A comparative study of coherent clones in the retinal epithelium is presented for mouse aggregation chimaeras and X-inactivation mosaics. There is a basic similarity in the number of coherent clones and the pattern of clonal development between mosaics and chimaeras. While this similarity is compatible with the difference in mean patch size reported by other authors this is at variance with some interpretations of previous work and suggests that no conclusive evidence on the timing of X-inactivation is provided by comparisons of patch sizes between chimaeras and mosaics.

The present clonal analysis also suggests that at days post coitum the cells in the retinal epithelium are distributed almost randomly while, in the adult, the cells are grouped into small coherent clones, which comprise an average of five or six nuclei. However, these data could also be explained by larger, irregular coherent clones.

The pigmented epithelium of the retina was one of the first tissues used to detect experimental chimaerism in the mouse (Tarkowski, 1964). This tissue forms a monolayer of cells between the neural retina and the choroid of the eye. In normal mice each cell of the retinal epithelium produces melanin granules which remain localized within the cell, whereas certain mutants such as albino (c/c) or pink-eye (p/p) fail to produce normal pigmentation. The combination of pigmented and unpigmentated cells in this tissue provides a two-dimensional system with a convenient cell-autonomous marker for the analysis of clonal growth by routine histological methods.

Several authors, including Tarkowski (1964), Mystkowska & Tarkowski (1968), Mintz & Sanyal (1970) and Mintz (1971) have used this system to detect chimaerism. Deol & Whitten (1972a) have compared patches in chimaeras and mosaics but few other detailed studies have been reported concerning the pattern of pigmentation in mouse chimaeras or mosaics.

The theoretical relationship between clone and patch sizes (Roach, 1968; West, 1975a), is used here in a comparative investigation of clonal development in the retinal epithelium of mouse aggregation chimaeras and X-inactivation mosaics. The patch size varies with the proportions of the two cell populations in the tissue and it is essential to allow for this source of variation between groups of mice. The analysis used here compares the mean size of the coherent clones in different groups of chimaeras and mosaics, and compensates for any differences, in proportions of the cell populations, between the groups.

Using this analysis it has been possible to demonstrate the basic similarity in the pattern of coherent clones between mouse chimaeras and X-inactivation mosaics.

(a) Mice

Eight-cell morulae from pigmented and unpigmented stocks were aggregated to produce chimaeric mice (McLaren & Bowman, 1969). The pigmented stocks of mice used included (C57BL/McL♀ × CSH/BiMcL♂)F1, and pigmented individuals from a closed, random-bred stock of Q-strain mice. Mice with unpigmented eyes were either albino (c/c) members of the Q strain or from a multiple recessive strain produced by Michie (1955), homozygous for non-agouti (a), brown (b), dilute (d), pink-eye (p), chinchilla (cch), waved-2 (wa-2), short-ear (se), vestigial tail (vt), supernatant-NADP isocitrate dehydrogenase type a (Id-Id) and glucose phosphate isomerase type a (Gpi–1a). This multiple recessive stock is designated ‘Recessive’ throughout. Chimaerism is indicated by the joining of the symbols for the two component genotypes or stocks with a double-headed arrow.

The X-inactivation mosaics used were flecked mice, heterozygous for Cattanach’s translocation (Cattanach, 1961). Cattanach’s translocation, T(7;X)Ct, involves the insertion of a large part of chromosome 7 (linkage group 1), carrying the wild-type alleles for albino (c), pink-eye (p), ruby-eye-2 (ru-2) and shaker-1 (sh-1), into the X-chromosome. The stock of flecked mice used in this study was derived from six mice obtained in November 1972 from Dr Bruce Cattanach at Harwell. These mice are of the unbalanced, duplication (type II) form and have a completely normal set of autosomes with no known deletions in chromosome 7, and are designated Dp(7; X)Ct. The original six females came from Dr. Cattanach’s ‘High line’ and had about 75% JU-strain genetic background. The stock was maintained by crossing flecked females to albino males; to retain fertility JU/Fa and albino Q-strain males were used to sire alternate generations. The mosaics (Dp (7; X)Ct) used in this study, therefore, have a complex genetic background which could affect the relative proportions of pigmented and unpigmented cells in the retinal epithelium.

