Brief Review of Pigment Generation, Part III (BIOL 5104 blog 7)

Migration of Melanoblast (continued)

In Xenopus embryo, contact inhibition of locomotion was found happens during neural crest cell migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, like metastatic cancer cells. Inhibition of non-canonical Wnt signaling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt signalling members localise at the site of cell contact, leading to activation of RhoA in this region. This contact inhibition of locomotion should be present in any species and breeds which have uniformly distributed pigmentation. But the I still don’t know whether this in chicken is similar like this in Xenopus.

Recently, two distinct types of melanocyte are described in mice, dermal (non-cutaneous) melanocytes and epidermal melanocytes (Aoki, et al., 2009). Experiments have shown that ectopically expression of Endothelin 3 (EDN3) affect the dermal pigmentation but not the hair color (Garcia, et al., 2008). These non-cutaneous like dermal melanocytes are incapable of contributing to epidermal hair follicle pigmentation further highlighting the functional differences between these two melanocyte populations (Aoki, et al., 2011). Similar dermal pigmentation phenotype is seen when EDN3 is ectopically expressed in the chicken, which is called dermal hyperpigmentation or Fibromelanosis (FM), a breed character of Silkie chickens. EDN3 is up regulated in Silkie chicken during migration of melanoblast and even in the adult Silkie chicken skin tissue. But Silkie expressing FM can be white in feather (so called feather, it looks like mammals hair or chick’s down) and also can be other feather color, which indicates that FM does not affect feather pigmentation (Dorshorst, et al., 2011). Previous studies have shown that in avian, EDN3 exerts trophic, mitogenic and melanogenesis-promoting activities on early neural crest cells in culture. EDN3-responsive precursors were identified in neural crest clonal cultures and include fate-restricted glial and melanocytic cells as well as bipotent glia–melanocyte (GM) precursors. The GM progenitor is thus a main target of survival and mitogenic activities of EDN3. Experiments have shown that EDN3 appeared capable for transition between glia and melanocytes in vitro (Douarin and Dupin, 2003). But it seems like glia, or at least its function, is not affected by high expression of in EDN3 Silkie chicken. The explanation need to be further investigated.

When we are talking about FM in Silkie, we have to mention another gene, inhibitor of dermal melanin (Id). The phenotype of Id gene is the abnormal migration of melanoblast, which will invade into the ventral pathway normally reserved for neuronal and glial cell lineages. So, Id causes pigmentation of internal connective tissue and the exterior (Dorshorst, et al., 2010). Similar like inhibitors prevent glial cell entering dorsolaterally pathway, something called barrier molecules (PNA-binding molecules) can stop melanoblast to migrate ventrolaterally. But what the barrier molecules exactly are is still unknown, we can only label them by PNA (Lection Peanut Agglutinin). In this way, we can know the distribution of barrier molecules is normal in Silkie during most of the time of melanoblast migration. But in later stage, they disappear and then melanoblast migrates from the dorsolateral space to the ventral part. So, the melanoblast in the ventral part is not because of their migration in the same way as the neural/glial precursors at the beginning. Some other experiments have shown that the abnormal migration of melanoblast is not an autonomous property of Silkie neural crest, but the environment difference around the pathways, which consists with previous observation of PNA-binding molecules’ down regulation (Faraco, et al., 2001).

The identification of the barrier molecules and the detail mechanism of Id gene will give us better understanding about neural crest cells’ path finding.


Carlos Carmona-Fontaine, Helen K. Matthews, Sei Kuriyama, Mauricio Moreno, Graham A. Dunn, Maddy Parsons, Claudio D. Stern, and Roberto Mayor. Contact Inhibition of Locomotion in vivo controls neural crest directional migration, Nature. 2008 December 18; 456(7224): 957–961.

Aoki H, Yamada Y, Hara A, Kunisada T. Two distinct types of mouse melanocyte: differential signaling requirement for themaintenance of non-cutaneous and dermal versus epidermal melanocytes. Development. 2009. 136: 2511–2521.

