Differentiation (1992) 49: 151-265 ontogeny, Neoplasis and Differentiation Therapy

0 Springer-Verlag1992

Monoclonal antibodies raised against pre-migratory neural crest reveal population heterogeneity during crest development Lindsay Heath, Arthur Wild, and Peter Thorogood * Department of Biology, Medical and Biological Sciences Building, Southampton University, Bassett Crescent East, Southampton, SO1 3TU, UK Accepted in revised form November 19, 1991

Abstract. In order to address the problem of when heterogeneity arises within premigratory and early migratory neural crest cell populations, mouse monoclonal antibodies were raised against quail premigratory neural crest. Due to the limited availability of immunogen an intrasplenic route for immunization was used. Three monoclonal antibodies (referred to as LH2D4, LH5D3 and LH6C2) were subsequently isolated which recognized subpopulations in 24 h cultures of both quail and chick mesencephalic and trunk neural crest in immunocytochemical studies. Subsequent investigations using a range of six antibodies, including LH2D4, LH5D3 and LH6C2, showed that population heterogeneity (which was not cell cycle related) could be detected as early as 15 h following mesencephalic crest explantation, a stage at which all the neural crest cells were morphologically identical. However, premigratory neural crest from the same axial level of origin was homogeneous, as judged by immunoreactivity patterns with these antibodies. Significant differences were found in the proportion of immunoreactive cells between populations of mesencephalic and trunk neural crest cultures. Double immunofluorescence studies revealed the existence of at least four separate cell populations within individual crest cultures, each identified by their unique antibody reactivity pattern, thus providing some insight into the underlying complexity of subpopulation composition within the neural crest. Immunocytochemical studies on quail embryos from stages 7-22 showed that the epitopes detected by LH2D4, LH5D3 and LH6C2 were not necessarily confined to the neural crest or to cells of crest derivation. All three epi topes displayed a spatiotemporal regulation in their expression during early avian ontogeny. Since the differential epitope expression described in this investigation was detectable as early as 15 h after premigratory neural crest explantation, took place in vi-

* To whom offprint requests should be sent at: Department of Oral Biology, Institute of Dental Surgery, Eastman Dental Hospital, Gray’s Inn Road, London WClX 8LD, UK

tro in the absence of any other cell type and changed progressively with time, we conclude that a certain degree of population heterogeneity can be generated very early in neural crest ontogeny and independently of the tissue interactions that normally ensue in vivo.

Introduction The problem of how cell diversity is generated during early embryonic development is exemplified by the differentiation of the neural crest. The cells comprising this tissue originate at the apices of the epithelial neural folds where they de-epithelialize, assume a mesenchymal morphology and subsequently undergo extensive migration throughout the embryo. This migration is arrested at defined sites where the crest cells differentiate into a wide variety of adult cell phenotypes. Neural crest-derived cells give rise to the neurones of the autonomic nervous system, the majority of the peripheral sensory neurones, neurosecretory cells of the carotid body, parathyroid glands and adrenal medulla, supportive cells of the peripheral nervous system, pigment cells, and skeletal and connective tissues of the head and face [23, 531. The generation of such a diverse range of cell phenotypes from an apparently homogeneous embryonic cell population raises the question “when and how do cell lineages become established and segregated during neural crest ontogeny ?” Much of the experimental work carried out on the neural crest emphasizes the important role played by the environment in providing cues for differentiation [28, 541. However, such local cues could act in two quite distinct ways: either by promoting the survival and proliferation of cells whose commitment is appropriate to that environment at the expense of other cells with inappropriate commitment states, or by acting on a homogeneous population of developmentally labile cells through induction of specific pathways of differentiation. The distinction between these two possibilities depends upon

