelling has been used in previous studies on chick retinal neurogenesis (Fujita and Horii, 1963; Moms, 1973; Kahn, 1973, 1974; Spence and Robson, 1989).

In ovo injection of [3H]thymidine White-Leghorn chick embryos were raised to the desired stage of development in a forced-draft incubator, at 37.5"C and 60% relative humidity. Embryos usually received single doses of 25 pCi of [6-3H]thymidine (Amersham; 23 Ci/mmol, at 1 mCi/ml) into the chorioallantois, through a window made in the shell. This window also facilitated the staging of young embryos according to Hamburger and Hamilton (1951). After the injection, windows were sealed with transparent cellophane tape, and the eggs returned to the incubator.

Tissue section autoradiographs Two independent series of labelling experiments were carried out. In the first series, two embryos were injected per day, at any one day between 2 and 11 days of incubation (E2-Ell). The injections were given late on the second day of incubation, and early on the other days. All embryos were removed at E14, when the layer structure of the retina is already complete. Pieces containing both eyes were cut from the heads with a razor blade and fixed in Bouin's solution. After a rinse in water, the tissues were immersed in ammoniated 70% alcohol, to eliminate any residual picric acid, and then dehydrated through a series of alcohols, and embedded in paraffin. Transverse serial sections (parallel to yy', Fig. 1, upper right) of 6- 10 pm were cut and mounted on albumin-coated slides. After deparaffinization, the slides were dried first at room temperature, and then at 37°C for 12 h. The slides were dipped in Kodak NTB2 emulsion (diluted 1 : 1 with distilled water), slowly dried in a humid atmosphere, and exposed for 3 -4 weeks, at 4"C, in light-tight boxes containing silica gel. After exposure, the autoradiographs were developed with Kodak D-19, stained wth 1 % thionin, dehydrated again, and coverslipped.

Dissociated retinal cell autoradiographs In the second series of experiments, chick embryos were injected at any one day between E5 and E12. The embryos were processed between E l 7 and E21. Retinas were removed and dissociated by enzymatic digestion, using a Streptomyces protease preparation (kindly supplied by Compaiiia Espaiiola de Penicilina y Antibibticos). Tissues were incubated at 35"C, for 30-60 min, keeping the protease concentration at -0.06 mglml, in a medium containing 6 % sucrose. Dissociation was aided by gentle pipetting using a Pasteur pipette with a fire-polished tip (a more detailed procedure will be published elsewhere). The resulting single-cell suspension consistently contained a mixture of intact (i.e. including all processes) photoreceptor, bipolar, and Miiller cells. Horizontal and amacrine cell types were also frequently seen, while entire ganglion cells were only obtained when dissociating El7 retinas. Drops of the suspension were spread onto gelatin-coated microscope

slides, quickly air-dried, and, after 6-8 h at room temperature, cells were fixed in ethanol-acetic acid (3: l ) , followed by three 15-min rinses in distilled water. The slides were dried again at room temperature, dipped in Kodak NTB2 emulsion (diluted 1: 1 in distilled water) for autoradiography, exposed for -4 weeks, at 4"C, and developed with D- 19. The slides were dried after development, provisionally mounted with a drop of water, and coverslipped for examination by phase contrast microscopy.

Quantitative evaluation of autoradiographs Birth dates for each class of retinal neurons were estimated by counting unlabelled (i.e. born) cells in section and/or dissociated cell autoradiographs, For birth date determinations in section autoradiographs, we have chosen an area, close to the posterior pole of the eye, slightly temporal, and dorsal to the optic nerve head (onh). and only sections passing through the onh have been considered (Fig. I , lower right). The small window in the figure, marks the approximate location within the dorsocentral sector (Fig. 1, lower left, DC) of sections where birth dates were actually estimated. A field of the retinal layers within ten perpendicular divisions of an ocular grid, using a 25 x objective, was selected for cell counting. In each case, the total number of cells (our 100% reference) was first determined in Feulgen- stained sections. Unlabelled photoreceptor and ganglion cells were counted in their respective layers; horizontal cells were counted in the outer row of the inner nuclear layer (inl), while the internal third of the inl was considered for amacrine cells. Counts were taken in at least two retinas, looking at different sections, and in several places within the dorsocentral sector (Fig. 1, lower left, DC). Miiller and bipolar cells could not be distinguished in section autoradiographs, and their birth dates were estimated in dissociated cell preparations. In these dissociated cell autoradiographs, microscopic fields were randomly selected, using again the 25 X objective. All labelled and unlabelled cells of each type were counted, up to 4000 cells per retina, in different slides. For any retinal cell type, the percentage of unlabelled cells has been plotted against the time of thymidine injection.

