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Development 122, 65-78 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV8292 65 Clones in the chick diencephalon contain multiple cell types and siblings are widely dispersed Jeffrey A. Golden1,2 and Constance L. Cepko1,3 1Department of Genetics, Harvard Medical School, 2Department of Pathology, Brigham and Women’s Hospital, and 3Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA SUMMARY The thalamus, hypothalamus and epithalamus of the vertebrate central nervous system are derived from the embryonic diencephalon. These regions of the nervous system function as major relays between the telencephalon and more caudal regions of the brain. Early in development, the diencephalon morphologically comprises distinct units known as neuromeres or prosomeres. As development proceeds, multiple nuclei, the functional and anatomical units of the diencephalon, derive from the neuromeres. It was of interest to determine whether progenitors in the diencephalon give rise to daughters that cross nuclear or neuromeric boundaries. To this end, a highly complex retroviral library was used to infect diencephalic progenitors. Retrovirally marked clones were found to contain neurons, glia and occasionally radial glia. The majority of clones dispersed in all directions, resulting in sibling cells populating multiple nuclei within the diencephalon. In addition, several distinctive patterns of dispersion were observed. These included clones with siblings distributed bilaterally across the third ventricle, clones that originated in the lateral ventricle, clones that crossed neuromeric boundaries, and clones that crossed major boundaries of the developing nervous system, such as the diencephalon and mesencephalon. These findings demonstrate that progenitor cells in the diencephalon are multipotent and that their daughters can become widely dispersed. INTRODUCTION of the diencephalon resembles the more caudal mesencephalon (midbrain) and rhombencephalon (hindbrain), which are parceled into discrete nuclei with mostly discrete projections. How the intermediate structure of the diencephalon develops is not clear. The development of some of the more rostral and caudal regions of the brain have been relatively well described such that we can now appreciate that these areas follow different rules in key aspects of their development. The rhombencephalon is transiently parceled into 8 morphologically identifiable units known as rhombomeres. The neurons in each rhombomere comprise groups of nuclei, each with defined functions and a stereotypical pattern of axonal projections (Lumsden and Keynes, 1989; Keynes and Lumsden, 1990; Lumsden, 1990). A molecular basis for the morphological and functional specification of rhombomeres has been established through studies of the expression and misexpression of Hox genes and other transcription factors (Guthrie and Lumsden, 1991, 1992; Hunt et al., 1991; Guthrie et al., 1992). Lineage analysis conducted in the hindbrain of chicks has shown that once the boundaries of the rhombomeres are established, the majority of clones appear to be restricted to a single rhombomere (Fraser et al., 1990; Birgbauer and Fraser, 1994) during the next 48 hours of development. Clones also appear restricted in cell fate in that most clones comprise siblings that adopt the same or a related neuronal cell fate (Lumsden et al., 1994). One of the hallmarks of the adult nervous system is the exquisite complexity of its cell types and synaptic connections. The mechanisms that generate complexity during development remain largely unknown. Early in development, the neural tube is thought to be parceled into unique units or segments (Lumsden, 1990; Puelles and Rubenstein, 1993; Rubenstein et al., 1994; Rubenstein and Puelles, 1994). Distinct morphological units first appear shortly before neural tube closure when multiple vesicular outpouchings develop rostrally. The primary vesicles are termed the prosencephalon, the mesencephalon and the rhombencephalon. The prosencephalon gives rise to the telencephalon (cerebral hemispheres) and the diencephalon. The diencephalon is the embryonic precursor to the hypothalamus, thalamus and epithalamus, and is anatomically situated between the cerebral hemispheres and more caudal areas of the brain. The diencephalon appears to be conserved structurally and functionally throughout evolution, although there is some debate about whether the organization within the diencephalon, and the thalamus in particular, is similar across species (Kappers et al., 1960; Jones, 1985). The embryological origin of the diencephalon appears to be conserved in disparate species (Kappers et al., 1960). While functional aspects of the diencephalon appear to mimic the rostral telencephalon, the anatomical organization Key words: cell lineage, central nervous system, diencephalon, thalamus, chick, thalamus, hypothalamus 66 J. A. Golden and C. L. Cepko The telencephalon, the most rostral part of the central nervous system (CNS), is not separated into morphologically defined, repeated units. Rather, the cerebral cortex, the largest component of the telencephalon, is organized into a diffuse laminated sheet of cells. Although the cortex can be parceled into functional domains, there is no discrete nuclear organization as is found in the hindbrain and diencephalon. Studies investigating the expression pattern of a variety of genes, mostly transcription factors, have uncovered several genes with nested (Simeone et al., 1992; Bulfone et al., 1993a,b) or lamina-specific (Frantz et al., 1994a,b; Leifer et al., 1994) patterns of expression. However, genes investigated thus far are generally not expressed within morphologically or physiologically defined areas. Lineage analysis in the telencephalon has demonstrated that siblings spread over great distances to give rise to neurons in functionally and anatomically unrelated parts of the cerebral cortex (Walsh and Cepko, 1992, 1993; Reid et al., 1995). Lineage analysis in the telencephalon has shown that individual progenitor cells are capable of generating neurons and glia (Price and Thurlow, 1988; Walsh and Cepko, 1992; Levison et al., 1993; Reid et al., 1995). In the retina (an outgrowth of the diencephalon) and the chick tectum (a derivative of the mesencephalon), lineage analysis has also shown that neurons and glia arise from a common progenitor cell (Turner and Cepko, 1987; Galileo et al., 1990; Turner et al., 1990; Gray and Sanes, 1992; Fekete et al., 1995). The diencephalon is organized into individual nuclei (collections of neurons with a specific projection or defined set of projections) with some groups of nuclei having similar functional properties and other neighboring nuclei having distinct functions. Functionally, however, the diencephalon and, particularly, the thalamus closely resemble the telencephalon (cerebral cortex and basal ganglia). The thalamus functions as the major integration and projection region to the cerebral cortex from more caudal structures, including the spinal cord, cerebellum, hindbrain and midbrain (Jones, 1985). The neurons of the thalamic nuclei project to different cortical areas with many thalamic nuclei projecting to overlapping cortical regions. Furthermore, the thalamocortical projections to the cortex appear to define the functional cortical units (see Jones, 1985 for review). During development, the thalamocortical projections show growth into specific cortical areas and thalamocortical axons may provide cues that help define the course of cortical neuronal projections (reviewed in O’Leary and Koester, 1993). Given that the telencephalon and rhombencephalon appear to use distinct mechanisms for patterning and perhaps generating cellular diversity, it was of interest to determine clonal relationships within the diencephalon. Classical