(b) Histological methods

Standard histological methods were used throughout this study. Adult eyes were removed for histological examination, but the eyes of embryos and newborn mice were sectioned in situ. Each adult eye was fixed in Bouin’s fluid over night, an incision made in the cornea and the lens removed with watchmaker’s forceps, under a dissecting microscope. Embryos and newborn mice were decapitated, the heads fixed overnight in a fixative based on Sanfelice’s fluid (Sanfelice, 1918) and rinsed in running tap water for at least 8h. Specimens from newborn mice were decalcified in 1 % nitric acid for three or four days to soften the skull before sectioning. All specimens were dehydrated in graded alcohols, cleared in toluene, embedded in wax and sectioned at 6 μm using either a Leitz or Cambridge rotary microtome. The sections were stained ‘With Ehrlich’s haematoxylin and eosin and mounted with D.P.X.

If the eye of an adult mouse is compared to a globe with the cornea in the north pole position, the two planes of section used are equivalent to ‘latitudinal’ sections, parallel to the equator, and ‘longitudinal’ sections, perpendicular to the equator. Both of the eyes from each mouse were sectioned in the same plane. Eyes from embryos and newborn mice were all sectioned in a plane para-sagittal, with respect to the body.

(c) Clonal analysis

The retinal epithelium of each adult eye was examined in three sections: the mid section, and one section about 500 μm each side of the middle. The two longitudinal sections either side of the mid-line were arbitrarily designated mid –500 and mid+ 500, whereas the latitudinal section nearest the cornea was designated mid + 500 and the section nearest the posterior pole (or ‘south pole’) was termed mid – 500. The retinal epithelium of a fixed adult eye normally has a diameter of about 2·5–3 mm.

The retinal epithelium of chimaeras and mosaics is patchy and appears in sections as a one-dimensional string of pigmented and unpigmented cells (Fig. 1). The length of each patch was measured using a microscope fitted with a calibrated eyepiece-micrometer. From these measurements the relative proportion of pigmented and unpigmented cells in the section was calculated, together with the mean patch length for each cell type. The mean one-dimensional patch length for each cell population varies with the relative proportion (p) of that population in the section, while the number of coherent clones per patch expected for a random string of coherent clones, can be estimated as 1/(1 – p) (see Roach, 1968; West, 1975a).

Fig. 1.

Histological sections showing the retinal pigment epithelium (PE) (Horizontal bar represents 25 μm in each case). (A) Adult pigmented-Q ↔ unpigmented-Q chimaera: patches of pigmented and unpigmented cells. (B–D) Retinal epithelium from mosaic embryo (D), a fully pigmented littermate (B) and an unpigmented control (C), 1212 days post coitum.

Fig. 1.

Histological sections showing the retinal pigment epithelium (PE) (Horizontal bar represents 25 μm in each case). (A) Adult pigmented-Q ↔ unpigmented-Q chimaera: patches of pigmented and unpigmented cells. (B–D) Retinal epithelium from mosaic embryo (D), a fully pigmented littermate (B) and an unpigmented control (C), 1212 days post coitum.

Our ‘coherent clone’ is that defined by Nesbitt (1974) as ‘a group of clonally related cells which have remained contiguous throughout the history of the embryo’. If cell mixing occurs during development these coherent clones will be smaller than the descendant clones observed by Mintz (1971), which more nearly represent the clonal pattern in the primordium of the retinal epithelium. Our ‘patch’ is defined by Nesbitt (1974) as ‘a group of cells of like genotype which are contiguous at the moment of consideration’, and may comprise several coherent clones.

The average coherent clone length for each cell type was calculated as the observed mean patch length divided by the expected number of coherent clones per patch in a linear array which is given by the formula 1/(1 –p) (see Table 1). The average coherent clone area was taken to be the square of the coherent clone length, and the number of cells per coherent clone was estimated from the calculated cell area. The cell area was calculated from measurements of mean length of unpigmented tissue per nucleus, and nuclear diameter, taking into account the section thickness (6 μm) and using a slight modification of the formula derived by Abercrombie (1946), described in the Appendix. This area was used for both cell types.

Table 1.

graphic
graphic

The mean coherent clone size, estimated in this way, represents the mean size of groups of cells which would produce the observed mean patch size by a random distribution. Our coherent clone is therefore a statistical estimate which in an ideal tissue represents a group of cells descended from a common ancestral cell which have remained adjacent throughout development. The accuracy of this statistical estimate will depend on the variation of coherent clone size and shape in the retinal epithelium. If the coherent clones are large and very irregular the analysis is likely to underestimate their mean size, but this limitation can be ignored for comparisons between different groups of mice.