When analysing the spatial and temporal patterns of neurogenesis, unlabelled cells were counted in all areas and sectors, following the same procedure outlined above. These counts have been used to visualize the progression of the neurogenetic gradients, and to establish the overall periods of neurogenesis for each retinal cell type. In the case of ganglion and photoreceptor cells we have also been able to draw neurogenetic maps on flattened retinal diagrams.

Reference system for the spatial analysis of retinal neurogenesis To help with the description of our results we have divided the chick retinal surface in areas, and the transversal sections in sectors, as indicated in Figure 1. The eye lens is considered as the anterior pole (ap), with the posterior pole (pp) in the opposite retinal surface (upper left). This posterior pole is the theoretical centre of the retina to which the concepts of central vs. peripheral refer. An equatorial plane (ee'), perpendicular to the axis joining the two poles, separates the central and peripheral 'halves', while another horizontal plane (xx') passing through the dorsal end of the onh (not the geometric centre) defines the dorsal and ventral areas. Finally, the vertical (transverse) plane containing both pps (yy', upper right) separates the nasal and temporal halves of the eye globe. The transverse sections used in this study are parallel to this yy' plane.

Neurogenesis in the chick retina 561

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FIG. 1. Spatial landmarks used in the description of chick retinal neurogenesis. Upper left: View of a chick eye globe showing the anterior pole (ap, at the centre of the lens I) , the posterior pole (pp). and two reference planes, namely, xx', a horizontal plane passing through the dorsal limit of the optic nerve head (onh, see below), which separates dorsal (D) from ventral (V) areas (the dorsal part being larger), and an equatorial (vertical) plane ee' separating the central and peripheral 'halves'. on (arrow), optic nerve. Upper right: Top (dorsal) view of a chick head showing the position of the vertical plane yy', which contains both retinal posterior poles, and separates the nasal (N) and temporal (T) halves. The transverse sections used in this study are parallel to this plane. Lower left: Transverse section of the eye showing the arbitrary limits of the different sectors considered for the analysis of section autoradiographs. These sectors are, from dorsal to ventral, DP, dorsoperipheral; DE, dorsoequatorial; DC, dorsocentral; VC, ventrocentral; VE, ventroequatorial; VP, ventroperipheral. We have a150 indicated the position of the plane xx' and of the optic nerve. Counts for birth date analysis were made in the DC sector, in sections passing through the optic nerve head. Lower right: Flattened retinal diagram, with reference planes (axes) xx' and yy'. Retinal neurogenesis begins in the enclosed area and spreads in all directions, as indicated by the arrows, albeit at different rates (see text). The narrow window marks the approximate position of the fields, within the dorsocentral sector of the sections, where counts for cell birth date determination were actually carried out. Transverse sections through this window pass through the optic nerve head (onh), as mentioned previously.

Within these sections, six different sectors have been considered for counting purposes, namely, dorsoperipheral, dorsoequatorial, dorsocentral, ventrocentral, ventroequatorial and ventroperipheral (Fig. I , lower left). The position of the onh in the chick is such that the dorsocentral sector in the sections is considerably larger than the ventrocentral one (Fig. I , lower graphs).