embryologists have described the diencephalon as arising from four horizontal strips (His, 1893; Herrick, 1910; Khulenbeck, 1973) or neuromeric units (Orr, 1887; Bergquist, 1952; Keyser, 1972) based on the presence of bulges and sulci along the medial walls of the third ventricle. These morphological units, labeled neuromeres or prosomeres (we have adopted the neuromere nomenclature in this paper), have been proposed to be analogous to the rhombomeres (Puelles et al., 1987; Figdor and Stern, 1993; Puelles and Rubenstein, 1993), although this analogy has not been completely tested (see Guthrie, 1995). Examination of the expression patterns of a variety of developmentally regulated genes has shown a correlation with the neuromeric units (Bulfone et al., 1993a,b; Rubenstein et al., 1994; Rubenstein and Puelles, 1994), similar to the correlation noted in the hindbrain. Short-term lineal relationships in the diencephalon have been analyzed using the method of single cell microinjection of a fluorescent dye (Figdor and Stern, 1993). The results indicated that the morphological neuromeres of the diencephalon are akin to the hindbrain rhombomeres in that clones were restricted to a single neuromere up to 48 hours after injection, the latest time point analyzed. However, this technique precludes analysis of the final patterns of clonal dispersion or the mature cell types within any one clone. Chick/quail chimera studies have also been performed in the diencephalon (Martinez and Alvarado-Mallart, 1989). In these studies, quail mesencephalon was transplanted into chick diencephalon, and thus the potential of diencephalic progenitors was not tested. Nonetheless, these studies indicated that mesencephalic progenitors dispersed to populate several, but not all, nuclei in the diencephalon. One interesting pattern of spread for the mesencephalic progenitors in these studies was that they selectively populated primary visual nuclei, suggesting a relationship with the tectum, the normal derivative of the mesencephalon. Using a complex retroviral library comprising DNA tags as lineage markers (Golden et al., 1995), we have evaluated 275 clones in the chick diencephalon. This technique allows analysis of clones in the mature diencephalon, after clonal dispersion is complete. We found that sibling cells can spread extensively within the diencephalon, sometimes occupying nuclei derived from more than one neuromere. Dispersion occasionally led to cells being located in both the diencephalon and the mesencephalon. Furthermore, siblings were found on both the left and right sides of the third ventricle in approximately 16% of the clones. We also found that progenitors of diencephalic cells were located in the ventricular zone of the third ventricle, as well as in the ventricular zone of the lateral ventricle. Clones frequently contained neurons, glia and radial glia, supporting the existence of multipotent progenitor cells. MATERIALS AND METHODS Production of the retroviral library CHAPOL Construction and characterization of the retroviral library used in this study have been described (Golden et al., 1995). Briefly, the library of retroviral vectors was constructed from a replication defective avian retrovirus, CHAP (Ryder and Cepko, 1994), that encodes the human placental alkaline phosphatase gene (PLAP). A pool of synthetic degenerate oligonucleotide tags with a theoretical complexity of >107 were cloned into the vector to create the vector library, CHAPOL. A large scale viral preparation yielded a stock with a concentration of 1.1×107 cfu/ml that was used for all experiments (Golden et al., 1995). Numerous experiments with this same stock of CHAPOL have led to recovery of >350 unique inserts (current paper and (Golden et al., 1995)). These data indicate that the CHAPOL library has a complexity of at least 105 members (Walsh et al., 1992). Furthermore, each insert has been recovered from only one infection event (i.e. only 1 brain or 1 well of an infected microtiter plate of tissue culture cells), indicating the library has an approximately equal distribution of inserts. In vivo infection The neural tube of fertilized virus-free White Leghorn chicken embryos was injected as previously described (Fekete and Cepko, Clones in the chick diencephalon 1993) using CHAPOL. Injections were performed at either stage 1012 or stage 16-17 (all staging according to Hamburger and Hamilton, 1951). Approximately 0.3-0.5 µl of CHAPOL stock was injected into the neural tube at stage 10-12 and 1.0-1.5 µl at stage 16-17. Infected embryos were incubated in a humidified, 37°C chamber until embryonic day 18 (E18) at which time the brains were harvested in PBS. After fixation overnight in 4% paraformaldehyde (in PBS, pH 7.4) at 4°C, brains were washed with three changes of PBS over 24 hours and then cryoprotected in 30% sucrose in PBS. Brains were oriented for coronal sections, embedded in OCT medium and cut on a Reichart-Jung CM3000 cryostat at 60 µm. Each section was collected and mounted sequentially on Superfrost/Plus microscopic slides (Fisher Scientific). On average, 3 sections/brain (approximately 45 sections included the diencephalon) were lost and the ordered position of each missed section was documented. Infected cells were identified histochemically by incubating the sections with X-phos (Research Organics, Inc.) and NBT (Research Organics, Inc.) for 60 to 240 minutes according to previously published protocols (Ryder and Cepko, 1994; Golden et al., 1995). Cells infected with the retrovirus were identified by the purple formazan precipitate. Coronal sections that included the diencephalon, from the anterior commissure (anterior limit of analysis) to the red nucleus (posterior limit of analysis) were photographed at 2× magnification with a Nikon SMZ-U stereomicroscope equipped with a Nikon DX FX-35WA Camera. Representative cells were photographed at higher magnification on a Zeiss Axiophot microscope. In addition, camera-lucida drawings of selected cells were made from a drawing tube mounted 67 on a Zeiss Axioskop. The 2× magnification Kodachromes were scanned into a Macintosh Quadra 650 using a Kodak 2035 plus slide scanner and Adobe Photoshop software. The location and cell type of each alkaline phosphatase positive (AP+) cell and/or cluster of cells was given a unique identification based upon the section number. Once documented, each cell or cluster of cells (with a small group of surrounding white AP− cells) was removed using heat-pulled glass micropipettes and transferred to a 96-well PCR plate with 10 µl of 400 µg/ml proteinase K solution (Golden et al., 1995). Approximately 50-60 AP+ cells were picked from sections and analysed on each 96well PCR plate. One or two picks from a similar stage uninfected brain that had been processed in parallel with the experimental brain were included on each plate as negative contrals. The cells were then digested, amplified and sequenced as previously described (Golden et al., 1995). Once all sequences were collected and stored, all common sequences could be grouped and the position and cell type(s) for sibling cells revealed. Each AP+ cell or cluster or AP+ cells was then reidentified on the computer images to obtain a map of the distribution of sibling cells in the diencephalon. The position of each clone was also transposed onto an atlas of the chick diencephalon (Kuenzel and Masson, 1988). AP+ cells were anatomically positioned on atlas sections by first defining their location within nuclei or white matter tracts on histological sections (Fig. 1). Since all nuclei and white matter tracts could not be defined on the AP-stained sections, cells outside anatomically definable structures were localized according to their position relative to defined structures. However, the majority of cells could be definitively assigned based upon the tissue morphol- Fig. 1. Examples