(a) Preliminary analysis

Figure 1 shows patches of pigmented and unpigmented retinal-epithelium in a pigmented-Q ↔ unpigmented-Q chimaera. The aabbcchcchddpp genotype of the Recessive stock also results in an unpigmented retinal epithelium and provides good contrast to the pigmented strains.

Three sections were examined in each plane (latitudinal and longitudinal) for each adult eye, as explained in the Materials and Methods section. Of these, the latitudinal section nearest the cornea was commonly entirely iris, so only data from the remaining two latitudinal sections are considered.

Data were collected from mosaics and several groups of adult chimaeras. Statistical analyses of the three longitudinal sections by analysis of variance, and the two latitudinal sections using Student’s t-test, showed no significant difference in the mean coherent clone lengths, between sections, for any of the four groups of mice. The mid sections are chosen as representative samples of the eye for subsequent clonal analysis.

The number of cells per coherent clone in two dimensions (coherent clone size) is estimated as

The estimation of the mean coherent length for each cell population is explained in the Materials and Methods section, and these estimates are averaged to provide one estimate of coherent clone length per eye. The mean coherent clone lengths for the two cell populations are not independent observations, and the hypothetical arrays G and H in Table 1 show that even if the two cell populations had widely differing mean coherent clone lengths, this would not be detected. The estimation of mean cell length is discussed in the Appendix.

Comparison between longitudinal and latitudinal mid-sections using Student’s Z-test, shows no significant difference in the mean number of cells per coherent clone. This suggests that, on average, the coherent clones are symmetrical, so the data from the two planes are pooled. Data from left and right eyes are also pooled. Although there is a significant negative correlation for clone size between left and right eyes for the small group of adult pigmented-Q ↔ unpigmented-Q chimaeras (r = –0·90; P < 0-01), the biological basis for this negative correlation in one group is unclear, and it is probably an artifact resulting from the small sample size.

Statistical analysis by Student’s Z-test shows a significant difference in the proportion of pigmentation between longitudinal and latitudinal sections for mosaics (t = 5·25; P < 0·01) but not in any of the three chimaeric groups. This difference between the two planes of section in mosaics is clearly shown in Fig. 2 and could be due to sampling error or reflect a non-random distribution of pigmented and unpigmented coherent clones in mosaic eyes. No significant difference in proportion of pigmentation, between the two planes, was found in a further sample of eight mosaic eyes, each of which was sectioned in both planes. (These eyes were sectioned to the equator and turned perpendicularly and sectioned again.) This suggests that the difference observed in the first group of mosaic eyes is not biologically significant and can be ignored.

Fig. 2.

Estimated two-dimensional coherent clone sizes in the retinal epithelium from mid-sections of mosaic and chimaeric eyes, showing independence of coherent clone size and proportion of pigmented cells (p). Both left and right eyes are shown in each graph. (A) Eyes from mosaic embryos, 1212 days post coitum. (B) Eyes from pigmented-Q ↔ unpigmented-Q chimaeras, 1212 days post coitum. (C) Eyes from adult mosaics. (D) Eyes from adult pigmented-↔unpigmented-Q chimaeras. In (C) and (D) • represents longitudinal mid-sections and ▪ represents latitudinal mid-sections. The proportion of pigmented cells (p) is estimated from the sum of the patch lengths for each population in the section. The coherent clone size for each eye is an average of the coherent clone sizes estimated from both pigmented and unpigmented populations.

Fig. 2.

Estimated two-dimensional coherent clone sizes in the retinal epithelium from mid-sections of mosaic and chimaeric eyes, showing independence of coherent clone size and proportion of pigmented cells (p). Both left and right eyes are shown in each graph. (A) Eyes from mosaic embryos, 1212 days post coitum. (B) Eyes from pigmented-Q ↔ unpigmented-Q chimaeras, 1212 days post coitum. (C) Eyes from adult mosaics. (D) Eyes from adult pigmented-↔unpigmented-Q chimaeras. In (C) and (D) • represents longitudinal mid-sections and ▪ represents latitudinal mid-sections. The proportion of pigmented cells (p) is estimated from the sum of the patch lengths for each population in the section. The coherent clone size for each eye is an average of the coherent clone sizes estimated from both pigmented and unpigmented populations.