Results

limits Of retinal neurogenesis T h e formation of the optic cup and optic fissure in the chick embryo takes place between stages 13 (E2) and 18 (E3) of Hamburger and Hamilton (1951). All cells are labelled, in section autoradiographs from


an E l 4 retina, after a single injection of tritiated thymidine given late on E2, except for some 10% of the ganglion cells (see Fig. 9) specifically located in a slightly dorsotemporal area, close to the posterior pole and to the optic disk (Fig. 1, lower right; see Fig. 7, shaded area). As already seen in our previous work (Prada et al., 1981), these ganglion cell neuroblasts are then the first to leave the cycle and start differentiation. On the other hand, retinal neurogenesis appears to end shortly after E12. Section autoradiographs from retinas injected at this time (Fig. 2) show a few labelled nuclei in the bipolar-Miiller (bi-M) layer, all throughout the retina, which cannot be easily ascribed to either cell type; however, dissociated cell autoradiographs from retinas injected at day 12 show clearly that all Miiller cells are unlabelled, and that only - 7 % of the bipolar cells in the whole retina show the tritium label (see Fig. 10). These observations demonstrate that bipolar cells are the last to leave the cycle. Neurogenesis in the chick retina therefore lasts for - 12 days.

Spatial pattern of neurogenesis When the sectors defined in the retina are compared, three components of the spatial pattern of neurogenesis become apparent. The first is a central-to-peripheral gradient. Retinal neurogenesis starts at E2 near the posterior pole of the retina, in an area slightly temporal, and dorsal to the onh (Fig. 1, lower right; see Fig. 7, shaded area), and gradually (during about 12 days) spreads towards the peripheral retina. During the entire period of neurogenesis, the two central sectors in transverse sections (DC and VC in Fig. 1, lower left) show more unlabelled cells than the two equatorial sectors (DE and VE) which, in turn, are more advanced in neurogenesis than the dorso- and ventroperipheral sectors (DP and VP). This is true for the entire population of cells and for cells in each layer, with the exception of a small population of cells in the bi-M layer that can be labelled up to E12, as shown in Figure 2. The central-to-peripheral gradient is already apparent in retinas injected at E6, and it is particularly evident in the autoradiographs of retinas injected at E7 (Fig. 3). Photoreceptors show the steepest central-to- peripheral gradient in the dorsal part of retinas injected at E7 (Figs 3 and 4), whereas horizontal cells show a less marked but quite similar gradient. Interestingly, both populations have the shortest generation periods (4 days) in the dorsal retina (Fig. 5) . The other classes of neurons have longer periods of neurogenesis (Fig. 5) and show consequently rather smooth gradients.

Besides this central-to-peripheral gradient of neurogenesis, we can distinguish a dorsal-to-ventral gradient. We have consistently found, throughout the different stages and layers, that the dorsal sectors have more postmitotic (unlabelled) cells than the corresponding ventral ones (compare for instance the dorsal with the ventral sectors in Figs. 3 and 6). Although this gradient is not so pronounced as the central-to- peripheral one, neurogenesis ends consistently earlier in the dorsal part of the retina (Fig. 5). Autoradiographic examples are given in Figure 6, where the left-side picture shows that ganglion, photoreceptor, horizontal and amacrine cell neurogenesis is complete by E8 in the central and equatorial sectors of the dorsal retina, where only -58% of the bi-M cells remain in cycle; in contrast, most photoreceptor, bipolar and Miiller cells, as well as significant proportions of horizontal cells in ventral retina, are yet unborn at E8 (Fig. 6 , right). Actually, in the VC sector, ganglion cells complete neurogenesis by E8, photoreceptor, horizontal and amacrine cells by E9, Miiller cells by E12, and bipolar cells shortly after E12.

Differences in neurogenesis can be equally observed when comparing the nasal and temporal portions of the retina (Fig. 3, N and T): many

DC v c

in1

ipl

gc l

FIG. 2 . Autoradiographs of fields in the dorsocentral (DC, left) and ventrocentral (VC, right) sectors, in retinal sections passing through the onh, obtained from E l 4 retinas labelled with [3H]thymidine on E12. The band of labelled cells seen in the inner nuclear layer ( id ) is found throughout the entire retina. In the VC sector the pigmented epithelium (pe) is detached from the neural retina. ph, photoreceptors; opl, outer plexiform layer; ipl, inner plexiform layer; gcl, ganglion cell layer. Bar = 20 pm.

more cells leave the cycle in the temporal than in the nasal side between E6 and E8, both in the dorsal and ventral areas of the retina. This temporal bias constitutes yet another component of the spatial pattern of retinal neurogenesis.