of distinct cell types and histological identification of diencephalic nuclei. (A-F) Representative examples of cell types identified on the basis of morphology. Neurons were identified by the presence of long thin processes. Glia (astrocytes) were identified by intense staining and indistinct borders, with occasional thick short processes. Radial glia were identified by a cell body apposed to the ventricular surface and a long thin process radiating into the parenchyma. Cells that could not be identified were generally small and round with no clear morphological feature. (G,H) Two representive sections of the diencephalon stained for AP activity. The left half of each figure is a schematic outline of the nuclei and white matter tracts that could be defined as a result of the AP histochemistry and anatomical landmarks. Note the AP+ cells in the nucleus rotundus in G and the nucleus ovoidalis in H. Scale bar 100 µm for A, B and E; 50 µm for C; 267 µm for D; 200 µm for F; and 450 µm for G and H. AL, ansa lenticularis posterior; DLAl, nucleus dorsolateralis anterior thalami (lateral); DLAm, nucleus dorsolateralis anterior thalami (medial); FPL, fasciculus prossencephali lateralis (lateral forebrain bundle); GLv, nucleus geniculatus lateralis, pars ventralis; ICT, nucleus intercalatus thalami; OM, tractus occipitomesencephalicus; OT, optic tract; OV, nucleus ovoidalis; ROT, nucleus rotundus. 68 J. A. Golden and C. L. Cepko ogy. The following structures were identified on histological sections: ansa lenticularis, anterior commissure, nucleus anterior medialis hypothalami, posterior commissure, nucleus dorsointermedialis posterior thalami, nucleus dorsolateralis anterior thalami (lateral), nucleus dorsolateralis anterior thalami (medial), nucleus dorsolateralis posterior thalami, nucleus dorsomedialis anterior thalami, nucleus dorsomedialis posterior thalami, fasciculus prossencephali lateralis (lateral forebrain bundle), nucleus geniculatus lateralis, pars ventralis, nucleus intercalatus thalami, optic chiasm, tractus occipitomesencephalicus, optic tract, nucleus ovoidalis, nucleus paraventricularis hypothalami, nucleus rotundus, tractus tectothalamicus. Three-dimensional reconstruction of the diencephalon was performed by transforming the Photoshop two-dimensional images using Spyglass Dicer. DiI labeling Radial glia were labeled as previously described (Gray and Sanes, 1992). Briefly, brains from uninfected E15-E18 chicks were dissected in PBS and fixed overnight in 4% paraformaldehyde. After washing in PBS, approximately 0.1 ml of a 2.5 mg/ml solution of DiI (Molecular Probes, Inc.) in 100% ethanol was injected into the right lateral ventricle of the brain using a 30-gauge needle. The DiI solution was allowed to passively fill the entire ventricular system. The brains were then placed in a 60 mm Petri dish filled with sterile water and 0.03% sodium azide and incubated for two weeks at 37°C. After incubation the brains were imbedded in 5% agar (in H2O) and sectioned on a vibratome at 100-200 µm. The sections were mounted on Superfrost/Plus microscopic slides, coverslipped with gelvatol and viewed with rhodamine fluorescent filters on a Zeiss Axiophot microscope. Photos were taken with Kodak Elite 100 film. RESULTS Summary of brains analyzed and recovery rates of infected cells The clones described here were isolated from seven E18 brains. Injection of the retroviral library was at stage 10 (n=1), stage 11 (n=1) or stage 16-17 (n=5). Small tissue samples containing a single alkaline phosphatase (AP+) cell or small clusters of AP+ cells along with surrounding AP− tissue were removed (each sample is referred to as a ‘pick’). The total number of picks was 1,537 from the diencephalon of the 7 brains. An additional 263 picks were collected from areas with no visible AP+ cells. PCR amplification yielded the predicted 121 base pair product from 1,088 (71%) of the 1,537 picks. Sequencing of the PCR products revealed that 70% (761) of the AP+ regions isolated contained a single insert and 17% (186) contained multiple inserts. (Those with multiple inserts were not analyzed further since identification of more than one sequence implies that cells that are members of more than one clone were present in the tissue sample analyzed. If one AP+ cell was present, it was impossible to define which sequence belonged to the AP+ cell and which belonged to the AP− cell(s). Similarly, if two or more AP+ cells were present, it was again impossible to determine which sequence belonged to which cell or cells. Furthermore, technical limitations have made it difficult to separate multiple PCR products, so that the sequence of each can be defined (Golden et al., 1995).) No sequence was obtained in 141 (13%) of the cases. Efficiency of recovery of PCR products and sequences was similar from single cells and small clusters of cells (data not shown, and Golden and Cepko, 1995). The 761 picks that gave a single sequence were classified into 275 clones (the same insert has never been recovered from more than one brain). Of these, 154 picks contained an insert that was recovered only once and were therefore defined as ‘single pick clones’. These 154 clones had from 1 to 8 cells (i.e. each pick had from 1 to 8 cells), 118 having a single cell. 121 insert sequences were obtained 2 or more times from the 761 picks and these were defined as ‘multiple pick clones’. Each pick from the multiple pick clones also had from 1 to 8 cells. The single pick clones and multiple pick clones are analyzed separately below. Single pick clones The 154 single pick clones contained an average of 1.52 cells of which 0.4 were neurons, 0.4 were glia and 0.1 were radial glia. An average of 0.6 cells in each clone was not classifiable on morphological criteria alone (Fig. 1). Oligodendrocytes were rarely found and difficult to identify morphologically in this study. Since they represented such a small subset of cell types identified, they have been lumped with astrocytes in the category of glial cells. The data derived from the single cell clones provide an approximation of the birthdates for different cell types (see Table 1 and Discussion). For example, a greater percentage of the single pick clones with more than one cell, compared to single pick clones with one cell, comprised glia only (31% vs 5%). This suggests that progenitors that ultimately gave rise only to glia were making some mitotic daughters at the time that they were infected with the retrovirus. In contrast, the percentage of neurons from single pick clones with multiple cells, compared to those with a single cell, was relatively similar (22% vs 27%). Thus neurons were being generated at the time of injection more often then glia and some progenitors produced mitotic daughters that gave rise only to neurons. Furthermore, the percentage of single pick clones with a single cell that was a neuron (27%) compared to the percentage of clones containing neurons in the entire data set (60%) was greater than the comparable numbers of clones containing glia (5% compared to 65%). Thus, neurons were far more frequently represented in the single cell clone category. Table 1. Cell type composition of single and multiple pick clones Single pick Multiple pick multiple cells single cell total 121 36 118 154 59(49) 6(5) 16(44) 8(22) − 32(27) 48(31) 40(26) Include glia Glia only 74(61) 9(7) 17(47) 11(31) − 6(5) 23(15) 17(11) Include radial glia Radial glia only 20(17) 0 3(8) 2(6) − 10(8) 13(8) 12(8) (a) Number of inserts recovered (b) Number (percent) clones with cell types Include neurons Neurons only The number (and percent) of clones that include the indicated cell type are grouped according to whether they are multiple pick or single pick clones. Multiple pick clones are the clones that gave the same insert from multiple tissue picks in the same brain. Single pick clones are