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THE ANATOMICAL RECORD 251:200–206 (1998)
Distribution Patterns of
Neural-Crest–Derived Melanocyte
Precursor Cells in the Quail Embryo
LAURA FAAS AND ROBERTO A. ROVASIO*
Cátedra de Biologı́a Celular, Facultad de Ciencias Exactas, Fı́sicas y Naturales,
Universidad Nacional de Córdoba, 5000 Córdoba, Argentina
ABSTRACT
Background: In vertebrate embryos, migration of trunk neural crest cells
(NCC) proceeds mainly in two streams: a dorsoventral path between the
neural tube and somites, and a dorsolateral one between somites and
ectoderm. This last pathway is taken by melanocyte precursor cells (MPC)
homing the skin, while pigment cells seeding internal organs and the
peritoneal wall follow the dorsoventral pathway. Early routes taken by
subpopulations of NCC have been well documented using the quail-chick
chimaera system and monoclonal antibodies to NCC. However, very little is
known about the advanced migratory behavior of MPC, which determines
their late distribution patterns at different embryonic axial levels.
Methods: Histological sections of neck, thorax, and abdomen of 6.5 to 9
day quail embryos submitted to DOPA reaction (tyrosinase activity) were
used. In four concentric areas—dorsal and ventrally subdivided—the relative
density of MPC was determined by morphometric methods.
Results: The relative regional density of MPC from their individualization as DOPA-positive putative pigment cells until their definitive seeding in
the epidermis showed a progressively higher cell density from deeper to
peripheral zones in all three levels studied, with peaks of cell density
suggesting a centrifugal pattern occurring in at least two waves of migratory
cells.
Conclusions: The spatial distribution of the MPC varies according to both
the axial level and the developmental stage of the embryo. Furthermore, the
general pattern of centrifugal distribution observed might be attributed to a
different timing of cell differentiation closely related to their migratory
behavior. Anat. Rec. 251:200–206, 1998. r 1998 Wiley-Liss, Inc.
Key words: cell migration; cell differentiation; melanocyte dispersion;
neural crest cells
Neural crest cells (NCC) of vertebrate embryos segregate from the dorsal neural tube, undergo epithelialmesenchymal transformation, migrate along complex pathways toward several target sites, and differentiate into
many cell types, including melanocytes of the skin (Le
Douarin, 1982).
In avian embryos, migration of trunk NCC proceeds
mainly in two streams: a dorsoventral path between the
neural tube and somites, and a dorsolateral one between
somites and ectoderm. This last pathway is taken by
melanocyte precursor cells (MPC) homing the skin (Erickson et al., 1992; Erickson and Goins, 1995), while pigment
cells seeding internal organs and the peritoneal wall follow
the dorsoventral pathway (Le Douarin, 1982).
r 1998 WILEY-LISS, INC.
Early routes taken by several subpopulations of NCC
have been well documented using the quail-chick chimaera system (Le Douarin, 1973, 1986; Couly et al., 1993;
Le Douarin and Dupin, 1993) and monoclonal antibodies
against NCC (Tucker et al., 1984; Newgreen et al., 1990;
Grant sponsor: CONICET; Grant sponsor: CONICOR; Grant
sponsor: SECyT-UNC (Argentina); Grant sponsor: The Third
World Academy of Sciences.
*Correspondence to: Dr. R.A. Rovasio, Cátedra de Biologı́a
Celular, F.C.E.F.N., Universidad Nacional de Córdoba, Av.Vélez
Sársfield 299, 5000 Córdoba, Argentina.
Received 19 September 1997; Accepted 21 January 1998
PIGMENT CELLS DISTRIBUTION IN QUAIL EMBRYO
201
Fig. 1. Transversal section of 7.5 day quail embryo at the neck (a) and
trunk (b) levels indicating the zones considered for cell counting. Boxed
areas in ventral and dorsal mesenchyme in b are enlarged in c and d,
respectively, to show DOPA-positive MPC. a: Hematoxylin-eosin, 328. b:
DOPA-reaction counterstained with Light Green, 319. c, d: DOPA-positive
MPC, 3556. DC, dorsal central zone; DD, dorsal dermis; DE, dorsal
epidermis; DM, dorsal mesenchyme; VC, ventral central zone; VD, ventral
dermis; VE, ventral epidermis; VM, ventral mesenchyme. Scale bars 5 1
mm (a, b) and 50 µm (c, d).