The existence of these dorsal-to-ventral and temporal-to-nasal gradients appears to depend not only on the eccentric (i.e. dorsotemporal) localization of the initial neurogenetic events, but also on the faster dorsotemporal propagation of cell birth waves up to about E7 (Figs 7 and 8). However, there is, from E7 onwards, a noticeable increase in the rate of spread of neurogenesis towards ventral and nasal areas, so that the more peripheral contours (100% born cells of a given type) in Figures 7 and 8 appear progressively more centred in the retinal map.

We have, then, been able to identify three distinctive components in the spatial pattern of chick retinal neurogenesis. The possible contribution of a fourth vitreal-to-ventricular gradient to the orderly development of retinal layering, was also examined, taking into account that ganglion cells, followed by amacrine cells, are the first types to start leaving the cycle. The sequence is, however, broken because horizontal and photoreceptor cells come next, finally followed by Miiller and bipolar (Figs 9 and 10).

Temporal pattern of neurogenesis Retinal neurons start to leave the cell cycle in the same order in every sector: ganglion cells first, followed by amacrine and horizontal cells, photoreceptor, and then Miiller and bipolar cells. However, these cell types reach their 50% and 100% population levels in a somewhat different order.

D P DE DC vc Neurogenesis in the chick retina 563

VP

N

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FIG. 3. Autoradiographs of transverse retinal sections from an El4 retina labelled with ['Hlthymidine on E7, showing the three gradients of neurogenesis described in the text. Series N comprises sector fields in a section nasal to yy' (Fig. 1, upper right); series YY' refers to sector fields in yy'; and series T include sector fields in a section temporal to yy'. The sectors considered have been defined in Figure 1 (lower left). Retinal layers as in Figure 2. The DC sectors contain more unlabelled cells than the DE ones, and these, in turn, more than the DP sectors. Clear-cut central-to-peripheral gradients can be observed in the case of photoreceptors and ganglion cells. Also, dorsal sectors show more unlabelled cells than their ventral counterparts, suggesting the existence of a dorsal-to-ventral gradient. Finally, within the central sectors, the relative number of unlabelled cells decreases as we go from temporal to nasal sections, with especially marked temporal-to-nasal gradients in the case of ganglion, photoreceptor and horizontal (the outermost row in the inl: arrowhead) cells. Bar = 20 pm.


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V FIG. 4 . Flattened chick retinal diagram showing the spatial distribution of unlabelled (born) photoreceptor cell neuroblasts at E7. The inner contour defines the area where 100% of the photoreceptor cells appear unlabelled. The surrounding contours define areas with decreasing proportions of unlabelled photoreceptors, thus establishing a spatial gradient of neurogenesis.

0 0 - V 0 - 1 Ganglion

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EMBRYONIC AGE (days) FIG 5. Overall periods of neurogenesis of the different chick retinal cell types, as estimated in [3H]thymidine autoradiographs. The left end in the segments (circle) marks the time when the first unlabelled cells of a given type can be identified in the retina, whereas the right end (arrowhead) shows the time when all retinal cells of that type have left the cycle. For ganglion, horizontal, photoreceptor and amacrine cells we have obtained independent dorsal (D) and ventral (V) neurogenetic time intervals (section autoradiographs). In the case of Miiller and bipolar cells, however, we show only one diagram per cell class because the neurogenetic periods were estimated in dissociated cell autoradiographs from whole retinas. A small proportion of bipolar cells (7%) appear labelled in El4 retinas injected at E12: bipolar cell neurogenesis should then end at about E13.

Retinal cell birth dates have been derived from data in section and dissociated cell autoradiographs. Section autoradiographs provide accurate counts of ganglion, amacrine and horizontal cell neuroblasts, and a reasonably good estimate of photoreceptor neuroblasts, while

FIG. 6. Autoradiographs of fields in the dorsocentral (DC, left) and ventrocentral (VC, right) sectors, in retinal sections passing through the onh, obtained from El4 retinas labelled with [3H]thymidine on E8. Whereas the label in DC is restricted to a band of cells in the in1 (note that the internal third, corresponding to the amacrine cell layer, and the outermost row of cells, occupied by horizontal cells, are nearly devoid of label), the VC sector shows considerable labelling in all cell layers, with the exception of the ganglion cell layer. Retinal layers as in Figure 2. Bar = 20 pm.

