Notes to Chapter 11

¹Beechy and Searle ( 1976) report that Sl^con is about 16 cM from grizzled ( gr). This is in reasonable accord with the estimate of 6-14 cM made by Cacheiro and L. Russell ( 1975).

²The most severe effect on erythropoiesis seems to occur between 15 days gestation and 1 week after birth when Sl/+ mice posses only about 65% of normal number of erythrocytes ( D. Bennett, 1956).

³Mintz ( 1957b, 1960) investigated the effect which Sl has on embryonic gametogenesis and found no apparent influence of this mutation on primordial germ cell number when present in a single dose. Thus Sl/+ embryos displayed similar numbers of primordial germ cells as W/+, W^v/+, and +/+ embryos. Moreover, she found no evidence that a simultaneous substitution of one mutant gene at the W and Sl loci (e.g., W/+;Sl/+) created any special difficulty in germ cell proliferation or viability. She concluded that the results suggest that each of these loci "makes a unique contribution to the growth and multiplication of primordial germ cells," and that it is possible that W/W;+/+ and +/+;Sl/Sl are defective at different points in a reaction sequence, each indispensable for the production of a further developmental stage ( Mintz, 1960).

⁴In the mouse the blood-islands of the yolk sac are the sole source of erythrocytes from the eighth through the eleventh day of gestation. From the twelfth through the sixteenth day of gestation the liver is the only hematopoietic organ and remains an important source of new erythrocytes up to birth. Bone marrow hemopoiesis first appears on the sixteenth day of development ( E. Russell and Bernstein, 1966)

⁵Not only are there about half the normal number of megakaryocytes in the marrow of Sl/Sl^d mice but the cells are larger than normal ( Ebbe et al., 1973). Moreover, even though these mice have a normal complement of circulating platelets, they may produce a thrombopoietic substance that stimulates the production of larger than normal megakaryocytes. Evidence for this stems from the observation that when normal (+/+) mice are parabiosed to syngeneic Sl/Sl^d animals, megakaryocytopoiesis is stimulated in the +/+ partner and the cells which are formed are larger than normal ( Ebbe et al., 1978).

⁶More recently Chui and Loyer ( 1975b) have shown that, in contrast to the unresponsiveness of Sl/Sl^d mice to erythropoietin in vivo, there are cells in the marrow of these mice capable of responding in vitro to erythropoietin in a normal fashion. They have also shown that erythropoietin present in Sl/Sl^d serum is biologically active in vitro.

⁷Sl/Sl^d mice also have more than 300-times normal specific activities of the enzyme nucleoside deaminase in their circulating erythrocytes ( Rothman et al., 1970; Harrison et al., 1975). There is also evidence that lymphocytic neoplasms arise much more frequently in the thymus of these anemics than in their heterozygous or normal littermates ( E. Murphy, 1969). On the other hand, there is no evidence that either Sl or W adversely affects the differentiation of the immune system ( Mekori and R.A. Phillips, 1969; but see Chapter 10, note 27).

⁸As in the case of W-genotypes which are sterile (see Chapter 10, note 24), the germ cell-deficient ovaries of Sl^d/Sl^d and Sl/Sl^d genotypes develop tumors in old age ( M.C. Green, 1966a).

⁹Melanin pigmentation has been found on the ovary, cervix, and related structures of Sl^con/Sl^con females ( Beechey and Searle, 1971).

¹⁰Recently it has been shown that Sl/Sl (or Sl/Sl^d) mouse embryos can be reliably identified in segregating litters at 11 and 12 days gestation by grafting skin from either the dorsal region between the levels of the fore- and hind limbs, or from the dorsal region just anterior to the level of the fore limbs, to the testes of histocompatible adults. The grafts are maintained in this location for 2-3 weeks and the absence of pigment in the hairs which form provide an excellent indication of their Sl/Sl (or Sl/Sl^d) genotype ( Chui et al., 1976).