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the differentiation state of the crest population during these interactions, and the degree of commitment of cells to their fully differentiated derivatives while still within the neural primordium. One of the strongest arguments in favour of an early developmental commitment of neural crest cells to disparate adult fates comes from observations dating back to the experiments of Raven [37]. Unlike cephalic crest, cells from the trunk crest seem unable to form bone or cartilage, even when transplanted into the head (see also [ l l , 34, 351). More recently, Kirby [26] has demonstrated that, unlike rhombencephalic crest, trunk and mesencephalic neural crest cells are incapable of generating ectomesenchyme competent to effect truncal septation during heart development. From these types of experiments it appears that the neural crest does not constitute a homogeneous population throughout the embryo. Apparently contradictory evidence, supporting the concept of neural crest homogeneity and developmental lability, comes from classic heterotopic grafting experiments that used either radioisotopically-labelled graft cells [36] or exploited the potential of the chick/quail chimaeric system (e.g. [29]). Grafted crest cells migrate and apparently differentiate according to a fate appropriate to that of crest cells at the site of grafting, not according to their site of origin. This developmental lability has long been assumed to show that not only do crest populations from every axial level contain a wide range of developmental potentialities but also that individual crest cells themselves are pluripotential. However, developmental potentiality or potency is a single cell (or clonal) concept and, although pluripotentiality is undoubtedly a property of the neural crest cell population at many axial levels, the developmental potentiality of individual cells (or their progeny) in such heterotopic grafts has not been established [33]. Recent investigations into neural crest differentiation have attempted to resolve this issue by examining the developmental potential of individual crest cells by clonal analysis in vitro [6] or by in vivo lineage tracing [9]. Using these two experimental strategies, again rather different conclusions have been reached. Analysis of clones derived from cells taken from the mesencephalic and rhombencephalic crest of quail embryos and cultured on feeder layers of growth-inhibited 3T3 cells, revealed striking differences between crest cells in terms of the different phenotypes characterizing the individual clones [6]. It was concluded that cephalic neural crest is not only highly heterogeneous in terms of proliferative ability and developmental potential but also that such heterogeneity is established very early in crest ontogeny. The influence of both intensive selection pressure (mean cloning efficiency was 24%), and the potential for interaction between crest cells and the feeder layer, must however be taken into account when interpreting the results of such experiments (also see Discussion). A rather different conclusion was reached from lineage tracing experiments. Bronner-Fraser and Fraser [9] used the microinjection of lysinated rhodamine dextran to label single cells on the dorsal aspect of the neural

tube. The dye is large and membrane-impermeable and, with the aid of sensitive imaging equipment, can still be detected intracellularly after 8-9 cell divisions. The majority of the resulting labelled clones apparently comprised multiple cell types, as determined by their location and morphology. However, due to progressive dilution of the tracer dye with successive mitoses, an inherent limitation of the technique is that the period over which lineally related cells can be recognized is restricted. Ultimate cell fate, as might be identified unequivocally later in development, cannot be determined and therefore, differentiative fate has to be extrapolated from location and morphology rather earlier in development. Nevertheless, the conclusion reached was that cell fate is established subsequent to or during, rather than prior to, crest cell migration [9]. A corollary of this is that the premigratory crest will largely comprise an homogeneous population of multipotential cells. A third strategy is to raise monoclonal antibodies (mAbs) against neural crest and look for heterogeneity of epitope expression within the crest population, with the underlying assumption that variation in epitope expression reflects differences in population composition. Previous attempts to raise monoclonal markers against crest cells have largely used crest-derived tissues as an immunogen because of the very limited cell numbers which comprise the premigratory crest itself and which proscribe immunization by conventional routes [2, 5, 121. The inherent drawback of this approach is that antibodies may be raised against subsequently expressed differentiation antigens and that these will consequently only recognise particular subpopulations of cells late in their development. However, using immunization strategies designed to overcome limited cell numbers, and using premigratory crest as an immunogen, we have successfully produced a number of mouse mAbs recognizing epitopes expressed by quail mesencephalic neural crest (hereafter referred to as LH2D4, LH5D3, and LH6C2). These antibodies have been used as tools to study the population composition of i) premigratory mesencephalic crest, ii) cultured mesencephalic crest and iii) cultured trunk crest. Four principal results emerge. Firstly, heterogeneity of epitope expression can be detected very early (15 h) in cultures of isolated crest but premigratory crest is virtually homogeneous with respect to expression of these epitopes. Secondly, expression of these epitopes is not necessarily mutually exclusive. Thirdly, epitope expression is not cell-cycle related. Fourthly, epitope expression is not solely confined to crest cells and immunocytochemical studies on subsequent developmental stages reveal clear spatio-temporal patterns of epitope expression. Methods Eggs of the Japanese Quail (Coturnix coturnix juponicu), from a breeding colony maintained by the School of Physiology & Biochemistry, University of Southampton, UK, were used throughout this work. The eggs were incubated in a humidified, forced draught incubator and staged according to the developmental table of Hamburger and Hamilton [22].