clones in which an insert was recovered only once. The single pick clones are further divided into the picks comprising a single cell or multiple cells. Clones in the chick diencephalon Analysis of the composition of small clones reveals that there are more radial glia than would be predicted based upon the overall frequency of radial glia in the entire data set. 10 of the 33 (30%) clones that contained radial glial cells were single cell clones. An additional 2 multiple cell clones from the single pick clones were composed of radial glia only, making a total of 12 of 33 (36%) clones with only radial glia. These two clones that contained radial glial cells as their only members indicate that there may be progenitor cells that can divide to give rise to 2 or more radial glia (also see Gray and Sanes, 1992). However, since there were no multiple pick clones composed entirely of radial glia, such a progenitor cell, if it exists, does not produce many radial glia. Analysis of the distribution of the 118 single cell clones in three dimensions suggested no particular bias for an anteriorto-posterior, medial-to-lateral, or dorsal-to-ventral birth order. However, the limited number of clones and injection times preclude definitive assessment of birthdating gradients in the diencephalon. Multiple pick clones 121 inserts were recovered from two or more picks in the same brain. The average number of cells in these clones was 7.8. The average numbers of neurons, glia and unidentified cell types in these clones were 1.4, 3.6 and 2.8 respectively. In contrast to the single pick clones, multiple pick clones frequently had multiple cell types (Table 1). It remains possible that some of the clones that contained unidentifiable cells would have been classified as clones with only one cell type. Nonetheless, 43 of the 121 (36%) clones had phenotypically distinguishable cells (Table 2), either neurons and glia, neurons and radial glia, glia and radial glia, or all three. These data indicate that multipotent progenitor cells were frequently infected. 101 of the clones were found exclusively on either the left or right side of the lateral ventricle. Table 2 is a list of all 121 multiple pick clones and includes the composition of cell types and the extent of dispersion in the anterior-posterior, mediallateral, and dorsal-ventral planes. Figs 2-4 are composite maps showing the distribution of cells within clones from a subset (44/121) of the multiple pick clones and Fig. 5 shows the anterior to posterior spread (also see Fig. 2, clone 3 and Fig. 3, clone 7). Clones were observed to spread from 2% (60 µm) to 54% (1140 µm) of the anterior to posterior extent of the entire diencephalon, with the average clone extending 11% of the anterior-posterior distance. Thus cells were separated by approximately 350 µm on average along the anterior-posterior axis. Spread of sibling cells was most pronounced in the medial-lateral axis, where the average extent of spread was 57.5% (between 1500 and 2000 µm; table 2), and spread ranged from none (cells located along the third ventricle) to spread across virtually the entire diencephalon from the third ventricle to the lateral margin (in Fig. 3G compare clone 8 to clone 5). Cells also spread along the dorsal-ventral axis across an average of 19% of the entire diencephalon, corresponding to approximately 1000 µm. The range of spread was from 1% (5 µm) to 74% (2900 µm; Table 2). The spread of cells in the dorsal-ventral plane showed a common bias toward the ventral side with progressively more laterally displaced cells. Siblings leaving the third ventricle, the most common site of birth, 69 appeared to disperse both laterally and ventrally (e.g. Fig. 2, clones 2 and 3; Fig. 3, clone 9; and Fig. 4, clones 12 and 14). Some clones showed a more limited dorsal to ventral dispersion with extensive lateral dispersion (e.g. Fig. 2, clone 12 and Fig. 3 clone 5), but few showed extensive dorsal-ventral dispersion without accompanying lateral dispersion. While the vast majority of clones showed lateral and ventral spread, several clones violated this general trend. Two clones were found to spread a significantly greater percentage along the dorsal to ventral axis compared to the medial to lateral axis (Table 2, asterisked clones). The dispersion of clones in three planes indicates that cells from an individual clone occupy >1 nucleus of the diencephalon (e.g. Fig. 2, clones 1 and 10; Fig. 3, clones 1 and 14; and Fig. 4, clone 3). Clonally related neurons and glia could be found in nuclei that are both adjacent to and distant from each other. Some clones remained localized to a single nucleus or cluster of nuclei (e.g. Fig. 2, clone 6; and Fig. 4, clone 13). Since sibling cells populated multiple nuclei, we attempted to define whether clones crossed neuromeric boundaries. However, detailed maps delineating which mature structures are derived from the developmentally defined neuromeres are not available for any species. Thus, in order to provide an estimate of the number of clones that occupy derivatives of >1 neuromere, we extrapolated from the data of Figdor and Stern (1993), identifying only those clones that clearly crossed neuromeric boundaries (Table 3). Cells that were in the region of a proposed boundary (Puelles and Zabala, 1982) were placed in the ‘could not determine’ category of Table 3. Based on this analysis, at least 8% of all clones crossed neuromeric boundaries. Because the precise boundaries have not been defined for the mature diencephalon, this represents the most conservative estimate possible. It is likely that some of the 47% of the clones that could not be definitively assessed did cross a neuromeric boundary. Furthermore, the fact that many clones were found within a single neuromere does not mean that they were restricted by the boundaries of that neuromere. Unusual clone types Several clones were found with unusual distributions of cells or an unexpected site of genesis. One type of clone had cells on both sides of the midline surrounding the 3rd ventricle. 20 clones (16%) in the data set were found with a bilateral distribution (Table 2; Fig. 3, clones 3, 6, 9, 11, 13 and Fig. 4, clone 2). These clones contained both glia and neurons, including neurons on both the right and left sides of the third ventricle in individual cases. Overall, the distribution of cell types was similar to that of the unilateral clones. Several presumptive clones in which cells appeared to be migrating across the anterior or posterior commissure have been found in brains infected at stage 16-17 with CHAP or CHAPOL and harvested at E8-E10 (Szele F, Golden J, and Cepko C, unpublished data and Fig. 6A). Analysis of the anterior-to-posterior distribution of bilateral clones showed no specific localization. Although most studies have focused upon the germinal zone of the third ventricle as the source of cells that populate the diencephalon, another rare clone type appeared to originate in the lateral ventricle. Several of these clones included radial glial cells projecting from the lateral ventricle medially and ventrally into the diencephalon (Fig. 4 clone 10; note that the cerebral hemispheres and lateral ventricles, located along the 3A. J. A. Golden and C. L. Cepko 2A. 70 4A. Fig. 5. Three-dimensional reconstruction of clones within the diencephalon. Representative clones have been transposed to the surface of a reconstructed diencephalon. Each cell was localized according to its anterior-posterior and dorsal-ventral position; medial-lateral position is not shown. Several clones spread widely in the anterior-posterior plane, while others remain relatively constricted. Dispersion along the dorsal-ventral axis also varied. The overall extent of dispersion of a clone did not always reflect the number of cells within the clone, as small clones showed similar dispersion patterns