Garcia RJ, Ittah A, Mirabal S, Figueroa J, Lopez L, et al. Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model. J Invest Dermatol. 2008. 128: 131–142.

Aoki H, Hara A, Motohashi T, Osawa M, Kunisada T. Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice. Pigment Cell Melanoma Res. 2011. 24: 125–135.

Ben Dorshorst, Anna-Maja Molin, Carl-Johan Rubin, Anna M. Johansson, Lina Stromstedt, Manh-Hung Pham, Chih-Feng Chen, Finn Hallbook, Chris Ashwell, Leif Andersson. A Complex Genomic Rearrangement Involving the Endothelin 3 Locus Causes Dermal Hyperpigmentation in the Chicken. PLoS Genetics. 2011. 7(12): e1002412.

Nicole M Le Douarin, Elisabeth Dupin. Multipotentiality of the neural crest. Current Opinion in Genetics & Development.  2003. 13:529–536.

Ben Dorshorst, Ron Okimoto, Chris Ashwell. Genomic Regions Associated with Dermal Hyperpigmentation, Polydactyly and Other Morphological Traits in the Silkie Chicken. Journal of Heredity. 2010. 101(3):339–350.

Cloris D. Faraco, Sonia A.S. Vaz, Maria Veronica D. Pastor, Carol A. Erickson. Hyperpigmentation in the Silkie Fowl Correlates With Abnormal Migration of Fate-Restricted Melanoblasts and Loss of Environmental Barrier Molecules. Developmental Dynamics. 2001. 220:212–225.

Brief Review of Pigment Generation, Part II (BIOL 5104 blog 6)

Migration of Melanoblast

In part I, we have reviewed how two types of neural crest cell, neural/glial precursors and melanoblast are differentiated from neural tube. After this process, the neural/glial precursors will migrate ventrolaterally and the melanoblast will migrate dorsolaterally. As we are focused on pigment generation, here we are going to talk about the migration of the melanoblast only.

There are several mechanisms that prevent nonmelanoblast to migrate dorsolaterally, one of them is ephrin. The expression of ephrin in posterior of somite and in dorsolateral pathway excludes the nonmelanoblast neural crest cells and only allows them to enter the anterior part of somite in ventrolateral way. So, why melanoblast can migrate dorsolaterally between the ectoderm and the somite in the presence of ephrin? The key receptor is EphB2 expressed only in melanoblast, which not only allow melanoblast to survive in ephrin, but also act as chemoattractant, to attract melanoblast to migrate dorsolaterally. Another inhibitor of nonmelanoblast is Slits. Similarly, the receptors of Slits, Robo1 and Robo2 in melanoblast allow them to migrate dorsolaterally. It is possible that ephrin and Slits act in conjunctly (Melissa and Carol, 2007). The last inhibiter of nonmelanoblast I want to mention here is endothelin3 (ET3), the ligand of EDNRB2. EDNRB is up regulated in neurons and glial precursors but cannot help them to survive in the presence of ET3. Then, melanoblast down regulates EDNRB and at the same time up regulates EDNRB2. Some experiments have shown that without these inhibitor, nonmelanoblast will migrate dorsolaterally even they do not have these receptors (this receptors act in the role of path finding). Interestingly, although the ligand of EDNRB2 and EphB2 are different, the overexpression of EDNRB2 can maintain normal dorsolateral migration of melanoblast in the absence of EphB2, and vice versa (Melissa, et al., 2008), suggesting they may have overlap in pathway. Other repulsive cues present in the dorsolaterally pathway that restrict the migration of neuronal precursors include spondins, chondroitin sulfate proteoglycans and PNA-binding molecules (Jia, et al., 2005).