(b) Estimation of coherent clone size in the developing retinal epithelium

Figure 1 shows that patches in the retinal epithelium of mosaic eyes can be detected as early as days post coitum, and mosaics can be clearly distinguished from fully pigmented or unpigmented individuals of the same age. The mean coherent clone size was estimated in mosaics and pigmented-Q ↔ unpigmented-Q chimaeras at various stages of development, and the results are summarized in Table 2 and Fig. 2. The results suggest that at days the mean coherent clone size in both the mosaic and chimaeric samples is close to one cell, so the cells of the two populations are distributed almost randomly in the retinal epithelium. At this stage the mean coherent clone size in the chimaeric group does not significantly differ from that in the mosaics, (t = 0·70; P > 0·05). The marked predominance of the pigmented population in the day chimaeras is most probably an artifact due to the small number of animals studied, and is considered more fully in the Discussion section below. The coherent clone size increases during development to an average of just under three cells in mosaics one day after birth, and nearly five cells in the mature adult. Chimaeric coherent clones are significantly larger both at days post-coitum (t = 3·12, P < 0·01) and in the adult (t = 2·92; P < 0·01), although in each case this difference is only about one cell. It is unlikely that any difference in the age structure between the two groups of adults influences the coherent clone size, as there is no correlation between coherent clone size and the age of the adult for either mosaics or pigmented-Q ↔ unpigmented-Q chimaeras.

Table 2.

Number of cells per two-dimensional coherent clone in the retinal epithelium (mean ± standard error of mean) for mosaic and pigmented-Q ↔ un-pigmented-Q chimaeric eyes at various stages of development

Number of cells per two-dimensional coherent clone in the retinal epithelium (mean ± standard error of mean) for mosaic and pigmented-Q ↔ un-pigmented-Q chimaeric eyes at various stages of development
Number of cells per two-dimensional coherent clone in the retinal epithelium (mean ± standard error of mean) for mosaic and pigmented-Q ↔ un-pigmented-Q chimaeric eyes at various stages of development

Table 3 gives a crude comparison of the various parameters in the adult pigmented epithelium between mosaics and the chimaeras, and shows approximate increases in size between days and maturity. It seems likely that the area of the retinal epithelium increases by both cell expansion and cell multiplication. The increase in coherent clone size does not keep pace with the increase in cell number and there is an increase in the estimated number of coherent clones indicating cell mixing during development.

Table 3.

Approximate dimensions of cells and coherent clones of the retinal epithelium in adult mice and comparison of growth between mosaic and chimaeric retinal epithelia

Approximate dimensions of cells and coherent clones of the retinal epithelium in adult mice and comparison of growth between mosaic and chimaeric retinal epithelia
Approximate dimensions of cells and coherent clones of the retinal epithelium in adult mice and comparison of growth between mosaic and chimaeric retinal epithelia

(c) Comparison of adult coherent clone size between different groups

The mean coherent clone size estimated from adult chimaeras of various strain combinations are shown in Table 4. Statistical analysis of the four groups of animals, using Student’s Z-test, showed no significant difference in coherent clone size between left and right eyes within each group, but a significantly larger coherent clone size for (C57BL × C3H)F1 ↔ Recessive chimaeras than for mosaics and possible other groups (Table 5). The mean coherent clone sizes estimated for the left and right eyes of this group are 8·37 and 12·65 respectively. The mean for the right eyes includes an estimate of 42·79 cells per coherent clone for chimaera XI8, which is almost three times the next largest for the group. The proportion of pigmentation in the retinal epithelium estimated from the midsection of the right eye of XI8, is only 0·01. The mean coherent clone length estimate is, therefore, based on a very small number of pigmented patches and so is more prone to inaccuracies from sampling error. A two-dimensional reconstruction of part of the retinal epithelium, from serial sections either side of the mid-section, revealed two pigmented patches. One patch was about the size of eleven cells and was assumed to be a single coherent clone, whereas the other was equivalent to about seventy cells and may have represented a patch of six or seven coherent clones. A coherent clone size of eleven cells agrees more closely with the other estimates from right eyes in this group. It is assumed that the original high estimate is due to the small number of patches sampled in one dimension, and data for this eye is omitted from the statistical analyses shown in Table 5.

