This article is from the Winter 1999 AFRMA Rat & Mouse Tales news-magazine.

By Debra Mauzy Melitz, September 1998

Colors are light. To understand how colors are formed, we need to understand some basic principles of light. Light has two main physical characteristics, a wavelength and a frequency. Both are related to each other, so we will focus just on the wavelength characteristic. White light is composed of varying wavelengths. A wavelength is referred to by its length in nanometers (nm). There are 100,000,000 or 100 billion nanometers in a meter. A human eye can see light between the wavelengths of 800 to 380 nanometers (red light to violet). See Figure 1.

What we see is the light that is absorbed by cells in the back of our eye in the retina. When we see a mixture of all the wavelengths between 800 to 380 nanometers, we see white. When we do not see any light, we see black. Individual colors are seen as the absorption of select wavelengths of light. For an object to appear colored, it must absorb only certain wavelengths and reflect back to our eyes the remaining light. For an example, if an object appears red, that means that the object absorbed all wavelengths in the 650 to 380 nanometer range (orange to violet) and reflected the wavelengths in the 800 to 650 range. The object is red because it did not absorb the wavelengths that correspond to red.

Several physical factors affect how the wavelengths of light are absorbed or reflected. They are:

1. Simple Reflection
   Simple reflection occurs at surfaces that contain many small crystals. All the light waves are reflected off the surface. The surface appears white.
2. Refraction
   Refraction occurs when some wavelengths of light are slowed down. The longest wavelengths (red) are slowed more than the shorter ones (violet). What we see is a rainbow.
3. Scattering
   Scattering occurs when very small particles are suspended in an otherwise transparent media. The light hits the small particle and changes its direction. The degree of scattering depends on the wavelength of the light (shorter wavelengths scatter better) and the size of the particle. The smaller the particle, the greater the refraction or change in direction. The blue color of the sky is a result of this effect. Numerous impurities exists in the atmosphere and when light hits these particles, blue light is reflected at a 90 degree angle. Blue light is affected strongly because of the high density and the small size of the particles. The other wavelengths of light are also refracted, but the degree or angle of refraction is less. This effect was first described by a 19th century physicist, John Tyndall. John Tyndall demonstrated his “why the sky is blue” to Queen Victoria and since then the phenomenon has been named Tyndall scattering.
4. Diffraction
   Diffraction occurs when light passes through minute openings. The red lightwaves are most effected. You can see the diffraction effect if you look at the colors surrounding a pinhole.
5. Interference
   Interference is a result of asynchrony between the wavelengths of light. Waves are slowed down in a medium that has a refractive index greater than air. If light waves enter a region of higher refractive indexes, they slow down. If they are then reflected back out of this medium and into air, they are out of phase with the other light. If light is out of phase where the wave crests meet wave troughs, the light is eliminated and we get black. If the wave crests and troughs coincide, the light is in phase and the color is very pure and bright. Anything in between weakens the color. Since white light is a mixture of wavelengths, only one wavelength will be exactly in phase and only one color will appear extra vibrant. The other wavelengths will be weakened or eliminated. How much out of phase the light will be depends on the distance it had to travel within the medium. Common occurrences of interference is the iridescence of soap bubbles and oil slicks. In both cases, the light passes through and is reflected back through a thin film. Most of the vibrant blues and violets of bird feathers are all caused by interference. Interference colors are the purest and brightest colors known.
6. Panchromatic Absorption
   Light may enter a medium and be internally reflected till it runs out of energy. If none of the light manages to leave the medium, the result is black. If some light is not caught in the internal reflection, the resulting light will be gray. Shiny black is the result of a small amount of reflection occurring at the surface and the rest of the light getting trapped within the medium.
7. Selective Absorption
   Certain compounds have the ability to selectively absorb certain wavelengths of light. The wavelength of light absorbed is directly related to the exact chemical composition of the material. These materials are called pigments.