affording only a rough quantitation of the bi-M cell layer as a whole. Dissociated cell autoradiographs were necessary to precisely determine birth dates and time courses of accumulation of Miiller and bipolar cells, and have also proved convenient to confirm the results on photoreceptor cells obtained in section autoradiographs. The results obtained in section autoradiographs from the dorsocentral sector of the temporal retina are summarized in Figure 9. As said, ganglion cells start leaving the cycle at the end of E2 (or before: 10% are already postmitotic at this time), followed by amacrine and horizontal cells (3 and 2%, respectively, are postmitotic at E4), and then by photoreceptors (7% postmitotic at E5). In dissociated cell autoradiographs (Fig. 1 I), we have also found that photoreceptors start to leave the cycle at E4: 2.5% of all retinal photoreceptors appear unlabelled when the isotope is given at E5 (Fig. 10). Bipolar and Miiller cell birth dates could only be unambiguously evaluated in dissociated cell autoradiographs (Fig. 11). Muller cells start to leave the cycle at the same time (E4) than photoreceptors (Fig. 5); we counted 10% unlabelled (postmitotic) Muller cells in retinas injected at E5 (Fig. 10). whereas in the same retinas no unlabelled bipolar cells were found. In retinas injected at E6, 4% unlabelled bipolar cells were counted (Fig. 10). Therefore, bipolar cells are the last to start leaving the cycle, at E5 (Fig. 5).

Concerning the time course of accumulation of born (unlabelled) cells, Figure 9 (results from section autoradiographs) also shows that the rate of production of each class of neurons, in a given sector, is not constant during the period of neurogenesis. In the dorsocentral sector, the rates for all classes of cells evaluated are highest during


N

D

T

V FIG. 7. Flattened chick retinal diagram illustrating the progression of ganglion cell neurogenesis. The different contours enclose areas where 100% of ganglion cells appear unlabelled when [3H]thymidine is injected at the days shown. The first unlabelled ganglion cells can be found within the shaded area at E2.

D

N

v FIG. 9. Time course of accumulation of born (i.e. unlabelled) ganglion ( O ) , amacrine (A), horizontal (0) and photoreceptor (A) cells, as counted in the DC sector (see text and Fig. 1) of section autoradiographs. Points in the curves represent percentage of unlabelled cells in E l 4 retinal sections, when [3H]thymidine is injected at the dates shown.

E6*: 40% ganglion, 64% horizontal, 66% amacrine and 77% photoreceptor cells are produced at this stage. The 50% postmitotic cell level is attained by ganglion cells during E5, by horizontal, photoreceptors and amacrine cells during E6, and by the bi-M cell layer during E7

*Following the established practice, expressions such as 'during E6' and 'at E6' refer to the period of time when the embryo is already 6 days old but not yet 7, i.e. the 7th day of life.

100

15

50

25

0

EMBRYONIC AGE (days)

FIG. 8. Flattened chick retinal diagram illustrating the progression of photoreceptor cell neurogenesis. The different contours enclose areas where 100% of photoreceptors appear unlabelled when [3H]thymidine is injected at the days shown. The first unlabelled photoreceptor cells can be found within the shaded area at E4.

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5 6 7 8 9 1 0 1 1 1 2 EMBRYONIC AGE (days)

FIG. 10. Time course of accumulation of born (i.e. unlabelled) photoreceptor (A), Muller (0) and bipolar (I) cells, as counted in dissociated cell autoradiographs. Points in the curves represent the percentage of unlabelled cells in retinas injected with [3H]thymidine, at the dates shown in the Figure and dissociated between El7 and E21 (see Materials and methods).

(not shown in Fig. 9: approximate estimation from section autoradiographs; precise data for these cell types, obtained in dissociated cell autoradiographs, are given in Fig. 10). The 100% postmitotic cell levels are attained by ganglion, horizontal and photoreceptor cells at E7, by amacrine cells at E9 (Fig. 9) and by cells of the bi-M layer at El2