Table 4.

Mean number of cells per two-dimensional coherent clone (mean ± s.E.) for adult mosaic and chimaeric retinal epithelia

Mean number of cells per two-dimensional coherent clone (mean ± s.E.) for adult mosaic and chimaeric retinal epithelia
Mean number of cells per two-dimensional coherent clone (mean ± s.E.) for adult mosaic and chimaeric retinal epithelia
Table 5.

Comparison of number of cells per two-dimensional coherent clone of the retinal epithelium, between unrelated groups of chimaeras and mosaics, showing t-values

Comparison of number of cells per two-dimensional coherent clone of the retinal epithelium, between unrelated groups of chimaeras and mosaics, showing t-values
Comparison of number of cells per two-dimensional coherent clone of the retinal epithelium, between unrelated groups of chimaeras and mosaics, showing t-values

The comparison of clone size estimated in two planes suggests that the clones of the retinal epithelium are, on average, symmetrical. Figure 3 (B–F) shows eyes from one chimaera and three mosaics. Longitudinal stripes, as described by Mintz (1971), can clearly be seen in some of the eyes, although, when present, these are normally restricted to the equatorial region. Figure 3 (E and F) shows two views of the left eye from one mosaic, which illustrate prominent striping near the equator but no evidence of any stripes over most of the eye. (The four prominent stripes radiating from the posterior pole in Fig. 3F, are regions of pigmentation in the overlying choroid.) The failure of the clonal analysis to detect any pattern of stripes from sections of the retinal epithelium probably reflects the apparent restriction of any stripes to a relatively small part of the retina.

Fig. 3.

(A) Tangential section of chimaeric eye showing two cell populations and both binucleate and uninucleate cells in the retinal epithelium. (The horizontal bar represents 50μm). (B–F) Low power photomicrographs of unstained mosaic and chimaeric eyes showing variegation in the pigmentation of both the dense choroid layer and the underlying retinal epithelium. (Actual diameter of eyes is about 3 mm). No obvious stripes are seen in the retinal epithelium of the chimaeric eye (B) or mosaic eye (C). Striping is clear in mosaics (D) and (E). Comparison of equatorial (E) and polar (F) views of the same eye suggests that striping may be restricted to the equatorial region. The four radiating stripes of dense pigmentation seen in (F) are due to pigmentation in the overlying choroid, (t marks a region of the retinal epithelium which was torn during the removal of the muscle from the eye.)

Fig. 3.

(A) Tangential section of chimaeric eye showing two cell populations and both binucleate and uninucleate cells in the retinal epithelium. (The horizontal bar represents 50μm). (B–F) Low power photomicrographs of unstained mosaic and chimaeric eyes showing variegation in the pigmentation of both the dense choroid layer and the underlying retinal epithelium. (Actual diameter of eyes is about 3 mm). No obvious stripes are seen in the retinal epithelium of the chimaeric eye (B) or mosaic eye (C). Striping is clear in mosaics (D) and (E). Comparison of equatorial (E) and polar (F) views of the same eye suggests that striping may be restricted to the equatorial region. The four radiating stripes of dense pigmentation seen in (F) are due to pigmentation in the overlying choroid, (t marks a region of the retinal epithelium which was torn during the removal of the muscle from the eye.)

The investigation of the mean coherent clone size during development suggests that at days post coitum the cells of the two populations are distributed almost randomly in both mosaics and pigmented-Q ↔ unpigmented-Q chimaeras. This indicates that cell movement mixes the two cell populations sufficiently to prevent significant coherent clonal growth at this stage, and further suggests that Nesbitt’s assumption of ‘limited coherent clonal growth in the developing mouse embryo’ (Nesbitt, 1974) may not be true for the retinal epithelium, at this stage of development. The analysis also predicts that limited clonal growth occurs after days. The estimate of mean coherent clone size is really an estimate of the mean number of nuclei per coherent clone. Ts’o & Friedman (1967) observed a high frequency of binucleate cells in flat preparations of rat and rabbit retinal epithelium, and inspection of tangential sections of the retinal epithelium from adult mosaics and chimaeras shows that these contain both binucleate and unicleate cells (see Fig. 3A), whereas there is no clear evidence of binucleate cells in similar sections from newborn mosaics and chimaeras. Part of the increase in the mean number of nuclei per coherent clone after birth may be due to formation of binucleate cells.