¹¹It should be noted that when 9-day-old +/+ neural tube was combined with 13- to 18-day-old embryonic albino ( c/c) skin, 20 of 23 of the recovered grafts lacked pigment although the possessed a full complement of hairs. Mayer believes these grafts failed to become pigmented because they already were populated by albino ( c/c) melanoblasts. To support this contention he cites that there was always a large population of pigment cells in the tissues of the host chick embryo immediately surrounding these grafts (see Mayer, 1973b).

¹²At this stage of development both the dermal mesoderm and the epidermal ectoderm are populated by melanoblasts — at least in pigmented and albino genotypes. This was shown to be the case by Mayer ( 1973b) who separated C57BL/6 and congenic albino ( c/c) embryonic skin, obtained from the dorsolateral side of the trunk midway between the limb buds, into their epidermal and dermal components and, after recombining the dermis of one with the epidermis of the other, placed them in the chick coelom. Whereas recombined skin from embryos 11 days of age formed pigment only when the mesodermal component stemmed from a genetically black embryo, i.e., black ectoderm-albino mesoderm combinations always failed to become pigmented, skin recombination made from 12-day-old embryos indicated that the ectodermal component had been populated with melanoblasts and by 13 and 14 days this was even more evident. From these observations Mayer concludes that the migration of melanoblasts into the developing skin occurs through the dermal mesoderm, "with a later secondary migration from the dermis into the overlying epidermal ectoderm." These results are in accord with Rawles ( 1940, 1947) observations as well as with the observations of A. Zimmermann and Becker ( 1959) who examined the positions of pigment cells in fetal Negro skin. They are not in accord with the observations of Weston ( 1963) who studied the migration of labeled neural crest cells in the chick and concluded that these cells entered the ectoderm almost immediately after leaving the neural tube.

¹³Mayer ( 1975) also attempted to determine if melanoblasts were present in the skin of 13-day-old Sl/Sl embryos by assuming that if they were they might be induced to express themselves if their environment was "enriched" with dominant spotting ( W/W) skin cells. Accordingly he dissociated skin from both Sl/Sl and W/W embryos, mixed them together in various proportions, and grew them as aggregates for 2 weeks in the chick coelom. Because this procedure failed to reveal the presence of any melanocytes, he concluded that either steel prevents "the migration of melanoblasts into the mutant tissue environment, perhaps at the time of initial migration of melanoblasts from the neural crest," or Sl/Sl melanoblasts have a normal migratory pattern but are in some way eliminated as viable cells in the skin by 13 days.

¹⁴Although Mayer ( 1970, 1973a) cites Silvers ( 1956) as having reported that steel skin lacks amelanotic melanocytes, no Sl genotypes were included in Silvers' investigation.

¹⁵Nevertheless there are some aspects of Mayer's efforts which raise some interesting questions. For example, while some of his observations are interpreted as indicating that Sl adversely affects melanoblasts differentiation not only within the skin, but in regions adjacent to it, other observations suggest the influence of Sl to be quite localized. Thus, whereas his results following combining +/+ melanoblasts with Sl/Sl^d or Sl^d/Sl^d skin suggest that these skin inhibit the differentiation of melanoblasts which it is assumed must have migrated into the surrounding tissues of the host, his mesodermal and ectodermal skin recombinant results indicate a strictly localized effect of Sl. Moreover, if Sl/Sl^d skin has such a strong influence in regions adjacent to it, why were some cases found, albeit very few, in which some +/+ melanocytes occurred in Sl^d/Sl^d hairs?

¹⁶It is likewise difficult to reconcile with the similar synergistic effect that Sl/+ (or Sl^d/+) has on the amount of white spotting when combined with other spotting determinants (see Figures 10-4c and 10-8d) which are known to act via the melanoblast and which interact with each other as they do with Sl/+ (or Sl^d/+).