FIG. 11. Identification of labelled and unlabelled retinal cell types in dissociated cell autoradiographs. 1, Photoreceptors dissociated from an E l 8 retina labelled at E6 (Nomarski). The series has been arranged, from left to right, in decreasing degree of labelling, the last one being totally unlabelled. The arrows point to the inner segment, and the arrowheads to the axon terminals. 2, Bipolar cells (h) from an E20 retina labelled at E5. The arrows identify the outer processes and the arrowheads the inner processes. 2a, Phase contrast observation to allow easy observation of inner processes. 2b. Transmitted-light observation for adequate visualization of the label. 3, Strongly labelled Muller cell (M) dissociated from an El9 retina labelled at E7 (Nomarski). The arrow points to the outer process and the arrowhead to the ramifying inner process; ph, photoreceptor cells. 4, Miiller cells (M, one labelled, one unlabelled) dissociated from an El8 retina labelled at E l 0 (Nomarski). 5 , Muller cell (M) from an El7 retina labelled at E8. The thin arrows point to the small processes arising from the outer process (thick arrow). The arrowheads identify the inner processes; gc, ganglion cell. 5a, Phase contrast observation. 5h, Transmitted-light observation. Note that the Muller cell is weakly labelled while the ganglion cell is not labelled. All bars = 20 pm.


(approximately as above; data not shown). At this point, it is worthwhile to note: (i) that, at every stage, the most postmitotic cells are found in the ganglion cell layer, followed, between E5 and E7, by horizontal cells, photoreceptor cells and the remaining inner nuclear layer cells (Fig. 9); and (ii) that the rates of accumulation of ganglion, amacrine, horizontal and photoreceptor cells change with the stages, being maximal at day 6 in the dorsocentral sector. This temporal pattern seems to be repeated in all sectors.

In the case of photoreceptor, Muller and bipolar cells, the rates of accumulation of postmitotic cells have been further analysed by use of dissociated cell autoradiographs (Fig. 10). Slopes in Figure 10 are noticeably smaller than in Figure 9 because they refer to the whole retina. A small percentage (2.5%) of photoreceptors in the whole retina has left the cycle before E5, while the highest number (34%) of photoreceptors are generated during E6. Postmitotic photoreceptors reach the 50% point at E7, and the 100% at E10. Miiller cells start to leave the cycle at the same time as photoreceptors (9.8% by E5), and attain their highest rate of production (30%) on E8. Fifty per cent of all retinal Miiller cells are also postmitotic at E8, and 100% at E12. As said before, bipolar cells are the last to start leaving the cycle (just at E5), reaching their highest rate of production (35%) at E7, the 50% level at E8 and the 100% mark presumably soon after E12. Ganglion, amacrine and horizontal cells were not quantified by this method because they are easily evaluated in sections, and also because the conditions for the dissociation of these cells are not the same as for photoreceptor, Miiller and bipolar cells.

Finally, when independent evaluations are carried out in dorsal and ventral retina to analyse the time-course of production of each cell type, it is quite clear that for all cell types-except a small population of Muller and bipolar cells (Fig. 2)-neurogenesis starts and ends in the dorsal retina earlier than in the ventral retina (Fig. 5).

Discussion Spatial pattern of neurogenesis Chick retinal neurogenesis starts in a dorsotemporal area of the central retina, close to the posterior pole and to the onh (Fig. I , lower right; Figs 7 and 8, shaded areas), and gradually spreads to the periphery, ending at about E12. This eccentric outbreak of the cell birth process, together with an initially faster dorsotemporal propagation of the neurogenetic waves, determine additional dorsal-to-ventral and temporal-to-nasal gradients, which become less marked after E7. We have considered the three gradients as three components of the spatial pattern of neurogenesis, in agreement with the observed distribution of unlabelled cells in the different retinal areas and sectors (Figs 3 and 6). The central-to-peripheral gradient has been found in all the vertebrate species analysed so far (Xenopus, Jacobson, 1968; Holt e t a / . , 1988; chick, Kahn, 1974; mouse, Young, 1985 and cat, Zimmerman et a/. , 1988). whereas the dorsal-to-ventral gradient has been detected only in Xenopus retina (Holt et a/., 1988), and the temporal-to-nasal gradient has not been reported before. Neurogenetic gradients have also been described in other brain areas, such as hippocampus (Jacobson, 1978).

