How The Cat Got Her Spots

No two calico cats have the same coat pattern. The calico dons a coat of white fur splotched with large and vivid patches of orange and blackish-brown fur. For over a 100 years, scientists have puzzled over how this tri-colour pattern comes to be.

Two research teams independently solved the final piece of this puzzle, and reported their findings this May in the journal Current Biology. One team was from Kyushu University, Japan, and the other was from Stanford University, US. 

One piece of the calico puzzle was solved in 2014, with the discovery of the mutation responsible for creating the background of white fur.  It was in a gene named KIT located on cat chromosome B1. The mutation was named white spotting.  

Mary Lyon’s prediction

A second piece of the puzzle was solved more than 60 years ago by the geneticist Mary Lyon. She explained why some of the pigmented patches were orange while the others were blackish-brown. Her explanation also accounted for why most calicos are females, although male calicos make exceptionally rare appearances. 

In mammals, females have two X chromosomes (XX), while males have one X and one Y chromosome (XY).  Lyon inferred that in the early development of female mammals, one of the two X chromosomes is randomly selected and inactivated in each cell. Thereafter, the cells descended from these cells maintained the same inactivation pattern as their parent cell. This is called X-chromosome inactivation (XCI).  It equalises the dosage of X chromosomal genes in males and females.

The Kyushu and Stanford teams solved the third and final piece of the puzzle. They discovered that in calico cats the two X chromosomes carried different variants of the ARHGAP36 gene. Let’s call them XO and XB, for orange and blackish-brown. 

And as predicted by Lyon, skin cells in the orange patches showed inactivation of the XB chromosome, and those in the blackish-brown patches had inactivated the XO chromosome.  

Calicos and tortoiseshells

The relevant skin cells are called melanocytes. Melanocytes migrate into the epidermis during early development. There they multiply and give rise to differentially-coloured patches of fur.  

The KIT mutation on chromosome B1 acts to slow the migration of melanocytes to the epidermis. Failure of melanocytes to reach the epidermis results in white (unpigmented) fur. The slower the migration, the larger are the white patches, and the less time there is for the coloured patches to intermingle.

What about XOXB females that do not carry the white spotting mutation?  In them, there is hardly any white fur and the orange and blackish-brown patches are more intimately mixed.  The cat has a more brindled appearance called tortoiseshell.

XXY males

The overwhelming majority of males has only one X chromosome.  Thus a male cat can be either XO Y or XB Y.  The former is orange, or, if it also carries the white spotting mutation, orange and white, and the latter is blackish-brown or blackish-brown and white.

Occasionally however, a male is produced bearing two X chromosomes and one Y chromosome (XXY).  This happens either when an egg (X) is fertilised by a XY-bearing sperm, or when an XX egg is fertilized by a Y-bearing sperm.  

Aberrant XY sperm and XX eggs are rare and result from failure of the sex chromosomes to separate during the formation of sperm or egg cells.  XXY males undergo XCI, and this generates their calico or tortoiseshell coat patterns.

The XXY condition is also encountered in humans. In males with a condition called Klinefelter syndrome.  Klinefelter syndrome is often not diagnosed until adulthood. Klinefelter males are taller than average, have smaller testicles, and make less of the hormone testosterone. They may also have less muscle mass, less body and facial hair, extra breast tissue, and produce little or no sperm.

Eumelanin versus pheomelanin

The O variant of the ARHGAP36 gene was missing a 5100 base-pairs long segment of DNA, whereas the B variant retained this segment. The O variant is the mutant form of the gene, whereas the B variant is the original non-mutant version. The missing segment contains a regulatory element that is conserved across mammals, and its absence in cat skin tissues resulted in over-expression of the ARHGAP36 gene.  

Additional studies suggested that aberrant ARHGAP36 protein over-expression in melanocytes led to suppression of synthesis of eumelanin (a dark, brown to black pigment) and promoted the synthesis of pheomelanin (a yellow to reddish pigment).

The Kyushu team attempted to produce calico mice by deleting the 5100 base-pair segment from the mouse genome.  However, the attempts were not successful.  This might be because the regulation of melanogenesis by ARHGAP36 protein might be specific to the cat or to felines.

CC, not a carbon copy

Why are we so confident that no two calicos have the same coat pattern? Which X chromosome is selected for XCI in different cells, and melanocyte movement to the skin to produce pigmented and un-pigmented patches are both essentially random processes.  This was borne out by experiments to clone a cat in 2001.  

The nucleus of an ovarian cell from a calico cat, named Rainbow, was transplanted into an enucleated feline egg. The re-nucleated egg cell was then implanted into a surrogate mother, which happened to be a tortoiseshell cat. The surrogate mother gave birth to a kitten named CC (copy cat). DNA analysis confirmed that CC and Rainbow were genetically identical. However, their coat patterns were markedly different.  

This finding dampened the excitement of cat-lovers who hoped to use cloning to posthumously ‘revive’ their beloved companions, and the business of pet cloning became a non starter. 

Durgadas P Kasbekar is a retired scientist.

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