Structural colors are due to the simple reflection, refraction, scattering, diffraction, interference, or panchromatic absorption of light. Structural colors occur because some compounds form crystals that have the ability to act like prisms and diffract or bend light in different ways. Interference and diffraction can lead to iridescence. The bright blues and purples of butterfly wings and peacock tails are all due to diffraction and interference. Diffraction colors are uncommon in mammals. One example is the eyeshine of some mammals due to diffraction at the tapetum lucidum at the back of the eyes. Eyeshine colors can vary from fluorescent green of the bush baby, to the blues and violets of cats and humans. The simple scattering of light is more common in mammals. The blue colors of some cat’s, human’s, and horse’s eyes are due to the (Tyndall) scattering of the light by very fine crystalline particles in the iris. The whiteness of a body is due to scattering of incident white light. Hair is white if it contains air spaces that scatter light, but no pigment granules that would absorb light. You can not extract the color for any color that is a structural color.

Most of the colors in mammals are the result of pigments. A compound that absorbs light is called a pigment. The word pigment comes from the Latin word pingere, to paint.

Just like an artist has a range of colors available to work with, nature also has a palette. The range of pigmented colors available to mammals range from white via gray to black and from black via brown to yellow and orange red. The range for other animals is much greater. Apart from carotene and bile, almost the only pigments found in mammals are hemoglobin and melanins. The pinkish skin color is due to the hemoglobin of the red blood cells. Another example of color due to hemoglobin is the faces of some monkeys that are a vivid red when threatened. But the melanins are the most important producers of hair color in mammals. They are also widespread throughout living things. Melanin is present in mushrooms, molds, flatworms, insects, and practically all vertebrates. It has even been found in fossils over 150 million years old. One other mammalian pigment is trichosiderin, a phenolic iron compound which can be extracted from human red hair.

In mammals, melanin exists in two forms: eumelanin (brown or black) and phaeomelanin (yellow or reddish).

Eumelanin is an insoluble polymer which is always attached to protein in mammals. A polymer is a compound made up of multiple subunits. The eumelanin subunits are derived or made from tyrosine, one of the essential amino acids. Tyrosine is converted to “dopa” (3.4-dihydroxyphenylaline) the first compound on the pathway to the eumelanin subunits by the enzyme tyrosinase (Figure 2). Dopa is then converted to Dopaquinone by the enzyme tyrosinase and another enzyme, dopa oxidase. The Dopaquinone undergoes further changes and polymerization before becoming melanin. At one point, Dopaquinone can either become Dopachrome or Cysdopas. If it becomes Dopachrome, then the final pigment will be eumelanin. If Dopaquinone becomes Cysdopas, then the pigment will be Phaeomelanin. The production of Cysdopas increases if another enzyme, glutathione reductase, is abundant. (Prota, 1992)

Eumelanin polymer can be composed of four different kinds of subunits, all are made from Dopachrome. The most common product is the indoles. The relative amount of each subunit can effect the packaging of the polymer which in turn effects the color seen. The eumelanin polymer is bound tightly to a peptide (protein) component. Two other enzymes are known to be involved with eumelanin subunit formation. They are TRP1 and TRP2.

Phaeomelanin is also a polymer composed of many subunits, but there is only one class of subunit that polymerizes to form phaeomelanin. Tyrosinase is also needed for phaeomelanin production, but there is a switch at one point in the biochemical pathway and the result is the production of the phaeomelanin subunit. Phaeomelanin also has a peptide component but it is not as tightly bound.

The cell that makes melanin is the melanocyte. Once the melanin is made, it may leave the melanocytes as granules called melanosomes. Other cells will absorb these granules. A melanocyte can be thought of as a unicellular gland, secreting pigment granules into surrounding cells. There are three basic types of melanocytes:

1. Retinal melanocytes
2. epidermal melanocytes
3. follicular melanocytes

Retinal melanocytes arise from the developing eye. All other melanocytes are derived from neural crest cells and are known as dendritic melanocytes because they have a very irregular shape. The dendritic melanocytes compose the epidermal (the melanocytes responsible for secreting melanin granules into the skin) and the follicular (the melanocytes responsible for secreting melanin granules into hair or hair-like structures).