153 Culture techniques. Mesencephalic neural crests were removed from st. 8-9 quail embryos as previously described [49]. For culture, individual neural folds were transferred to a 30 mm petri dish containing a sterile glass coverslip and 2 ml of a-modification of Dulbecco's minimal essential medium (MEM) with 10% foetal calf serum (FCS). The cultures were incubated at 37" C in 5% C 0 2 in air until required. Trunk neural crest cultures were obtained from trypsin/pancredtin-isolated neural tubes of St. 14 quail embryos according to the technique first described by Cohen and Konigsberg [14] and cultured in x-MEM + 10% FCS as described above. Cytospinpreparations. Isolated mesencephalic neural folds were dissociated for 15 min at room temperature in 0.1 YOtrypsin/0.2% EDTA. Cells from 5-6 neural folds, resuspended in 50 p1 orMEM + 10% FCS, were used for each cytospin slide. Cytospins were made onto ethanol-cleaned glass slides at 350 g for 10 min, allowed to dry at room temperature and fixed in 4 % paraformaldehyde. To control for the possible effects of enzymatic dissociation removing membrane antigens, crest fragments were also physically dissociated; this gave identical results (ectodermal contamination is negligible in this type of dissection - see later). Preparation of tissue sections. Embryos ranging from st. 7-22 were briefly washed in phosphate-buffered saline (PBS) before being fixed in 4% paraformaldehyde at 4" C for 4-12 h, depending upon the size of the specimen. All specimens were equilibrated in 20% buffered sucrose for 5 h at 4" C, mounted in Ames OCT compound (Miles Labs Inc., USA) and 'quick-frozen'. Transverse 6-8 pm sections were cut from freshly-prepared material on a Reichert Jung Cryocut E cryostat, mounted on gelatin-chromalum-coated slides and stored at -70" C for a maximum of 4 weeks before use. Slides were allowed to reach room temperature and washed in PBS before immunostaining. Monoclonal antibody production. For each immunization approximately thirty mesencephalic neural crest were dissected from st. 8-9 embryos, pooled in serum-free a-MEM and cut into small fragments using tungsten needles (total cell number was estimated to be 3 x lo4). Female Balb C mice were anaesthetized by intraperitoneal injections of 0.5 mg Diazapam/100 g body weight (Roche Products Ltd., UK), followed by an intramuscular injection of 0.1 m1/30 g body weight of a 1:10 solution of Hypnorm in normal saline (Janssen Biotech, Belgium), and immunized intrasplenically using a method based on that of Spitz et al. [43]. Fur on the left side of the abdomen was shaved, and an incision, approximately 1 cm long, made through the skin and body wall just below the rib cage in order to expose the spleen. To minimize the injection volume of the immunogen, and to avoid unnecessary damage to the spleen, hand-drawn micropipettes were used for injection. Pipettes were drawn from capillary tubing, plugged with cotton wool and heat sterilized; silicon tubing was used to attach the pipette to a glass mouthpiece. The spleen was held stable by its tip and the micropipette, containing the immunogen, inserted along the length of the spleen. The cells in suspension were expelled as the pipette was slowly withdrawn, to ensure that the immunogen was distributed throughout the spleen. The peritoneal incision was closed with one or two sutures of silk thread (3/0 mersilk, Ethicon Ltd., UK) and the skin incision closed in the same way. Two such injections separated by an interval of four weeks were given to each mouse. Three days after the second injection the mice were sacrificed and their spleens removed. Splenocytes were fused with NSI mouse myeloma cells (at a ratio of 1 : 5, myeloma: spleen) using 50% polyethylene glycol 1500 in 75 mM Hepes buffer (Boehringer Corporation Ltd., UK) according to manufacturers instructions. Growth of hybridomas was effected in Kennett's medium [25] containing HAT and using peritoneal fluid macrophages as feeder layers. Hybridomas giving positive supernatants on immunofluorescence were weaned onto normal Kennett's medium and subjected to three limiting dilutions to ensure monoclonality.