Comparison between the coherent clone sizes for developing mosaic and pigmented-Q↔ unpigmented-Q chimaeras show a marked similarity at days, and only a small difference after birth. The high proportion of pigmented cells, shown in Fig. 2B, for the -day chimaeric group might suggest that the eyes are non-chimaeric and the development of pigment is incomplete. Although Tarkowski (1964) found unpigmented cells in some of his control stocks up to 13 days, this seems an unlikely explanation here. The chimaeric eyes were at least as well developed as the mosaics shown in Fig. 2D, and the chimaeras were readily distinguishable from fully pigmented embryos of the same age. Of the six -day chimaeric embryos used, five were littermates and it is possible that either genetic or developmental differences between the pigmented and unpigmented embryos used as aggragants are responsible for the high proportion of pigmentation. Even if the comparison between the -day embryos is ignored, the results from chimaeras at later stages, which show no marked bias in proportion of pigmentation, suggest similar patterns of development for mosaics and this chimaeric group. While the present analysis clearly indicates a basic similarity in clonal development between chimaeras and mosaics, the mean size of the coherent clones may be consistently underestimated if they are very irregular in shape.

Comparisons between adult chimaeras of different strain combinations suggest that (C57BL × C3H)F1 ↔ Recessive chimaeras have more nuclei per coherent clone that the other groups considered, although crude estimates indicate that the number of nuclei per retinal epithelium is very similar in all chimaeric groups. Presumably coherent clonal growth in this strain combination has been less disrupted by cell mixing than in the other groups studied. One possibility is that the association between cells of unlike genotype is less stable than associations of like genotype in this particular strain combination.

The smaller differences between other groups may also be due to differences in cell interactions. The two cell populations in a mosaic will only differ genetically by the activity of X-linked genes, and so differences in interactions between cells of the two populations are less likely in mosaics than in chimaeras. The smaller mean coherent clone size shown by mosaics than by any chimaeric group is consistent with a slightly greater tendency of cells of like genotype to stay together in chimaeras. However, the difference may be simply an artifact of the method of analysis. It has already been noted that some chimaeras have very unequal proportions of the two cell populations, and in these cases the coherent clone size estimation is based on fewer patches, and so is less reliable than usual. Mosaics, as Deol & Whitten (1972 a) have also noted, tend to have more equal proportions of pigmented and unpigmented cells.

Deol & Whitten’s one dimensional analysis of histological sections of the retinal epithelium also revealed that the mean number of patches in mosaics was three times that in C57BL/10Wt ↔ SJL/Wt chimaeras (Deol & Whitten., 1972A). If this reflects a similar difference in coherent clone number, the results suggest a far greater difference between mosaics and chimaeric coherent clones than any seen in the present study. Interpretations of these results have been made to suggest either late X-chromosome inactivation (Deol & Whitten, 1972A), or coherent clonal growth, in both developing chimaeric and mosaic embryos, following X-inactivation earlier in development (Nesbitt, 1974).

Much of the difference between patch length in mosaics and the C57BL/ 10Wt ↔ SJL/Wt chimaeras is probably due to the observed differences in the proportions of the two cell-populations. More mosaics have nearly equal proportions which, in a one-dimensional analysis, will result in a larger number of patches even if the coherent clone sizes are equal to the chimaeric coherent clones. Strain specific interactions between C57BL/10Wt and SJL/Wt might also occur and reduce the degree of cell mixing in these chimaeras, and account for part of the difference between the results from Deol & Whitten’s analysis and the present study. A combination of strain-specific interactions between C57BL/ lOWt and SJL/Wt cells and a difference in proportions of the two cell-populations between the mosaics and chimaeras, would probably reconcile Deol & Whitten’s results with early X-inactivation without the need to postulate limited coherent clonal growth.

The present observations agree with Deol & Whitten’s finding that chimaeric retinal epithelia tend to have less equal proportions of the two cell-populations. The more equal proportions seen in the mosaic pigmented epithelia may partly reflect more equal proportions of the two cell-populations in the whole body (see Nesbitt, 1971), which might be expected if X-inactivation occurred fairly soon after the formation of the inner cell mass, as is widely believed (Lyon, 1972).