Erickson and Goins, 1995). The most active migration of
MPC toward the skin takes place before the sixth day of
incubation, seeding the epidermis at the end of the fifth
day and the feather buds of chimaeric embryos during the
sixth day (Teillet and Le Douarin, 1970; Teillet, 1971).
Although several studies have focused on early migratory
behavior and differentiation of MPC, very little is known
about the advanced migratory behavior determining their
late distribution patterns and differentiation at definite
axial levels of the embryo. Moreover, it has been shown
that the onset of avian melanogenesis is breed-specific
(Hulley et al., 1991), suggesting that melanocyte development is under the influence of factor(s) differentially
expressed, and that temporal and spatial pattern(s) of
normal MPC development should be specifically defined.
The aim of the current study was to determine the
relative regional density of NCC-derived MPC from their
individualization as DOPA-positive putative pigment cells
until their definitive seeding in the epidermis, as well as
their pattern of distribution.
Fig. 2.
PIGMENT CELLS DISTRIBUTION IN QUAIL EMBRYO
MATERIALS AND METHODS
Fertile quail eggs (Coturnix coturnix japonica) were
incubated at 38°C in a humidified incubator until they
reached embryonic stages 22, 23, 24.5, and 25 (6.5, 7.5, 8,
and 9 day embryos, respectively) (Zacchei, 1961).
DOPA Reaction (Tyrosinase Activity)
Quail embryos fixed in phosphate-buffered saline (PBS)
formaldehyde (4%) at pH 7 (Mishima, 1960) at 4°C for 24
hr were sectioned in segments corresponding to different
axial levels (neck, thorax, and abdomen). After washing in
several changes of PBS (pH 7.3), the segments were
incubated in 0.1% L-DOPA (Sigma, ST. Louis, MO) in PBS
(pH 7) at 37°C for 12 to 18 hr according to Mishima’s
procedure (1960). Equivalent segments incubated in PBS
were used as controls. Treatment with exogenous L-DOPA
induces the synthesis of ‘DOPA-melanin‘ in melanocyte
and premelanocyte cells that can then be identified as dark
(Mishima, 1960) or electron dense (Mishima, 1964; Hirobe,
1982) cytoplasmic granules.
After DOPA reaction, the segments corresponding to
each axial level were dehydrated in graded ethanol, embedded in paraffin, sectioned (7 µm), and counterstained with
Light Green.
Cell Counting
On the basis of histological criteria, four concentric
zones were delimited in each embryo section, considering
separately the dorsal and ventral regions (Fig. 1a,b). Thus,
DOPA-positive cells were counted in eight different zones:
dorsal (DE) and ventral (VE) epidermis, dorsal (DD) and
ventral (VD) dermis, dorsal (DM) and ventral (VM) mesenchyme, and dorsal (DC) and ventral (VC) central zone. The
central zone involved mainly the visceral region. At least
30 paraffin sections of each axial level were used for cell
counting.
Area Measuring
Each zone considered for cell counting was digitalized by
means of a graphic tablet Summasketch II (Summagraphics, Seymour, Conn.), and the corresponding areas were
calculated with the SigmaScan (Jandel Scientific, San
Rafael, CA) software. Statistical comparison between data
was performed by means of the Kruskal-Wallis test (Montgomery, 1991).
RESULTS
The earliest embryonic stage considered was day 6.5,
when the first operative evidence of melanocyte commitment (DOPA-positive reaction) takes place.
Neck Level
At the 6.5 day stage, we saw MPC located only in deeper
zones of the dorsal region (Fig. 2a), whereas in older stages
the cells observed were progressively peripheral. Thus, in
Fig. 2. Distribution of MPC in the epidermis (E), dermis (D), mesenchyme (M), and central zone (C) in the dorsal region of neck, thorax, and
abdomen of 6.5 (a), 7.5 (b), 8 (c), and 9 (d) day quail embryos. Numbers
over columns indicate the mean value of cell density (number of
MPC/mm2). All differences between equivalent zones were statistically
significant (Kruskal-Wallis test, P , 0.001).
203
7.5 day quail embryos, MPC were located in the epidermis
but not in the dermis, whereas some of them were in the
mesenchyme and the central zone (Fig. 2b). In older stages
(days 8 and 9), MPC were found in every zone studied,
showing a progressively higher cell density from deeper to
peripheral zones.
The comparison between stages in the ventral region
indicated that there were MPC in deeper zones in each
stage studied (Fig. 3a–c), but none has been observed in
peripheral zones at this level except in 9 day embryos
(Fig. 3d).