to large clones. Figs 2-4. Schematic representation of 44 clones. Diagrams of the diencephalon and associated structures were traced from an atlas of the chick brain (Kuenzel and Masson, 1988). Cells from approximately 7 serial 60 µm sections were entered onto each of seven coronal images, (A-G) oriented from anterior to posterior, respectively, based on the location of the cells in the histological sections (see Methods). Each color represents a separate clone that has been randomly numbered. All of the cells within a clone are represented regardless of cell type. For illustrative purposes, the clones from several brains, regardless of their location in the brain, have been placed on the left side of each figure except bilateral clones which are shown on both sides. White matter tracts are colored light grey and nuclei are clear. AC, anterior commissure; ALP, nucleus ansae lenticularis posterior; AM, nucleus anterior medialis hypothalami; CP, posterior commissure; CPa, commissura pallii; DIP, nucleus dorsointermedialis posterior thalami; DLAl, nucleus dorsolateralis anterior thalami (lateral); DLAm, nucleus dorsolateralis anterior thalami (medial); DLP, nucleus dorsolateralis posterior thalami; DMA, nucleus dorsomedialis anterior thalami; DMP, nucleus dorsomedialis posterior thalami; FPL, fasciculus prossencephali lateralis (lateral forebrain bundle); GCt, substantia grisea centralis; GLdp, nucleus geniculatus lateralis, pars dorsalis; GLv, nucleus geniculatus lateralis, pars ventralis; ICT, nucleus intercalatus thalami; IH, nucleus inferioris hypothalami; LA, nucleus lateralis anterior thalami; LHy, lateral hypothalamic nucleus; OC, optic chiasm; OM, tractus occipitomesencephalicus; OT, optic tract; OV, nucleus ovoidalis; PHN, nuclus periventricularis hypothalami; PVN, nucleus paraventricularis hypothalami; ROT, nucleus rotundus; SCE, stratum cellulare externum; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; T, nucleus triangularis; Te, tectum; TeO, optic tectum; TT, tractus tectothalamicus; VLT, nucleus ventrolateralis thalami. Clones in the chick diencephalon 71 72 J. A. Golden and C. L. Cepko Table 2. Cell type composition and dispersion of all multiple pick clones * * B B B B B B B B B B N 0 2 2 8 0 0 0 0 0 3 0 1 1 1 1 0 0 1 1 0 3 1 1 6 5 0 0 1 0 0 3 2 0 3 1 0 0 8 0 0 0 0 0 1 3 5 18 0 0 0 4 1 0 1 0 4 7 0 0 0 2 G(RG) 0 0 18(1) 5 0(2) 1 8(1) 0 1 0(2) 4 0 1 1 4 3 0 10 10 1(4) 28 0(1) 5 2 4 2 13 0 5(3) 15 0 0 2 21 17 2(1) 0 0 1 1 2 6 3(2) 13(1) 3 0 0 0 0 0 2 6 5 6 3 7 1 3 0 1 0 U APµm(%) 2 60(2) 0 120(4) 4 540(16) 1 660(20) 2 60(2) 2 120(4) 0 360(11) 2 60(2) 1 0 60(2) 1 300(9) 2 360(11) 1 0 60(2) 0 840(25) 1 120(4) 3 60(2) 0 120(4) 0 300(9) 0 0 480(15) 0 60(2) 0 300(9) 2 1020(31) 0 60(2) 0 60(2) 2 300(9) 1 480(15) 2 420(13) 2 1140(35) 0 60(2) 0 420(13) 2 60(2) 1 300(9) 0 1020(31) 0 120(4) 5 420(13) 2 300(9) 1 360(11) 2 360(11) 0 60(2) 1 360(11) 2 720(22) 0 360(11) 2 3 900(40) 5 900(40) 42 60(3) 7 480(20) 4 60(3) 1 4 540(25) 7 180(8) 2 0 0 1 0 22 10 4 1 1 n= 1 6 4 405(30) 3 4 0 Range µm % ave% DVµm(%) 1515(42) 61(3) 667(16) 1152(19) 2273(54) 152(3) 667(15) 212(5) MLµm(%) 3030(74) 3152(83) 1667(70) 2273(71) 1758(48) 2667(80) 1212(36) 1970(52) 455(10) 2121(56) 91(2) 364(10) 2879(63) 1000(30) 91(2) 1758(39) 1152(25) 242(8) 697(14) 2667(70) 1121(30) 2000(73) 1424(39) 1364(50) 2697(70) 2727(45) 1515(36) 2212(46) 303(6) 2273(47) 667(16) 60(1) 1485(32) 1303(28) 1091(22) 1727(38) 394(10) 1394(30) 154(4) 3030(68) 1000(21) 2636(82) 2182(64) 2273(63) 2576(79) 1364(59) 1667(46) 1606(57) 2848(76) 1606(27) 1970(57) 2152(62) 3333(92) 3364(74) 2667(97) 2242(62) 636(13) 303(7) 394(8) 1364(31) 364(8) 818(23) 1788(87) 1455(44) 1970(54) 2636(76) 1000(22) 455(11) 1300(30) 185(4) 460(18) 185(4) 185(4) 850(30) 370(15) 3061(76) 1152(32) 2200(90) 750(27) 1850(95) 400(15) N G(RG) U APµm(%) 0 0 2 2 2 0 60(2) 1 0 1 60(2) 0 7(1) 3 0 5 0 120(4) 0 0 3 0 3 3 120(2) 0 0 1 0 8 0 300(11) 1 0 2 60(2) 0 4 1 120(5) 8 0 7 240(9) 1 0 1 120(5) 0 0 2 60(2) 0 0 2 60(2) 1 0(1) 0 60(2) 0 3 1 180(7) 1 0(1) 0 300(11) * 1 2 0 840(32) 0 0 3 360(14) 1 0 3 300(11) 1 0 1 60(2) 1 2 3 360(14) 0 4 0 120(6) 0 2 0 60(3) Many Many 180(9) 0 2 4 1140(54) 11 14 2 4 480(23) 2 15 0 240(10) 0 2 1 60(2) 0 3 1 60(2) 2 20(2) 1 3 660(24) 0 0 2 210(7) 3 0(2) 1 60(2) 0 4 0 240(9) 8 1 5 120(4) 2 0 0 60(2) 1 0 2 360(13) 1 0(1) 1 240(9) 2 3(1) 4 900(33) 0 3 1 240(9) 0 2 3 840(31) 0 2 0 60(2) 0 0 3 2 0 0 60(2) 0 9 3 1020(38) 0 2 3 60(2) 3 0 0 60(2) 0 0 4 60(2) 0 0 2 60(2) 2000(98) 1750(67) B B B B B B B B B B 1 13(1) 1 0(1) 0 3 0 0 0 2 0 8 1 4 3 5 1 0(1) 0 0 Clones: 60-1140 2%-54% 11.10% 5-2939 1%-74% 18.90% DVµm(%) MLµm(%) 520(12) 273(7) 2881(75) 2515(75) 182(5) 636(28) 394(8) 455(14) 394(10) 515(12) 152(3) 303(7) 2394(53) 61(1) 91(2) 1576(63) 1606(76) 2515(73) 1061(78) 1455(55) 485(19) 545(29) 667(16) 1303(31) 2939(69) 2061(74) 212(5) 273(6) 2455(54) 50(1) 250(5) 550(13) 930(20) 930(20) 580(15) 2939(94) 91(8) 1606(65) 2455(79) 1303(41) 667(22) 2606(82) 93(26) 355(53) 2930(76) 2470(67) 2900(82) 2090(77) 600(22) 500(12) 300(7) 200(7) 300(7) 650(15) 0 200(7) 300(8) 1900(48) 80(3) 200(5) 150(4) 1900(72) 2350(80) 1850(63) 500(37) 800(26) 600(24) 0 1700(58) 500(19) 1900(62) 1950(70) 1900(70) 2350(84) 180(4) 1000(25) 150(5) 410(11) 1000(24) 300(7) 1750(66) 1470(54) 0 400(14) 1320(69) 1320(41) 2 5 2 3 1 3 2 11 4 1 n=121(44) 0-3550 0-98% 57.50% The table is a complete listing of the cell types and the dispersion of all multiple pick clones (n=121). Dispersion is given in µm and in parenthesis is the percent of the distance from anterior to posterior (AP), dorsal to ventral (DV), and medial to lateral (ML) at the level of the brain where the clone was located. If dispersion distances are not listed, this indicates that either these clones arose from the lateral ventricle, or includes cells from AP− areas that also gave the same insert and thus the full extent of dispersion could not be accurately determined. An asterisk identifies the clones that showed a greater dorsal-to-ventral spread compared to the medial to lateral. B indicates that the clone is bilateral. N, neurons; G, glia; RG, radial glia; U, unidentified cell. Clones in the chick diencephalon Table 3. Clones crossing neuromeric boundaries Clone type n Cross Don’t cross Could not determine Unilateral Bilateral 90 18 5 (6%) 4 (22%) 47 (52%) 1 (6%) 38 (42%) 13 (72%) 108 9 (8%) 48 (44%) 51 (47%) Total The number (and percent) of clones that were observed in >1 neuromere (crossed) was determined by comparing the location of all members of a clone to the boundaries proposed by Figdor and Stern (1993). Only clones with siblings clearly located in more than one neuromere were included as crossing. All clones that had cells near borders were classified as “could not determine”. dorsal border, are not included on the illustration). Other cells from these clones included neurons and glia that were within the body of the diencephalon. A total of 5 of the 275 (2%) clones appeared to have arisen from the lateral ventricles, making the contribution of progenitors from the lateral ventricles to the final cell numbers in the diencephalon relatively small during the time period studied. No specific composition of cell types within these clones was found (Table 2). Another unusual clone type had siblings in both the diencephalon and the mesencephalon (tectum) (Fig. 3, clone 12 and Fig. 4, clone 10). Since only a few AP+ cells were analyzed from the mesencephalon, and no cells from the telencephalon, an estimation of the frequency of clones that cross from the diencephalon to adjacent brain regions cannot be made. However, the presence of siblings in both the diencephalon and mesencephalon indicate that the earliest subdivisions of the CNS do not form absolute lineage boundaries, as all infections were made after the prosencephalon and mesencephalon boundaries were established. One of the clones