Although molecules directly responsible for neurogenetic gradients have not yet been found, the notion of a spatially organized distribution of molecules involved in the generation of spatial cell distribution patterns is not new and is compatible with accepted evidence. For instance, in the same case of the chick retina, early differentiation also proceeds in gradients: the central-to-peripheral and vitreal-to-ventricular

ones are well documented in morphological studies, and we have furthermore observed dorsal-to-ventral and temporal-to-nasal gradients of differentiation in well-impregnated Golgi preparations (C. Prada et al., unpublished data). Molecules distributed in gradients and supposedly related to these differentiation gradients have actually been found in the chick and rat retinas. In the chick retina, some cell surface molecules termed TOP (for toponymic) are distributed in a dorsal-to- ventral gradient from E5 onwards; in addition, an inverted gradient of these molecules is present in the tectum from E6 to El8 (Trisler and Collins, 1987), suggesting a role for TOP antigen as a positional marker involved in the generation of retinal and tectal gradients of differentiation, and even perhaps in the orientation of the retinotectal map. In the rat retina, the so-called JONES antigen has been found distributed in a dorsal-to-ventral gradient in the early stages of differentiation (Constantine-Paton et al., 1986). On the other hand, molecular differences between the dorsal and ventral retina (also expressed sometimes in gradients) have been found in the adult chick (Barbera, 1975; Gottlieb et al. 1976; Marchase, 1977; Irwin et a / . , 1985), where they have been shown to be involved in the preferential adhesion of dorsal retinal cells to ventral tecta, and ventral retinal cells to dorsal tecta, in vitro. The development of some cellular properties in the chick retina, such as kainate sensitivity (Catsicas and Clarke, 1987), uptake of lucifer yellow (Layer and Kotz, 1983) and the binding of peanut agglutinin (Liu et al., 1983) also follow spatial gradients very similar, if not identical, to the neurogenetic ones presented in this paper. Neurogenetic gradients could then be-at least in the chick visual system-the first manifestation in a chain of events leading to an asymmetric distribution of molecular and cellular properties responsible for positional reference and interactive specificity in organized neuronal fields, layers or circuits.

Temporal pattern of neurogenesis The temporal progress of neurogenesis has been monitored by analysis of unlabelled cells in section and dissociated cell autoradiographs. Birth dates of different chick retinal cells had been previously determined, in section autoradiographs, by Fujita and Horii (1963); Morris (1973); Kahn (1973, 1974) and Spence and Robson (1989). Our birth date determinations (Fig. 9) show that ganglion cells are the first to start leaving the cycle, at about the end of E2, in agreement with the results of Kahn (1973, 1974), and with the fact that ganglion cell neuroblasts can be first stained by the Golgi method at 2.5 days of incubation (Prada et al . , 1981) and can be labelled by the RA4 monoclonal antibody at E3 (McLoon and Barnes, 1989). Our Figure 9 shows that most ganglion cells in the field of the dorsocentral sector selected for this study (Fig. 1. lower graphs) leave the cycle between E5 and E7. These results fully agree with reports by Fujita and Horii (1963), Kahn (1973) and Spence and Robson (1989). However, Kahn later (1974) concluded that: ‘the majority of the ganglion, amacrine, horizontal and photoreceptor precursors begin to withdraw from the cycle on E3, completing the process by E5’ in apparent contradiction with his own earlier results on ganglion cell neurogenesis (Kahn, 1973). Our results on the birth dates of photoreceptor cells are also at variance with the above statement. Photoreceptors do not start leaving the cycle on E3, but rather on E4 (Figs 9 and lo), and their neurogenetic period does not end on E5, but on E7 in the central retina (and even later in the more equatorial and peripheral areas), our results being in complete agreement with the findings of Fujita and Horii (1963). and of Morris (1973). Furthermore, most of the horizontal and amacrine cell populations do not leave the cycle between E3 and E5, but rather


between E6 and E7. As for Muller and bipolar cells, they were usually considered to be the last types to become postmitotic, although no concrete experimental results were available until Kahn (1974) reported that they would leave the cycle from E6 onwards. Our dissociated cell autoradiographs show however quite clearly the first unlabelled Muller cells in retinas injected with ['Hlthymidine as early as E4, just at the same time when photoreceptors start leaving the cycle (Fig. 10).