¹⁷Although each time a hair is produced the same basic dermal-epidermal connection is retained, and dormant melanocytes persist ( Silver et al., 1977), almost an entirely new follicle is formed (see Chase and Silver, 1969). For this reason one question concerning hair growth which remained unanswered was whether a melanocyte could deposit pigment in more than one hair generation, or whether it "shoots its wad," so to speak, after it has delivered pigment to a single hair cell (see Chapter 3, Section I, G, 2)? To resolve this question we determined how the secondarily pigmented Sl/Sl^d hairs which arose just outside the border of pigmented ear skin implants responded to plucking. It follows that if after plucking these hairs (and it should be noted that their numbers varied from a few to a dozen) the regenerated hairs were white (nonpigmented) this would indicate that the melanocytes of the previous hair bulbs had been lost, i.e., they were able to function only in one hair. On the other hand, if a pigmented hair replaced the plucked pigmented one, this would provide strong evidence that at least some a/a;B/B melanocytes (or their mitotic descendents) can pigment more than one hair generation. The latter situation prevailed. Indeed, some secondarily pigmented hairs were plucked (at the end of each cycle) as many as 6 times and still regenerated pigmented (Poole and Silvers, unpublished).

¹⁸Similar findings have recently been duplicated in vitro. Thus when W/W^v bone marrow cells were fed to an adherent layer of previously established Sl/Sl^d marrow cells, cell production was markedly less than when either marrow cells from completely normal donors were employed, or when Sl/Sl^d marrow cells were fed to an adherent layer of previously established W/W^v cells ( Dexter and Moore, 1977).

¹⁹Bernstein ( 1970) also found that hematologic values were elevated when Sl/Sl^d histocompatible spleens were grafted to W/W^v anemics. However, he notes that whereas these transplants are effective because they provide a source of stem cells which can function normally in the hematopoietic organs of the W/W^v host, W/W^v spleens transplanted to Sl/Sl^d mice are effective because they pick up Sl/Sl^d circulating stem cells and provide them with a suitable environment for their reproduction and normal development. He also notes that in the case in which +/+ spleens reside in Sl/Sl^d recipients, migration does not appear to be necessary. Rather in this situation an organ which is entirely normal to begin with continues to function after grafting without the necessity of acquiring any new operational capacity. Mintz and Cronmiller ( 1978) have produced allophenic mice composed of both Sl/+ and +/+ cells and the clinical blood picture of these mosaics is indistinguishable from that of normal (+/+) controls, even when only a small minority of cells in all tissues are of the +/+ type. Indeed, because such a surprisingly small complement of normal cells is able to prevent the expression of the anemia, they propose "that relatively short-range diffusible substances, produced by cells in the microenvironment and required for normal erythropoiesis" are operating. For other hematological investigations on steel mice see Cole et al. ( 1974), McCarthy ( 1975), McCarthy et al. ( 1977), and Adler and Trobaugh ( 1978).

²⁰From 12 days gestation to at least 60 days of age f/f mice are also smaller than their normal littermates ( E. Russell and McFarland, 1966; E. Russell et al., 1968). Whether this is attributable to the transitory anemia, even though this growth defect persists long after the anemia is over, or whether it reflects another independent primary effect of the f/f genotype, is not known. There is also evidence that f/f mice are more susceptible to induced leukemia than +/— animals. How this effect is mediated also remains to be determined, but it appears to be a specific gene effect on susceptibility, rather than to linkage of susceptibility genes and f (Law, 1952).

²¹Unlike the flexures of the tail and the belly spot, which may often fail to manifest themselves on account of inhibitory modifiers and unknown environmental conditions, the anemia appears to be fully penetrant. Indeed, E. Russell and McFarland ( 1966) report that it is conceivable that at birth one dose of the flexed-tailed gene ( f/+) has some effect on erythrocyte number.

²²The erythrocytes of f/f mice have a reduced hemoglobin concentration, but they contain "free" iron in granular form as shown by the Prussian Blue test. This type of erythrocyte has been termed a "siderocyte." These cells are not peculiar to f/f mice as they are found also in normal mice, albeit in smaller numbers with significantly less free iron per cell (Grüneberg, 1941, 1952).