Several authors have compared chimaeric and mosaic phenotypes in order to investigate the validity and timing of X-chromosome inactivation in the mosaics. In a number of cases larger patch sizes have been claimed for chimaeras. These differences can only be interpreted on the basis of late X-inactivation if the larger chimaeric patches are known not to result from differences in the proportions of the two populations, or from a tendency for cells of like genotype to remain together in the chimaeric group. These possibilities were not excluded in the studies of the retinal epithelium (Deol & Whitten, 1972 A), migratory melanocytes of the eye and inner ear (Deol & Whitten, 1972 b) and tail banding patterns caused by the tabby gene (TA) (McLaren, Gauld & Bowman, 1973).

In conclusion, the retinal epithelium of X-inactivation mosaics and chimaeras show a broad phenotypic similarity and it is suggested that there is no basic difference in the coherent clone size or the pattern of clonal development between mosaics and chimaeras. This similarity may be masked by cellular interactions in chimaeras of some strain combinations and observed differences can be attributed either to cell selection or to early sampling processes (such as the formation of the inner cell mass) which occur after the two chimaeric populations are aggregated but before X-inactivation. Comparisons between mosaics and chimaeras provide no conclusive evidence on the timing of Xchromosome inactivation. The present analysis suggests that at -days postcoitum cell mixing in the retinal epithelium is sufficient to disrupt any clonal growth but after this time limited coherent clonal growth occurs in both mosaics and chimaeras until, in the adult, coherent clones comprise an average of five or six nuclei.

I wish to thank Dr Anne McLaren for generous help, supervision and encouragement given to me throughout this study. I am also grateful to Drs Anne McLaren, Patricia Bowman and Mrs Janet Carter for providing the chimaeras, to Mr B. Doyle for reliable technical help, and to Dr W. K. Whitten of the Jackson Laboratory, Bar Harbor, Maine, for reading the manuscript and making many helpful suggestions. This study was supported by a Science Research Council Studentship, and by the Ford Foundation.

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Estimation of mean cell area in retinal epithelium

Abercrombie (1946) showed that the estimation of the nuclear population density from histological sections is only possible if the mean nuclear size is taken into account. Some of the nuclei visible in a section are whole nuclei, whereas some are nuclear fragments, so extrapolation of crude counts per area to apparent number of nuclei per volume will result in over-estimation. This is relevant to the estimation of the mean cell area over the surface of the retinal epithelium, where the extrapolation is one of counts per length to counts per area. Abercrombie derived the following equation to correct for the exaggeration of nuclear counts :
where P is the average number of ‘nuclear points’ per section, A is the crude count of visible nuclei per section, M is the section thickness (in μm) and L is the average length of the nuclei (in μm) perpendicular to the plane of the section. A ‘nuclear point’ is any geometrical point of the same relative position in all nuclei and cannot overlap two adjacent sections. The function M/(L + M) is the proportion of visible nuclei whose ‘nuclear points’ lie within the section.

In chimaeric or mosaic retinal epithelia the nuclei can only be clearly seen in unpigmented patches, so the estimation is based entirely on one population of cells. The length of the nuclei was not measured in a plane perpendicular to the section as it was shown that the nuclei were symmetrical. The mean nuclear length, based on twenty nuclei, for each of ten mosaic eyes, sectioned in the ‘latitudinal’ plane (6·31 ±0·11 μm) did not differ significantly from the equivalent mean nuclear length derived from sections in the ‘longitudinal’ plane (5·96±0·16μm). (Values from a Student’s t-test: t = 1·94; P > 0·05.)

In tangential sections, cells of the retinal epithelium appear roughly symmetrical, and this assumption is supported by the similarity of estimates of cell length from longitudinal and latitudinal sections of the same eyes (West 1975 b). If the estimated mean cell length (parallel to the epithelial surface) is termed C the cell area is C2. The length of unpigmented retinal epithelium considered will be called R. The cell area to be considered is the surface area, perpendicular to the plane of the section, and is equal to the area of retinal epithelium (R · M) divided by the number of nuclear points (P), or :
As
cell area
cell length
where L is the mean nuclear length, visible in the section and parallel to the surface of the epithelium, Mis the section thickness (6 μm), and A is the number of nuclei counted in the length of unpigmented retinal epithelium, R. This estimate of cell area (C2) is a measure of the area per nucleus and is equivalent to the cell area only if each cell is uninucleate.
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Edinburgh University
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