Thoracic Level
In the dorsal region, we observed that MPC have already
reached the epidermis at the 22nd embryonic stage (6.5
day), although there were no cells in the dermis (Fig. 2a).
The following stage (7.5 day embryos) showed two peaks of
cell density in the epidermis and in the mesenchyme,
respectively (Fig. 2b; see Fig. 1b, boxed areas); in 8 and 9
day embryos, the peak of high density was observed only at
the epidermic zone (Fig. 2c,d). The oldest stage embryos
showed the same relative distribution of MPC as their
equivalents at the neck level, with a progressive cell
density from deeper to peripheral zones (Fig. 2d).
As in the neck level, we observed MPC only in the deeper
zones in the thoracic ventral region of 6.5 day embryos
(Fig. 3a). However, in 7.5 day embryos, the cells had
already reached the epidermis and were present in every
zone studied, although in decreasing density toward the
dermis (Fig. 3b). At the 8 day stage, the increase in MPC
density was observed in the epidermis as well as in the
dermis (Fig. 3c), and the highest cell density was seen in
the epidermic zone of 9 day embryos (Fig. 3d).
Abdominal Level
The relative distribution of MPC observed in the dorsal
region of 6.5 day embryos was similar to that described for
the thoracic level. However, cell density was higher in the
mesenchyme than in the central zone (Fig. 2a). The 7.5 and
8 day quail embryos showed a progressive distribution of
MPC toward the peripheral zones (Fig. 2b,c). By day 9, the
relative distribution of MPC was equivalent to the other
levels considered, with a stabilized progression of cell
density from the central zone to the epidermis (Fig. 2d).
The ventral abdominal region of 6.5 day embryos showed
only scarce MPC in the mesenchyme (Fig. 3a). At the
following stage (day 7.5), MPC were present in all zones
studied, with two peaks of cell density at the epidermis
and mesenchyme (Fig. 3b). As in the dorsal region, by day 8
the MPC increased progressively toward the periphery
(Fig. 3c), and by day 9 the main peak of cell density
appeared in the epidermic zone (Fig. 3d).
DISCUSSION
It is well known that definitive pigmentary pattern in
the quail involves the final cell localization and concentration of MPC in the epidermis (Le Douarin, 1982). Our data
on DOPA-positive cells observed in serial sections of four
consecutive stages of quail development indicate that the
pattern of MPC is established in the dorsal region of the
quail embryo by day 9 of development at the three levels
studied, as a result of the stabilization of relative cell
distribution. Our results suggest that to reach such a
Fig. 3.
PIGMENT CELLS DISTRIBUTION IN QUAIL EMBRYO
definitive pattern, at least two main processes must be
taken into account.
First, the distribution patterns observed may represent
a different timing of cell differentiation. It has been
demonstrated that the NCC that colonize the epidermis
and differentiate into pigment cells migrate following the
dorsolateral pathway (Teillet and Le Douarin, 1970; Teillet, 1971); these NCC also show a delay of approximately
24 hr with respect to the NCC that follow the dorsoventral
pathway (Weston, 1963; Oakley et al., 1994). In heterochronic tritiated-thymidine–labeled neural tube graft studies, it has been suggested that NCC migration is temporally ordained, being the ventral derivatives colonized
previous to the lateral ones (Weston and Butler, 1966). In
vivo studies using tracking of the vital dye Di-I confirmed
these observations (Serbedzija et al., 1989). Those MPC
that colonize deeper zones of the embryo are already
settled in their final locations, exposed to a possible
differentiative influence of local environment, while the
subectodermal NCC population is still migrating in the
dorsolateral way. Hence, the first DOPA-positive cells in
the deeper zones of the embryo would represent MPC of
internal organs; concomitantly, the absence of DOPApositive cells in peripheral areas would indicate that
differentiative metabolites have not yet developed in precursor cells. In this regard, histochemical localization of
DOPA-positive cells has been reported from day 7 of
development in dorsal (pigmented) feather buds of quail
embryos wings, whereas ventral (unpigmented) ones were
DOPA-negative (Richardson et al., 1989). However, there
is no evidence about cells with DOPA activity at earlier
stages or other embryonic regions and axial levels, although staining with the monoclonal antibody Mel-EM
specifically revealed the presence of MPC in the dorsolateral path as early as 4 days of development (Nataf et al.,
1993).
