Title: Establishment of Spatial Pattern: Pigment Pattern Development in the Zebrafish
1Establishment of Spatial PatternPigment Pattern
Development in the Zebrafish
Presented by Tasha Hanes March 20, 2002.
2Pattern formation
- process by which cells in a developing embryo
acquire identities that lead to a well ordered
spatial pattern of cell activities
(Wolpert 1998) - five stages
- defining the body axes
- formation of germ layers
- morphogenesis
- cell differentiation
- growth
-
3Pigment cells are derived from neural crest cells
- Neural crest cells originate at the edge of the
neural tube
4Neural crest cells are pluripotent
5Neural crest cells migrate
- Requires disruption of the basal lamina
surrounding the neural tube - Requires loss of adhesion molecules on neural
crest cells - Two migration pathways
6Pigment cell precursors take the dorsolateral
pathway
- Migration between the ectoderm and the somites
7- Mutational Analysis of Endothelin Receptor b1
(rose) during Neural Crest and Pigment Pattern
Development in the Zebrafish Danio rerio
David M. Parichy, Eve M. Mellgren, John F. Rawls,
Susana S. Lopes, Robert N. Kelsh, and Stephen L.
Johnson Developmental Biology, 227, 294-306 (2000)
8Study organismZebrafish (Danio rerio)
9Pigment Patterns in the Zebrafish
- Zebrafish exhibit alternating stripes of
melanocytes, iridophores and xanthophores
Black melanocytes Silver iridophores Yellow
xanthophores
10Zebrafish undergo metamorphosis
- Pigment pattern changes during metamorphosis
- early larva
- stripes of melanocytes and iridophores in a
background of xanthophores - adult
- dark stripes of melanocytes and iridophores with
light iridophore and xanthophore stripes in
between
11How is gene activity controlling pigment pattern
partitioned across life cycle stages?
- Pigment patterns specified by genes
- Some genes affect pigment pattern at only one
life cycle stage
12Rose mutants
- Wild type pattern during early larval stage
- Fewer melanocytes during late metamorphosis
- Adults
- melanocyte deficits
- iridophore deficits
- ventral spots
13D. rerio ednrb1 may be responsible for the rose
phenotype
- G-protein coupled receptors for ET-1, ET-2 and
ET-3
- Endothelin receptors (ednr) initially found to
mediate vasoconstriction in amniotes - Mice ednrb1 knockouts exhibit melanocyte
deficiencies - rose mutation and endothlelin receptor b1
(ednrb1) have been mapped in the vicinity of each
other
14Goals of this paper
- 1) Test the correspondence of rose and endothelin
receptor b1 (ednrb1) - 2) Identify cell populations expressing edrnb1
- 3) Test the role of endothelin-1 in signaling to
ednrb1 in pigment cell precursors
15Materials and Methods
- Fish rearing
- spontaneously-occurring or ethylnitrosourea-induce
d mutant rose alleles - Cloning and sequencing
- reverse transcriptase PCR to clone a D. rerio
ednrb1 cDNA - compared exons from wild-type and rose mutant
backgrounds to test the correspondence of ednrb1
and rose - In situ hybridization
- digoxygenin- and fluorescein-labelled riboprobes
16Does ednrb1 correspond with the rose mutant
phenotype?
- Mapped rose to within 0.1 cM of ednrb1
- Discovered ednrb1 lesions in three rose alleles
17Does the neural crest-melanocyte lineage express
ednrb1?
- ednrb1 cells present at the pre-migration
location of neural crest cells - ednrb1 cells present along the migration pathway
- two-colour in situ hybridization
- to stain simultaneously for ednrb1 and for three
melanocyte-lineage markers - result co-expression of ednrb1 and the markers
18Does endothelin-1 signal to ednrb1 neural crest
cells?
- Expression diminishes anteriorly and increases
posteriorly through development - Detectable before expression of ednrb1
- However, endothelin-1 mutants did not exhibit
pigment pattern abnormalities - Endothelin-1 may promote but is not sufficient
for development of melanocytes
19Is ednrb1 expressed in non-melanocyte lineages?
- Xanthophores
- two colour in situ hybridization to stain
simultaneously for ednrb1 and for a xanthophore
marker - result co-expression of ednrb1 and the marker,
but only in xanthophore precursors
- Iridophores
- coincident pattern of iridophores and ednrb1
cells
20Is ednrb1 expressed during pigment pattern
metamorphosis?
- Rose phenotype may result because
- 1) early larval pigment cells fail to develop
into adult cells due to a lack of ednrb1 - 2) ednrb1 is required only during adult pigment
pattern formation (ie. after metamorphosis)
- Iridophores and melanocytes expresses ednrb1
during early metamorphosis - Expression continues through late metamorphosis
21Summary
- ednrb1 and rose correspond
- ednrb1 is expressed in the neural crest-pigment
cell lineage - ednrb1 mutations affect adult but not early
larval pigment pattern development
22Future Directions
- How does ednrb1 promote pigment cell development?
- Why does ednrb1 affect adult but not early larval
pigment pattern phenotypes - D. rerio may provide an important system for
studying amniote development - metamorphism decoupling of traits
- relevant model for post-embryonic and fetal
development?
23References
- Beauvais-Jouneau, A., Pla, P., Bernex, F.,
Dufour, S., Salamero, J., Fassler, R., Pathier,
J., Thiery, J.P., and Larue, L. (1999). A novel
model to study the dorsolateral migration of
melanoblasts. Mechanisms of Development. 89,
3-14. - Hayashi, H., Nakamura, S., and Fujii, R.. (1996).
The endothelin receptors that mediate aggregation
of pigment in fish melanophores. Comparative
Biochemistry and Physiology. 115, 143-152. - Kelsh, R.N., Brand, M., Jiang, Y., Heisenberg,
C., Lin, S., Haffter, P., Odenthal, J., Mullins,
M.C., van Edden, F.J.M., Furutani-Seiki, M.,
Granato, M., Hammerschmidt, M., Kane, D.A.,
Warga, R.M., Beuchle, D., Vogelsang, L. and
Nusslein-Volhard, C. Zebrafish pigmentation
mutations and the processes of neural crest
development. Development. 123, 369-389. - Parichy, D. M., Mellgren, E. M., Rawls, J. F.,
Lopes, S. S., Kelsh, R. N., and Johnson, S. L.
(2000). Mutational analysis of endothelin
receptor b1 (rose) during neural crest and
pigment pattern development in the Zebrafish
Danio rerio. Developmental Biology.227, 294-306. - Wolpert, L. (1998). Principles of Development.
Current Biology Ltd., New York.
24Questions?