Notes to Chapter 9

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1Although one might conclude that certain mutation affect only certain pigment centers, e.g., the lumbar centers by bt, the coronal center by Wa, the sacral centers by Rw (rump-white) etc., Schaible believes his selection experiments support Wright's ( 1942) gradient concept of pigment formation in spotted mammals. Thus, not only does bt appear to restrict the proliferation of melanoblasts in the lumbar region but, to a lesser extent, it also appears to restrict proliferation of melanoblasts in pigment areas located adjacently and farther away. Moreover, while Schaible contends that white spotting results from a failure of pigment cells to migrate out from one or more of his proposed "pigment centers," he also believes that the primary mechanism responsible for this migratory failure differs among the various mutants.

2s/+ mice as a rule are pigmented normally but may have a white tail tip and digits.

3Previously Gates ( 1926) and Dunn and Mohr ( 1952) had reported that piebald spotting affected the pigmentation of the choroid. Gates found that all the s/s mice he examined microscopically lacked choroidal pigment, including the pigment in the outer layer of the iris; retinal pigment was unaffected. Dunn and Mohr noted the choroidal pigment of their piebald mice was generally defective. In some of these mice (which were only partially inbred) one or both eyes appeared light ruby or pink. When these eyes were examined with an ophthalmoscope the commonest finding was an enlarged pupil (aniscoria), varying all the way from a slight enlargement to what appeared to be complete absence of the iris (aniridia). This enlargement of the pupil was usually accompanied by a reduction of the amount of pigment in the iris. A smaller proportion of lighter colored eyes had pupils of normal or nearly normal size but lacked a part or all of the dark iris pigment. In the stock examined, about 80% of the mice and 63% of the eyes had iris defects.

4In most cases Deol observed that the choroid had one large spot and several small ones, and in some areas there was an intermingling of very heavily pigmented and lightly pigmented regions. The borders of the spots were generally indented but very occasionally they ran along blood vessels and so were reasonably straight. The intensity of pigmentation in most pigmented regions seemed to be normal. Also, there was more pigment in the ventral half of the choroid, and no regular pattern or symmetry was evident.

5The incidence of this condition is very much less in s/sl animals which, at least on the genetic background studied by Lane, were heavily spotted and indistinguishable from s/s mice. Lane found that of 12 s/sl heterozygotes maintained for breeding, three died of megacolon at 11, 13, and 23 months, respectively, while the others were free of the condition when autopsied between 12 and 23 months of age.

6There is another simple autosomal recessive piebald mutation which provisionally has been designated pb. This mutation, which occurred at Albert Einstein in the ot12 stock, is of special interest because when homozygous, it causes a progressive dilution of pigmentation so that "some pb/pb animals become completely white, and are distinguishable from albino mice only by their pigmented eyes" ( Gluecksohn-Waelsch, 1965). Even more remarkable is the fact that this progressive loss of pigment starts early in life and is rapid (Gluecksohn-Waelsch, unpublished). Moreover, during the period of pigment loss many pb/pb mice bear a striking resemblance to c/c <--> +/+ allophenics. Indeed, because these mice display conspicuous striping patterns while undergoing pigment loss, it is tempting to speculate that they possess not only some inviable clones of melanoblasts, which are preprogrammed to die prenatally (see Chapter 7, Section VII), but also some which are preprogrammed to die postnatally after they have differentiated and produced pigment. This mutation also induces a high incidence of megacolon ( Gluecksohn-Waelsch, 1964a). Symptoms observed in homozygotes include abnormalities of fecal excretion, abdominal distention, and stunting of growth and are reminiscent of those described for the human syndrome known as Hirschsprung's disease ( Gluecksohn-Waelsch, 1964b). As far as the author is aware, the relationship of this mutation to either s or sl is not known.

For the pb allele:
pb Allele (MGI) Gene (MGI) All Alleles (MGI)

7The all-white line employed possessed from 85 to 100% dorsal white (mean 99.3%) and the dark line had 5 to 25% dorsal white (mean 9.9%).

8K. Zimmerman ( 1941) mentions a wild population from Berlin-Buch in which approximately 70% of the individuals were homozygous for s in the absence of any " k" genes. These mice possessed either a few white hairs on their belly or one or two central spots with an occasional white tail tip (see Keeler, 1933).

9Grüneberg ( 1936b) states that "genes affecting the pigmentation of the tail tip are very common in tame mice and are also not rarely found in free living mouse populations." From a breeding study involving an agouti line, in which most of the animals had dark tail tips, and a nonagouti stock, in which about 75% of the animals had light tail tips, Grüneberg concluded that dark tail tip was dominant, though possibly not completely so, and that a single gene pair was involved.

10As pointed out by Grüneberg, the gene flexed-tailed (see Chapter 11, Section II) also fits into this category of "minor spotting genes."

11To rule out a third possibility, namely that s/s melanoblasts do not reach all areas of the integument because they are unable to migrate, Mayer ( 1967a) studied the migration of +/+ and s/s neural tube cells by labeling them with tritiated thymidine before combining them with unlabeled +/+ or s/s skin. Although the maximum incubation time of 96 hours precluded the development of melanocytes from these labeled grafts, Mayer found that there was a good correlation between the presence of mouse melanocytes in the host tissues surrounding the +/+ grafts after 125 days incubation, and the presence of labeled +/+ or s/s labeled neural tube cells in this same area after an incubation of 96 hours, the only difference was that at 96 hours the area covered by the labeled cells was not as great as the pigment spread after 15 days. It therefore seemed most likely that the labeled cells were in fact melanoblasts and that their migratory capacities were the same regardless of their genotype.