In the view of spatial expression, while melanoblast is migrating between ectoderm and dermamyotome, ET3 is expressed in both ectoderm and dermamyotome (Nagy, et al., 2006). But Steel Factor (SLF) only up regulated in dermamyotome after melanoblast are well into the dorsolaterally pathway, at the same time of the expressing of c-KIT in melanoblast (Reedy, 2003). In the mouse, Kit is necessary to maintain the survival of melanoblast, and for their consequent dispersal onto the dorsolaterally pathway (Wehrle-Haller, 2001). But it is different in chickens. c-KIT knocking out does not affect the migration of melanoblast, just affect the maintenance of melanoblast function.

Here I have present the main part of melanoblast migration. Besides, there are some other interesting processes happened during migration, which I will introduce in the following part.


Melissa L. Harris, Ronelle Hall, Carol A. Erickson. Directing pathfinding along the dorsolateral path – the role of EDNRB2 and EphB2 in overcoming inhibition. Development. 2008. 135, 4113-4122

Melissa L. Harris, Carol A. Erickson. Lineage Specification in Neural Crest Cell Pathfinding. Developmental Dynamics. 2007. 236:1–19.

Jia, L., Cheng, L. and Raper, J. Slit/Robo signaling is necessary to confine early neural crest cells to the ventral migratory pathway in the trunk. Dev. Biol. 2005. 282, 411-421.

Nagy, N. and Goldstein, A. M. Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system. Dev. Biol. 2006. 293, 203-217.

Reedy, M. V., Johnson, R. L. and Erickson, C. A. The expression patterns of c-kit and Sl in chicken embryos suggest unexpected roles for these genes in somite and limb development. Gene Expr. 2003. Patterns 3, 53-58.

Wehrle-Haller, B., Meller, M. and Weston, J. A. Analysis of melanocyte precursors in Nf1 mutants reveals that MGF/KIT signaling promotes directed cell migration independent of its function in cell survival. Dev. Biol. 2001. 232, 471-483.

Polydactyly (BIOL 5104 blog 5)

During the Christmas vacation of last year, I have driven to Key West, FL. That’s my first trip in the U.S. and really wonderful. What most impressed me are the Hemingway cats in Key West.

The Hemingway’s Cats (Fig. 1), descended from a single polydactylous cat given to the American author Ernest Hemingway by a ship’s captain in the 1930s. Around 40 descendants populate the grounds of the Ernest Hemingway Home in Key West ( of which about half have extra toes. Polydactylous cats, also called ‘mitten’ cats, are a well-known curiosity of the pet world. Valued as good luck charms by sea captains on sailing ships in the past centuries, polydactylous cats are frequently found along the northeastern coastal regions of North America.


Fig.1 Hemingway’s Cat (Photo taken by me)