Second, the different patterns of cellular density observed may also be explained as being the result of cell
translocation. Although the initial stages of MPC migration and colonization have been well documented (Teillet
and Le Douarin, 1970; Teillet, 1971), there are few reports
about the highly invasive behavior of differentiated melanocytes (Weiss and Andres, 1952). Moreover, it has been
shown that melanogenesis does not signify the end-stage
in the migration process (Hulley et al., 1991). From our
present work, we may consider that the observed pattern
of MPC distribution could result from a centrifugal and
directional cell migration, insofar as cellular density increases progressively from deeper to peripheral zones at
the three axial levels. Related results were reported in two
breeds of pigmented chicks, suggesting a movement of the
deeper melanocytes toward the epidermis (Hulley et al.,
1991). On the other hand, the results we obtained about
the relative cell distribution in the same region (dorsal or
ventral) at successive stages of development suggest that
this centrifugal pattern is carried out in at least two waves
of migratory cells (Figs. 2, 3), thus conforming a multistep
Fig. 3. Distribution of MPC in the epidermis (E), dermis (D), mesenchyme (M), and central zone (C) in the ventral region of neck, thorax, and
abdomen of 6.5 (a), 7.5 (b), 8 (c), and 9 (d) day quail embryos. Numbers
over columns indicate the mean value of cell density (number of
MPC/mm2). All differences between equivalent zones were statistically
significant (Kruskal-Wallis test, P , 0.001).
205
invasive behavior of MPC. A multistep pattern for migration of NCC derivatives has also been described in relation
to corneal development, in which a first wave of migratory
NCC gives rise to the corneal endothelium and a second
wave develops the stromal structure (Hay and Revel,
1969). This double wave of migratory NCC appears to
depend on a favorable extracellular environment (Toole
and Trelstad, 1971; Hay, 1980). Notwithstanding, we
cannot correlate our results with extracellular matrix
variations but only with spatial and temporal constraints.
Complementary mechanisms, probably acting concomitantly with cell differentiation and translocation, might be
due to a selective cell proliferation. This could account for
the increase of MPC density in peripheral zones between
days 8 and 9 of development. It has been reported that
melanocyte differentiation is preceded by a period of active
division of epidermal melanoblasts (Teillet and Le Douarin, 1970; Teillet, 1971). Moreover, cultures of early NCC
maintain their high cell density while they migrate (Rovasio et al., 1983; Rovasio and Thiery, 1987), and recently we
showed the conspicuous proliferative behavior of this cell
population in vivo (Paglini and Rovasio, 1994a,b). Nevertheless, the decrease of cell density observed at several
levels may be a contribution of selective cell death, as
reported with respect to the early development of rhombencephalic NCC (Graham et al., 1993, 1994).
With respect to the distribution patterns in the ventral
region, our data suggest that the developmental pattern
seems delayed when compared with the dorsal region. In
this connection, it has been shown that a delay of approximately 1 day exists between the onset of NCC migration
along the dorsolateral pathway with respect to the dorsoventral pathway (Erickson et al., 1992; Erickson and
Goins, 1995). These findings and our observations of scarce
variations in the relative cell distribution at each level
studied suggest that the ventral cell distribution may
represent an ‘‘in progress’’ pattern. The delayed colonization of the ventral area by MPC may also be attributed to
the long pathway that MPC must travel from the dorsal
aspect of the neural tube along the dorsolateral pathway.
Furthermore, it has recently been suggested that nonepidermal melanoblasts migrate more slowly and/or retain
their migratory capacity for a longer time than epidermal
melanoblasts (Brand-Saberi et al., 1993). These and our
data about the presence of MPC in peripheral zones at the
ventral neck region only in the oldest stage studied, as well
as the scarce number of pigment cells in ventral regions of
all the embryonic stages studied, lend support to the
opinion of an in progress ventral pattern.
The present study supports the view that the distribution patterns of MPC depend on the axial level and
developmental stages of the avian embryo. It might be
argued that changes in local cell density are due to passive
dragging in a rapidly growing embryo. However, the
intermittent appearance of MPC progressively from depth
to the surface and the stage-related distribution in definite
histological zones do not support this hypothesis. Spatial
and temporal distribution of MPC suggests the expression
of a differentiative pattern of MPC in close relation to their
migratory behavior, although we are aware that complementary participation of proliferation and cell death cannot be ruled out.
206
FAAS AND ROVASIO
ACKNOWLEDGMENTS
The authors thank Vı́ctor H. Tomasi for technical assistance; Diana Abal for graphic designs; Natalia Laura
Battiato, José Barcelona, and Nacho Quiroga for photographic work; and Dr. Martha González-Cremer for critical reading of the manuscript.
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