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. 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