Recent studies have shown that the polydactyly in cat is caused by point mutations in the 778 bp conserved noncoding element of the limb-specific cis-regulator of Shh termed the ZRS (Lettice et al. 2008). In the developing limb, Sonic Hedgehog (Shh) is normally expressed in a region of the posterior mesoderm, called the zone of polarizing activity (ZPA), and is required for proper anterior/posterior limb patterning (reviewed in Mariani and Martin, 2003). A highly conserved long-range limb-specific Shh enhancer is present within intron 5 of the LMBR1 gene (Dorshorst et al. 2010). This enhancer is called the ZPA regulatory sequence (ZRS) and is also known as mammalsfishes-conserved-sequence-1 (MFCS-1). The function of ZRS and Shh in variety of vertebrates’ digit development is quite similar. In normal birds, 4 toes is common and is thought as loss of the fifth toe from their ancestor. But evidence has shown that the polydactyly in chicken is not the reappear of the lost fifth toe, but has the same mechanism as 6 toes human, mice and cats. Chicken polydactyly is the existence of one or more extra most anterior toes developed preaxially (Fig. 2), some polydactyly cause the extra toe and also cause the loss of the normal most anterior toe. Different from other vertebrates, polydactyly can be a kind of breed character that is fixed in some breeds (e.g. Silkie, Sultan, Houdan, Dorking et al. We are not thinking Hemingway’s Cats as a breed, so I don’t think there are some breeds that fixed with polydactyly in vertebrates other than chickens). So the study of polydactyly in chicken is much earlier. In 1944, Warren has studied the genetics of polydactyly of chicken, the results is so complex that we will not talk about that here. Because of its variety of expression, it is hard to apply defined mapping. Some studies around 2000 showed the association of SNP within intron 5 of LMBR1 is strongly associated with polydactyly in Silkie chicken. In the meantime gain-of-function and loss-of-function analysis confirmed that the Lmbr1 gene is required for limb formation and reciprocal changes in levels of Lmbr1 activity can lead to either an increase or decrease in the number of digits in the vertebrate limb (Clark et al., 2001). Then researchers started to study the relationship between the expression and the function of Lmbr1 and polydactyly. But little progress is made in that area. Till recent work in the mouse has identified the ZRS region in the intron of LMBR1 which has nothing to do with the expression of Lmbr1 but can cis-regulate and repress Shh expression in the limb, (Maas and Fallon 2005), and similar mechanism found in human, researchers started to focus on the study of Shh in polydactyly chicken. Shh expression in the posterior of polydactyly chicken limbs is abnormal and demonstrates that ZRS long-range Shh control element is not an anterior specific element but general to regions of the limb that have the potential to express Shh. In ZRS mutated chickens, the size of the Shh expression domain and the range of Shh signaling increased. Interestingly, this increased range of Shh signaling played a role in induction of anterior Shh expression in the polydactyly chicken leg bud. A loss of programmed cell death in the anterior of the leg bud has been implicated in the formation of preaxial polydactyly in the Dorking chicken which is thought to lead the polydactyly (Ian et al. 2011).


Fig.2 Polydactyly in Chicken (Photo taken by me)

But two interesting things are shown by Warren that low temperature during incubation can inhibit the expression of polydactyly and when asymmetry of digit number individuals are found, the left leg are always be the side has more digit. This phenomenon was repeated later (Huang et al. 2006) but no one can give explanation till now. Mammal (like mouse) has some advantages on studying the digit development than chickens, but not in these two aspects: 1. mammal embryo developed in low temperature will cause much more problem rather than the development of digit. 2. Asymmetry of digit number in mammals is so rare. But if that happened in mammals, it will be much easier to find the clue of the mechanism by compare the development process between deferent species, and that must help us to have a better understanding on vertebrates digit development.


Laura A. Lettice, Alison E. Hill, Paul S. Devenney, Robert E. Hill. Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly. Human Molecular Genetics, 2008, Vol. 17, No. 7 978–985

Mariani FV, Martin GR. Deciphering skeletal patterning: clues from the limb. Nature. 2003.423:319–325.

Ben Dorshorst, Ron Okimoto, And Chris Ashwell. Genomic Regions Associated with Dermal Hyperpigmentation, Polydactyly and Other Morphological Traits in the Silkie Chicken. Journal of Heredity. 2010. 101(3):339–350

Warren DC. Inheritance of polydactylism in the fowl. Genetics. 1944. 29:217–231.

Yan Qun Huang, Xue Mei Deng, Zhi Qiang Du, Xiangpin Qiu, Xiaohui Du,Wen Chen, Mireille Morisson, Sophie Leroux, F. Abel Ponce de Léon, Yang Da, Ning Li. Single nucleotide polymorphisms in the chicken Lmbr1 gene are associated with chicken polydactyly. Gene. 2006. 374 10–18.

Clark, R.M., et al. Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1. Genetics. 2001. 159, 715–726.

Ian C. Dunn, I. Robert Paton, Allyson K. Clelland, Sujith Sebastian, Edward J. Johnson, Lynn McTeir, Dawn Windsor, Adrian Sherman, Helen Sang, Dave W. Burt, Cheryll Tickle, Megan G. Davey. The Chicken Polydactyly (Po) Locus Causes Allelic Imbalance and Ectopic Expression of Shh During Limb Development. 2001. 159, 715–726.