12Mayer points out that because s/s produces rather small patches of white hair largely restricted to the belly on a C57BL/6J background, the +/+ skin sample used in grafting would actually have been pigmented in a C57BL/6J- s/s mouse.

13According to Mintz one would also have to assume that the older explants of s/s skin were more likely to possess a higher proportion of moribund (inviable) melanoblasts than the younger explants, and this too could affect the ability of viable cells to migrate into and become established in these transplants.

14Since both melanoblasts and myenteric ganglion cells are of neural crest origin, it appears that s and sl influence the neural crest before it differentiates into these components. Moreover, because the inner ear of sl homozygotes also displays severe abnormalities, with the neural epithelium always affected, Deol ( 1967) contends that it is reasonable to assume that there is a deficiency in the acoustic ganglion and that part of this ganglion too is derived from the neural crest.

15The latest mutation has been designated btJ; however we will refer to both as bt.

16Schaible ( 1969) contends that the synergistic effect which occurs when two spotting genes, e.g., bt/bt; Miwh/+, are incorporated into the same genome may be more apparent than real and he cites an experiment by Reams ( 1967), as well as some observations by Mayer and his colleagues ( Mayer and Maltby, 1964; Mayer, 1965; Mayer and M.C. Green, 1968), to support this contention. Reams inoculated leg buds of White Leghorn embryos with different numbers of chick melanoblasts. He found that when the number of cells introduced was large not only did the skin and feathers of the entire leg become heavily pigmented, but the underlying muscles likewise were pigmented. On the other hand, when the number of cells inoculated was small, the melanocytes remained in the skin without emigrating into either the feather follicles or into the muscle. Mayer and his associates compared the occurrence of melanocytes in the leg musculature of mice heterozygous or homozygous for lethal spotting ( ls), piebald ( s), viable dominant spotting (Wv), and steel ( Sl). They found that whereas melanocytes always occurred in the leg musculature of normal mice they were absent in all mutant homozygotes and reduced in numbers in all heterozygotes. From these findings Schaible argues that while each spotting gene by itself causes a reduction in the number of pigment cells so that few or no cells emigrate to the muscle, the skin population is only slightly reduced. As a result, the white regions of the coat are small. However, when two spotting genes are combined, a much greater proportion of the reduction in the cell population occurs in the skin. As a consequence, "the visible effect on the amount of white regions in the coat is much greater than the additive effects of the two mutant genes even though the effects on the total pigment cell populations probably were additive." While this explanation cannot be refuted it is difficult to reconcile with the fact that some spotting genes, such as belted ( bt), which affect only hair pigment [estimates of the number of melanocytes in the muscles of the leg, as well as in all other regions of the body which normally possess melanocytes, are the same in bt/bt and +/+ mice ( Mayer and Maltby, 1964)] nevertheless have a hyperadditive effect when combined with other spotting factors.

17In a systematic study of 11 inbred strains of pigmented mice, Mayer and Maltby ( 1964) found that in addition to the retina and choroid of the eye, harderian gland, and ankle skin, melanocytes also occur in the leg musculature and the membranous labyrinth of the ear (where they are associated with the membranes of the utriculus and the semicircular canals). For example, in the C3H/J strain they observed that the lateral and medial heads of the gastrocnemius plantaris, soleus, and the peroneal group were pigmented whereas the muscles comparing the anterior compartment of the leg were generally pigment free. They report that "the total number of melanocytes in the leg musculature is 390 +/- 47, and these cells are located in the endomyseal and perimyseal connective tissue between the muscle fibers."

18Schaible also believes that because in his and Mayer and Maltby's experiments the grafts were exposed to an embryonic environment much longer than is normally the case (until the equivalent of 6 days postpartum in Mayer and Maltby's experiments and 7 to 9 days postpartum in his), that melanoblast migration is enhanced; i.e., he contends that the stimulus for proliferation and migration of pigment cells is for some unknown reason much stronger in the chick coelom than in the normal environment of the mouse. As far as I am aware there is no evidence to support this assertion.

19Actually, when one considers that Mayer and Maltby found pigmented hairs in 50% (9/18) of the grafts which originated from the level of the belt, their findings are not too different from Schaible's. Moreover, the fact that five of these grafts possessed both pigmented and white hairs is undoubtedly significant. One could also argue that the reason why some of the 14-day-old embryonic grafts of Schaible were unpigmented was because they had one or two extra days of (intussusceptive) growth in vivo. Clearly if differences in growth are responsible for the discrepancy in results, grafts from the lumbar region of "wide-belt" embryos, of the size employed by Mayer and Maltby, should produce many more unpigmented hairs.

20In 1975 Kelly reported a recessive mutation which produced a narrow wide belt. The deviant occurred among the progeny of a male treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Since this mutation is not allelic with either bt or s it has been designated "belted-2" ( bt-2).

For the bt-2 allele:
bt-2 Allele (MGI) Gene (MGI) All Alleles (MGI)
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