There are many factors (temperature, natural variability of developmental progression among the embryos, counting methods, area of the retina selected for counting, among others) that can explain variations in the results concerning birth dates of chick retinal neurons, even when using the same technique of autoradiography. Some of these factors have been extensively discussed by Spence and Robson ( 1 989), and can explain small differences in the percent of unlabelled cells of any type obtained at different ages and in different laboratories. However, these authors did not consider these factors to be the only reason for the discrepancies observed between Kahn's results (1974) and those of Fujita and Horii (1963), or their own. Our point of view is that the disagreement between Kahn's results and those of Fujita and Horii (1963), Morris (1973), Spence and Robson (1989) and our own, reflects differences in the criteria used for autoradiograph interpretation. By consistent use of the unlabelled cell criterion, we have been able to reproduce the results of Fujita and Horii (1963) and Morris (1973), in the case of photoreceptor cells, and to extend these studies to the determination of birth dates of the other retinal cell types. We have not, however, included in this study any specific data on displaced amacrine and ganglion cells. Displaced amacrine cells are in fact mixed with ganglion cells at E l 4 and cannot be distinguished in our autoradiographs. Our estimates of ganglion cells in Figure 9 actually include all the displaced amacrine cells present in the gcl. Only when chicks are killed earlier (between E9 and E l 1) is it still possible to find them in the ipl. We used this approach to fix the birthdates of displaced amacrine cells, in the posterior pole, between E5.5 and E6.5 (Genis-Galvez ef al . , 1977), which agrees with the time-span of generation of all amacrine cells (Fig. 9). As for displaced ganglion cells, their very low incidence (some 10 OOO cells per retina, or 0.43 % of the total ganglion cell population (Prada et a / . , 1989) and their dispersion throughout the retina (being more numerous in the peripheral than in the central retina) preclude a precise examination of their temporal pattern of neurogenesis using the present analytical approach.

The technique of dissociated cell autoradiographs has been used here for the first time to validate our results on photoreceptors, which are not easy to count in section autoradiographs, and to accurately establish birth dates of bipolar and Muller neuroblasts. These cells have small somas, with uniformly stained, tightly packed cytoplasm, and are furthermore mixed together so that they cannot be distinguished in the thinnest (5-6 pm) paraffin sections or even in 1 pm plastic sections. These limitations were already pointed out by Hinds and Hinds (1978) in their studies on mouse retina. Although photoreceptors are not easy to count in sections, our approximate estimates are borne out by the results from dissociated cell preparations (compare Figs 9 and 10).

One of the most interesting results of our paper concerns the identity of the neuroblasts produced at the beginning and the end of chick retinal neurogenesis: whereas only ganglion cells appear during the first stages of proliferation (day 2-2 .5) , bipolar cells, and not other cell types, are produced at the close of the generation process (days 11 - 13). This suggests some kind of correlation between birth dates and retinal layers, in the chick retina, which has not been so clearly observed in other vertebrate species. Thus, in Xenopus, only a minimal correlation has

been ascertained because only ganglion cells are preferentially produced in the dorsocentral retina for a short period of time at the start of neurogenesis (Holt et al., 1988). In the mouse retina, cones, amacrines, horizontal and ganglion cells are produced prenatally, whereas bipolar and Muller cells are essentially postnatal. Only rods are produced at all times, being also the last cells leaving the proliferative cycle and thereby closing the neurogenetic process (Young, 1985). Thus, retinal patterns of neurogenesis are species-dependent, with marked differences in the overall and cell-type-specific neurogenetic periods, and in birth date/layer relationships.

Acknowledgements This work was supported by the Direccidn General de lnvestigacidn Cientifica y Tecnica (Grant 0441184) and Fundaci6n Ram6n Areces. We thank Juan I . Medina for his help with the preparation of the photographic plates.

Abbreviations hi-M D DC DE DP En

in1 ipl M N onh

Pe

T V vc VE VP

gcl

OPI

Ph

bipolar-Miiller cells dorsal dorsocentral dorsoequatorial dorsoperipheral embryonic day rz ganglion cell layer inner nuclear layer inner plexiform layer Miiller cell nasal optic nerve head outer plexiform layer pigment epithelium photoreceptor cell temporal ventral ventrocentral ventroequatorial ventroperipheral

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Spatial and Temporal Patterns of Neurogenesis in the Chick Retina

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