with cells in the diencephalon and mesencephalon appeared to have its origin in the lateral ventricle. Thus it is possible that cells generated in either the lateral ventricle or third ventricle can migrate between these two brain regions. Examination of whole-mount E8 chick brains showed a relatively simple and direct possible pathway for the migration of cells into these two structures (diagramed in Fig. 6, cell C). The pattern of radial glia in the diencephalon The most common pattern of clonal dispersion in the diencephalon was from medial to lateral with a progressively more ventral location as members of the clone migrated more laterally. One common pathway for migrating newborn neurons in the CNS is along radial glial fibers. To investigate whether radial glia could account for the most common pattern of dispersion seen here, DiI was used to label radial glial cells. Fig. 7 shows the pattern of fibers revealed by the DiI staining in the diencephalon. From the third ventricle, radial glial fibers course laterally and ventrally in a pattern paralleling the distribution of sibling cells. Clones that did not disperse in this common pattern may be using other guides that were not identified in this study. Radial glial cells were also noted in the lateral ventricle extending into the diencephalon (Fig. 7). The processes of these cells showed a general downward trend as they moved from the lateral ventricle into the substance of the diencephalon. This pattern reflects the distribution of cells found in a few of the clones that appeared to originate in the germinal 73 zone of the lateral ventricle, supporting the finding that progenitors located in the lateral ventricle can contribute to the diencephalon. Analysis of alkaline phosphatase negative (AP−) areas A potential problem in retroviral mediated lineage analysis is the failure to detect infected cells. For example, transcription from the LTR promoter could be weak or absent or the AP protein could be mutant or unstable. To investigate this issue, the entire diencephalon from one heavily infected brain was systematically analyzed for the presence of non-expressing cells. 263 regions were isolated from histochemically negative areas (i.e. no purple cells). These picks were defined as either small (n=52), approximately the size of the picks from single AP+ cells, or large (n=211). The large picks involved removing from 1/8 to 1/4 of a section of diencephalon from a slide and subjecting the tissue to PCR, as was done with the AP+ areas. For the small AP− picks, an insert was amplified from 19 (37%) picks. Sequence analysis of the inserts from the small picks revealed a single insert in 17 of the 19 amplified products, and multiple inserts in 2 of the 19. The large picks had a higher recovery rate for inserts after amplification (178/211, 84%). Sequence analysis revealed 133 of the 178 inserts were actually multiple sequences and only 25 of the 178 inserts were single sequences. 20 amplified products yielded no sequencing product. The higher recovery rate and presence of multiple inserts in a greater proportion of the large picks was attributed to the large tissue volumes collected. In some cases, the sequences from the histochemically negative regions were unique indicating that entire clones either did not express active PLAP or turned off the expression at some time prior to E18. Among other clones (5/93, 5.4%), a subset of cells exhibited alkaline phosphatase activity while other cells did not at the time of analysis. In addition, 25 samples from uninfected brains were analyzed and an insert was never recovered from these brains. Several explanations for the AP− cells exist. Since we completed this study, we observed that some cells, particularly some neurons, require a staining time longer than used here to become positive by light microscopy (J. Lin, F. Szele, J. Zitz, J. Golden and C. Cepko, unpublished data). Studies from other brain regions have also shown that some cells can turn off AP activity (Halliday and Cepko, 1992) while other cells appear never to exhibit AP activity. Although we cannot determine the contribution of any one mechanism in our data, it remains likely that a combination of these factors play a role in recovering viral genomes from AP− areas. DISCUSSION We have analyzed lineal relationships in the chick diencephalon using a highly complex retroviral library. Although the rodent has served as a model organism for lineage analysis in the cerebral cortex (Price et al., 1987; Walsh and Cepko, 1988, 1992; Austin and Cepko, 1990; Parnavelas et al., 1991; Levison et al., 1993) and retina (Turner and Cepko, 1987; Turner et al., 1990), technical limitations prevent the use of rodents for studying structures such as the diencephalon that develop before E12-E13, the earliest age that the neuroepithelium is accessable for viral injections. The timing of 74 J. A. Golden and C. L. Cepko no characteristics that distinguished them from the clones of the animals injected later, the data from all 7 animals were pooled. Fig. 6. Generation of bilateral clones and clones that span the diencephalon and mesencephalon. (A) A 60 µm coronal section at the level of the anterior commissure (AC) of an E9.5 chick brain injected with CHAP at stage 17 is shown. A nearly continuous population of cells appears to trace back to an origin in the ventricular zone of the third ventricle. Migrating cells, with long leading processes, appear to move away from the ventricle, then appear to turn and enter the AC. A bundle of fibers crossing the midline clearly defines the dorsal and ventral limits of the AC. (B) A whole-mount of an E8 chick brain viewed from the dorsal side with the midline opened to display the ventricular surfaces. The left side of the brain was traced and the major subdivisions colored and labeled. The position of the anterior and posterior commissures (AC and PC, respectively) is shown on the diagram (arrows) as possible routes for cells to cross the midline. Cell A depicts one potential route in which a cell could migrate around one end of the third ventricle. Although this is only shown at the anterior end, there is a continuous surface from the anterior end to the posterior limit of the diencephalon, along the ventral surface. Cell B illustrates a model in which a midline progenitor cell is the source of cells that migrate into each side of the diencephalon. Cell C shows where a clone might originate that spans the diencephalon and the mesencephalon. Scale bar 200 µm. injection of the retrovirus was selected according to the time when neuromeric boundaries were established in the diencephalon (Figdor and Stern, 1993). 5 of the 7 chicks were injected at stage 17 after the major boundaries were established and the other two were injected prior to the formation of diencephalic boundaries. Since the two brains infected at stage 1011 contained a total of only 9 clones, and these clones showed Neuromeres and patterning of the diencephalon The developmental mechanisms governing segregation of the diencephalon into nuclei remain largely unknown. One hypothesis is that the diencephalon is parceled into segments, each giving rise to a set of functionally related nuclei, analogous to the rhombomeres in the hindbrain. Two models have been proposed for parcelling the diencephalon into segments. The first model divides the diencephalon into three units (prosomeres) and has been most extensively studied in the mouse (see Rubenstein and Puelles, 1994 and Rubenstein et al., 1994 for reviews). In the second model, four units (neuromeres) have been identified, with the most caudal two in this second model corresponding to the first prosomere in the first model (Figdor and Stern, 1993). Since the second model of four neuromeres has been proposed for the chick and the first primarily established in the mouse, we have chosen