Neural crest cells are the primary source of several structures and help in the formation of others. Examples of structures requiring or derived from neural crest cells are: visceral skeleton, head and trunk mesencyme, the cephalic, spinal, sympathetic and visceral ganglia, the melanocytes, and the sheath cells of the Schwann. Any mutations that would affect neural crest cells as a group would affect many other important cell types so these mutations are therefore usually lethal. Neural crest cells that form melanocytes migrate early in development. In adult mice, all the melanocytes that give rise to hair color come from just 34 original neural crest stem cells. They migrate from the neural crest and divide to produce clones that will become melanocytes in particular localized regions of the body. The epidermal and follicular melanocytes come from only 14 of the 34 original neural crest stem cells. Each original neural crest stem cell population migrates to a specific region of the body. In the mouse, these 14 regions are diagrammed below (based on the work by R.H. Schaible in 1969).

Epidermal melanocytes may vary in size and shape. In areas where there are few melanocytes, they tend to have long dendrites with a shape resembling a central nervous system nerve. Where the density of the melanocytes is greater, they have shorter dendrites and a more rounded cell body. They transfer the pigment they produce to surrounding cells called kerotinocytes. The numbers of melanocytes per unit area of the epidermis vary but density does not appear to be related to the final amount of pigmentation. Variations in skin color are due to differences in melanocyte activity rather than density of the cells themselves. The production and secretion of the pigments proceeds throughout life and can be triggered by both internal and external stimuli. An example of an external stimuli is sunlight.

Follicular melanocytes only synthesis melanin during a particular stage of hair development. The pigmental activity of the follicular melanocytes frequently differs from the surrounding epidermal melanocytes. Furthermore the follicular melanocytes may cease to function, such as graying, but the surrounding epidermal melanocytes will continue to produce melanin.

Two functional forms of melanocytes can be found—amelanotic (without melanin) and melanotic (with melanin). Both types are present in the hair follicle. Each form has the ability to change to the other form barring any major genetic problems. The amelanotic form is found in albinos. These melanocytes cannot produce melanin because the first enzyme in the pathway, tyrosinase, is not present. The dendritic class of melanocytes respond to the hormone, melanocyte stimulating hormone (MSH) produced by the pituitary gland in the brain, to begin or increase production of melanin.

One other role for melanocytes besides production of pigmentation is their involvement with the development of the mammalian inner ear. If melanocytes are not present as is the case in some mutations, the inner ear has a reduced amount of interdigitation in the cochlea duct. This appears to lead to the cochlea duct becoming dysfunctional (Strel and Barkway,1989).

Within melanocytes, pigment formation takes place in a distinct compartment known as the melanosome. There are two basic types of melanosomes, the eumelanosome and the phaeomelanosome. Usually, epidermal melanocytes will produce only one type of melanosome. Follicular melanocytes can produce both types. The number, type and distribution of melanosomes in the epidermis is genetically controlled. Typically, dark skinned individuals have larger melanosomes than lighter skin individuals. Light skinned melanosomes tend to clump together and eventually get degraded. The size of individual granules can be increased upon exposure to UV radiation.

Stages of Melanosome Development
For Eumelanin:

1. Synthesis of “pro-tyrosinase” enzyme occurs. The enzyme is not able to convert tyrosine to Dopa yet.
2. Arrangement of the pro-tryosinase into ordered structure called the pre-melanosome occurs and then the enzyme becomes functional.
3. The initiation of melanin synthesis with the accumulation of melanin within the pre-melanosome begins. The pre-melanosome is now called the melanosome. When the melanin formation is completed, the enzyme, tyrosinase, becomes inactive. The melanosome is now a fully formed ovoid melanin granule. When viewed under the microscope, eumelanin granules can have three different grades of color, four different shapes, and a wide variety of sizes.

For phaeomelanin:
Instead of an organized pre-melanosome matrix, there is a tangled mass of fine fibers deposited rather randomly in a rounded granule. When viewed under the microscope, phaeomelanin granules have a uniform shape, size, and intensity.

• Prota, G. Melanins and Melanogenesis. Academic Press, San Diego, 1992.
• Strel, K. P. and Barkway C., 1989. “Another Role for Melanocytes: Their Importance for Normal Stria Vascularis Development in the Mammalian Inner Ear.” Development 107(3):453–463.

Go to Part 2: Hair Development.
Go to Part 3: Genetic Loci.

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