²³Erythrocyte number and size are normal in adult f/f animals, although Grüneberg ( 1942c) reported 3% siderocytes in their blood in contrast to none in normals over 1 week of age.

²⁴There is evidence ( Dickie, 1964b) that some Sp/+ embryos may die in utero, with or without any observable anomaly.

²⁵Evidently the longevity of Sp homozygotes can be selected for as in a stock selected for minimal white such homozygotes lived to term usually with rudimentary tail, sacral spina bifida, and cranial hernia ( Hollander, 1959).

²⁶The genotypes of these abnormal embryos were confirmed by transplanting ovaries from them, shortly before they were expected to die, to histocompatible (but appropriately genetically marked) +/+ hosts and mating these hosts to normal (+/+) males. The observation that all the offspring produced from such matings were phenotypically identical with Sp/+ mice made it evident that the donor ovaries must have been Sp/Sp ( W. Russell and Gower, 1950).

²⁷It is interesting to note that whereas the two Sp mutations ( Sp^J and Sp^3J) which occurred in the C57BL/6J strain were characterized only by a large belly spot, the one ( Sp^2J) which occurred in the C3H/HeJ strain was characterized by a head blaze as well. This blaze, however, is occasionally not evident in Sp^2J/+ C3H/HeJ mice and is never present in Sp^2J/+ offspring derived from C3H/HeJ ( Sp^2J/+) x C57BL/6J (+/+) matings. This occurrence of a head blaze in strain C3H/HeJ and its absence when outcrossed also has been observed with various steel ( Sl) alleles ( Dickie, 1964b).

²⁸The other semidominant mutation did not involve the Sp-locus. It occurred in a C57BL/6J male and is called "belly spot" ( Bs). Bs/Bs homozygotes die prior to the twelfth day of gestation ( Dickie, 1964b).

²⁹Searle ( 1968a) finds it difficult to think of any other explanation for the variegation of Va mice than somatic mutation (see Chapter 10, note 28), and, if he is correct, this would imply that this gene is highly mutable. He cites as possible evidence Schaible's ( 1963a) observation that Va/+ males had significantly more non-Va than Va offspring on outcrossing. Searle also notes that "an alternative explanation might be the existence of some physiological threshold during development with respect to some aspect of pigment production, so that only very occasionally does a melanoblast acquire the potentialities necessary for full pigment production in its descendant melanocytes." Schaible ( 1962) concluded that each wild type area of pigment in Va/+ (or in W^a/+ or Mi^wh/+) mice resulted from a population of cells descended from a single altered melanoblast, a contention which certainly seems most reasonable, but whether this alteration is due to something other than a mutation, such as somatic crossing-over, remains to be determined.

³⁰If Grüneberg is correct and the influence which Va has on pigmentation is secondary to abnormal hair structure one would anticipate most of the hairs of Va homozygotes to be grossly abnormal since they are unpigmented. This remains to be determined. Moreover, if the pigmentary effect of Va always is mediated via the hair it is somewhat surprising that the small patches of pigment which occur in homozygous animals are of unaltered color as one might expect at least some of these hairs to be moderately abnormal and appear grey.

³¹According to Deol ( 1954) the anomalies of the labyrinth are not congenital as the first defect is not observed until the animals are 4 days old. The tectorial membrane of the cochlea, the first structure to be visibly affected, is delayed in thinning out, loses all contact with the organ of Corti, and eventually shrivels up. During the second week after birth the spiral ganglion and the organ of Corti also begin to display a progressive degeneration and, during the third week, the stria vascularis degenerates. In Va/+ mice the cristae ampullares are severely affected in Va homozygotes. The average cell size in the vestibular ganglion also is significantly reduced. In general all of these lesions are more pronounced in Va homozygotes than in heterozygotes.