to use the neuromeric model in analysis of the data. Figdor and Stern (1993), utilizing single cell injection and DiI and DiO labeling techniques, found that clones did not cross the morphological boundaries between the neuromeres of the diencephalon by stage 25 in development of the chick. These findings are similar to those of lineage analysis in the hindbrain rhombomeres. In contrast, however, using the boundaries of neuromeres defined by Figdor and Stern in the stage 40 chick (see Fig. 2 in Figdor and Stern, 1993) at least 8% (a minimum defined by conservative criteria) (see Table 3) of the clones in the current study cross the boundaries of neuromeres. Furthermore, in the prosomeric model (Rubenstein and Puelles, 1994; Rubenstein et al., 1994), the hypothalamus and thalamus are derived from distinct prosomeres, thus the clones spanning these two regions identified in this paper would also have to be considered boundary crossers (see below). In the calculations performed for Table 3, hypothalamic-thalamic clones were not included as crossing boundaries. Several explanations exist for the difference in dispersion between the two studies. The first, and most likely, is that the tracer labeling studies were analyzed after only 48-72 hours, whereas in the current study, embryos were harvested 16 days beyond the time of infection. Thus it seems likely that progenitor cells give rise to daughters that are initially restricted to within a single neuromere, but that later some siblings escape restrictions and move according to other mechanisms or cues. Alternatively, a relatively small subset of clones cross neuromeric boundaries early in development; such a subset of clones may have been missed by the techniques used in the previous study. This scenario is similar to the recognition of a small percentage of clones that violate rhombomere boundaries early in development (Birgbauer and Fraser, 1994). We have also analyzed retrovirally marked clones in the ventral forebrain, focusing on the hypothalamus, in brains harvested at E8-E10 (Arnold-Aldea and Cepko, 1995). Approximately 96% of the clones were simple radial columns, which appeared to respect neuromeric boundaries. However, approximately 4% of the retrovirally marked clones were widely dispersed, with distinctive patterns of dispersion at E8E10, including bilaterally symmetric clones in the hypothalamus. Ongoing experiments in our laboratory are seeking to Clones in the chick diencephalon 75 Fig. 7. DiI labeling of radial glia. (A) Overview of a DiI-labeled, 200 µm section through the diencephalon of E15 brain. The third (3 V) and lateral (LV) ventricles of a fixed embryo were filled with a DiI solution; periventricular regions show bright labeling. Single fibers are seen radiating into the body of the diencephalon from both ventricular locations. (B) A schematic diagram of (A) with a tracing of several of the radial glia arising along the third and lateral ventricles. The two boxes indicate the location of both C and D. (C) A view at higher magnification from the region where the radial glial fibers decussated. Fibers are seen coming from the lateral ventricle (upper left) and third ventricle (upper right). (D) The radial glial fibers located along the third ventricle showed a slight ventral slope as they projected from medial to lateral. Scale bar 1000 µm for A; 125 µm for C; and 250 µm for D. identify the timing of the dispersion that occurs in the majority of clones reported in the current study, which should clarify the relationships of the patterns reported in all of the aforementioned studies Clonal dispersion and the generation of diencephalic nuclei Data from birthdating studies using [3H]thymidine have been used as the basis for a proposal of gradients of cell generation and other properties of the germinal zone of the third ventricle. A study in Xenopus led to a proposal of an overall gradient from ventral-lateral to dorsal-medial in the generation of neurons in the diencephalon (Tay and Straznicky, 1982). In mammals, where the majority of work on the diencephalon has been conducted, a detailed analysis of the birthdates of each nucleus or small cluster of nuclei has been reported (Angevine, 1970; Altman and Bayer, 1978a-c, 1979a-c, 1988a-c, 1989ac). Together these studies have indicated that some nuclei within the diencephalon have fairly short periods of genesis while the neurons of other nuclei are born over a longer period of time. Furthermore, the birthdays of particular nuclei did not precisely correlate with the anatomical location of the nuclei. These birthdating studies led to the hypothesis that the germinal epithelium is a mosaic of patches, each giving rise to neurons in a nucleus or specific region of the diencephalon (Altman and Bayer, 1988a). The clonal distributions reported here included clones where the sibling cells tended to cluster in one or a small group of nuclei of the diencephalon. These clones would be consistent with the [3H]thymidine data, supporting the idea that progenitors within the ventricular zone of the third ventricle generate specific regions in the diencephalon. However, other members of some of these clones as well as many other clones were widely dispersed, indicating that most progenitors are not dedicated to producing cells for one nucleus or a small group of nuclei. Since siblings did not strictly populate one region of the diencephalon, we wanted to investigate how clonally related cells disperse. To examine one possible mechanism, the architecture of the radial glia in the diencephalon was explored using DiI labeling. The labeling of radial glial fibers arising from the third ventricle revealed that fibers projected into the diencephalon from the medial-to-lateral direction, with a dorsal-to-ventral slope, paralleling the most common patterns of distribution within clones. It is worth noting that not all clones showed this pattern of dispersion. Several clones showed marked displacement in the dorsal-ventral plane with relatively little dispersion from medial to lateral. This suggests that other mechanisms for clonal dispersion in the dorsalventral plane exist in the diencephalon. In addition, a mechanism that could explain the marked anterior-to-posterior dispersion of clonally related cells is currently unknown. Generation of cell types within clones The majority of large clones and approximately 50% of small clones contained more than one cell type. This indicates that progenitor cells are frequently multipotent, capable of producing both neurons and glia, similar to progenitors within the rodent retina (Turner and Cepko, 1987; Turner et al., 1990) and telencephalon (Price and Thurlow, 1988; Walsh and Cepko, 1992; Levison et al., 1993; Reid et al., 1995) and the chick tectum (Galileo et al., 1990; Gray and Sanes, 1992). The distribution of cell types indicates that most progenitors generate relatively small numbers of neurons and large numbers of glia (see Table 2). This was not always the case, as several clones had large numbers of neurons with relatively few, or no, glia. The large number of glia in many clones, along with the small numbers of neurons in some of these same clones, is consistent with a multipotent progenitor that divides and first gives rise to one neuron at each cell division. Multiple cell divisions with one neuronal daughter at each division would account for the clones with multiple neurons. One or more mitotic daughters from these same progenitors could continue to proliferate and later produce one or many glial cells. Analysis of single cell clones (see below) supports this 76 J. A. Golden and C. L. Cepko order of genesis, as does [3H]thymidine birthdating (Angevine, 1970). The observation of two cell clones with a radial glial cell and a neuron might suggest that progenitor cells remain multipotent up to the last cell division, although cell death and/or inefficiency in amplification or sequencing could result in missing cells from such clones. Analysis of the single cell clones also provides insights into the timing of genesis of different cell types in the diencephalon. The 118 single cell clones could be derived by one of several mechanisms, which are not mutually exclusive. As retroviruses integrate during the M phase of the cell cycle, integration into a single chromosome during M-phase means that only one daughter cell from the first cell division will be marked (Roe et al., 1993). If this daughter cell does not re-enter the cell cycle, it will result in a 1-cell clone. Alternatively, clones of greater than 1 cell would appear as single cell clones if some siblings were AP−, died, did not amplify with PCR, or did not sequence. However, it appears that no particular cell type amplified or sequenced preferentially and thus these potential problems would not bias the data set. An estimation of the number of clones that contained AP+ cells and AP− cells is approximately 5% (see Results and unpublished data). Therefore, single cell clones can be interpreted as an approximation of cell birthdays. 27% of all single cell clones were neurons, indicating that neurons were being born near the time of injection of the retrovirus at stage 16-17 in development. No single cell clones of neurons were found in the two brains analyzed from the stage 10-12 injections, but only 9 clones were analyzed, making the data set too small to permit a definite conclusion as to whether neurons were being born at this time. In contrast, glial cells were rarely born at the stages of development that injections were performed. Despite their high frequency in the total data set, only 6% of the single cell clones comprised glial cells (other than radial glia). This is consistent with classical [3H]thymidine birthdating studies in the diencephalon of many other species (Altman and Bayer, 1979a-c, 1988b,c, 1989a-c; Tay and Straznicky, 1982). The fact that there were any single cell glial clones is of some interest. The siblings of these cells may have been missed due to the inefficiency of the PCR and/or sequencing, or death. Alternatively, the single cell glial clones may have arisen from radial glia. Several lines of evidence have indicated that radial glia transform into astrocytes later in development (Schmechel and Rakic, 1979; Levitt and Rakic, 1980; Pixley and De Vellis, 1984; Voigt, 1989; Cullican et al., 1990; Gray and Sanes, 1992). A large percentage of radial glial clones were in fact single cell clones, indicating that they were born near the time of the retroviral infection, consistent with birthdating of radial glia in other studies (Levitt and Rakic, 1980; Misson et al., 1988). Unusual clone types Several unusual clone types were identified in this study. One type was bilateral, with sibling cells on either side of the third ventricle, but unlike the bilateral clones observed in the diencephalon from another study (Arnold-Aldea and Cepko, 1995), the bilateral clones in this study were not symmetric. At least three possible migration pathways could result in bilateral clones. A cell could cross through one of the major commissures (see Fig. 6), which include the anterior, posterior and supraoptic decussation dorsalis. Analysis at E18 has led to identification of cells within each of these commissures. However, as yet, clonally related cells on each side of the diencephalon and within one of these commissures have not been observed within the infected brains in the current data set. A second pathway to the generation of bilateral clones is for cells to migrate around the anterior, inferior or posterior limits of the diencephalon (Fig. 6B, cell A). We have not seen this type of migration in the material that we have examined, but a more extensive examination of younger brains would be required to exclude these routes. A third possibility is that a population of progenitor cells exists along the midline that is capable of generating siblings that can migrate to populate both sides of the diencephalon (Fig. 6B, cell B). Similar midline cells are present in invertebrates (Crews et al., 1988; Nambu et al., 1990, 1991; Crews et al., 1992), and in vertebrates such as the zebrafish (Hatta et al., 1991), and have been proposed as the source of bilaterally symmetrical clones in the chick diencephalon seen at E8 (Arnold-Aldea and Cepko, 1995). We have no direct evidence from the current data set for this mechanism of generating bilateral clones. One rare subset of clones had their origins in the lateral ventricle and descendants in the diencephalon. Radial glia with their cell bodies in the wall of the lateral ventricle and processes radiating medially into the diencephalon were observed to have sibling cells, both glial and neuronal, within the body of the diencephalon. Several other studies have suggested that progenitors from the ventricular zone of the lateral ventricle could provide cells to specific nuclei in the diencephalon (Rakic and Sidman, 1969; Altman and Bayer, 1978b) Although no specific nuclei were populated by the clones originating in the lateral ventricles, the few clones observed in this study preclude a definitive analysis. Surprisingly, clonally related cells in the diencephalon and the mesencephalon were observed. Since the first cerebral vesicles that form are the prosencephalon, the mesencephalon and the rhombencephalon, the presence of clonally related cells in the diencephalon and mesencephalon indicates that even these very early segments of the nervous system do not form absolute lineage boundaries. Such clones are undoubtably more frequent than reported here as AP+ cells in all regions of the brain were not systematically analyzed. However, if they are rare, it is not clear what meaning one can ascribe to them. They could simply be due to progenitor cells that are situated exactly on the border between the two areas. Alternatively, they could play a more meaningful role in patterning or connections between brain areas. AP+ cells were not sampled from the telencephalon and therefore no clones would have been identified that crossed from the diencephalon into the telencephalon. However, clones were found in the hypothalamus and thalamus. In the model where prosomeres form the segments of the brain, the thalamus and hypothalamus are in distinct prosomeres, including the hypothalamus arising from the ventral telencephalon (Puelles and Rubenstein, 1993; Rubenstein et al., 1994; Rubenstein and Puelles 1994). Clones that occupy both the thalamus and hypothalamus thus would cross the diencephalon-telencephalon boundary if the model proposed for prosomeres held for the chick. In summary, lineage analysis has been conducted in the diencephalon of the chick using a complex retroviral library. We have characterized the patterns of dispersion of clones and found several novel distributions. Clones were found to spread Clones in the chick diencephalon in all directions. Dispersion of clones from medial to lateral and from dorsal to ventral was found to parallel the pattern of radial glial fibers, suggesting that migration along radial glial fibers is one mechanism for clonal dispersion. Furthermore, siblings were found to occupy multiple nuclei, including nuclei derived from more than one diencephalic neuromere. Characterization of the cell types within clones demonstrated a high frequency of clones containing neurons and glia, supporting the hypothesis that progenitor cells in the chick diencephalon are multipotential. In addition to the common clones, unusual clones showed bilateral dispersion, origins in the lateral ventricle and/or siblings in both the diencephalon and mesencephalon. The significance of these rare patterns of dispersion remains unknown. 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