Testing for successful immunization: Serum was obtained from a small quantity of blood taken from the tail tip of immunized mice, diluted 1:lO in PBSA and tested for immunoreactivity by indirect immunofluorescence on 24 h mesencephalic neural crest primary cultures. Negative control serum was obtained from nonimmunised mice. Screening for antibody production: Undiluted hybridoma supernatants were tested for secreted antibody by indirect immunofluorescence on 24 h mesencephalic neural crest primary cultures. Determination of immunoglobulin subclass : This was achieved using subclass-specific antibodies in a sensitive immunodiffusion assay (Serotec Ltd., UK). Immunocytochemistry. In addition to the mAbs described in the previous section, three others were also used. These comprised : HNK-1, a mouse monoclonal IgM raised against the human T-cell line HSB-2 [I] and reactive against carbohydrate moieties on surface adhesion proteins but also generally adopted as a marker of avian neural crest [50] ; Anti-PGP9.5, an affinity-purified rabbit polyclonal antibody raised against a soluble cytoplasmic protein isolated from human brain extract and found in neurones, nerve fibres and some neuroendocrine tissues from a wide variety of species [48, 581; PAI, a mouse monoclonal IgM reactive against a plasmalemmal lipoprotein of 195 kD expressed by a number of avian embryonic epithelia (Tucket, Hodivala, Wild, Thorogood, manuscript in preparation). Indirect immunofluorescence: LH2D4, LH5D3, LH6C2, HN K-3 and PA-1 were all used as undiluted culture supernatant; AntiPGP9.5 polyclonal rabbit antiserum was used at a 1 : 100 dilution. The secondary antibodies used were either a 1 :20 dilution of fluorescein isothiocyanate (F1TC)-conjugated sheep anti-mouse, or a 1:20 dilution o f FITC-conjugated donkey anti-rabbit immunoglobulin (both from Amersham International plc, UK). All crest cultures and cytospin preparations were fixed in fresh 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and washed in three changes o f PBS for a total of 15 min before immunostaining. Each culture/cytospin/section was treated with 25-30 p1 antibody solution and incubated in a humidified container for 3CL 60 min at room temperature, followed by washing for 15 min in three changes of PBS. A 20 pl aliquot of FITC-conjugated secondary antibody was applied and the slides/coverslips incubated and washed in the same way. The preparations were mounted in Citifluor (Agar Aids Ltd., UK) and examined by epifluorescence and phase contrast microscopy. Indirect double immunofluorescence : The following (fluorochrome-conjugated) class- and species-specific secondary antibodies were used to differentiate between primary antibodies bound to epitopes in the same or separate cells : 1. 1:20 dilution of FITC-conjugated sheep anti-mouse immunoglobulin (Amersham International plc, UK). 2. 1 :40 dilution of tetramethyl rhodamine (TR1TC)-conjugated goat anti-rabbit immunoglobulin (Nordic Immunological Labs., UK). 3. 1 :40 dilution of FITC-conjugated rabbit anti-mouse IgM (specific against the Fc portion of the molecule) (Nordic Immunological Labs., UK). 4.1 :20 dilution of TRITC-conjugated goat anti-mouse IgG (Southern Biotechnology Associates Tnc., UK). Before use in combination, these secondary antibodies were mixed in a ratio dependent on the optimal working concentrations determined previously for each antibody. These antibody cocktails were incubated overnight, in the dark, at 4" C and centrifuged at 8000 g to remove any antibody complexes formed as a result of crossreactivity between the conjugated antibodies. Primary antibodies were applied sequentially for the indirect immunofluorescence technique. Cultures were examined using filter systems giving either 520 nm and 580 nm wavelengths to detect fluorescence from FITC and TRITC respectively. Control tests consisted of using either PBS+ FCS as a substitute for both primary antibodies or as a substitute for the secondary antibody cocktail, or using one of the primary antibodies in combination with the cocktail of second-

154 ary antibodies. In all cases, levels of background staining at 520 nni and/or 580 nm were found to be negligible. Quuntitutive methods. Counts of positively and negatively stained crest cells in cytospin preparations were made on randomly chosen fields viewed with a X40 phase contrast objective. At least 1000 cells from five slides were counted under epifluorescence for each primary antibody used. In order to assess the extent of any ectoderma1 contamination in cytospin preparations, the mouse mAb CAM-5.2 was used; CAM-5.2 recognizes the lower molecular weight cytokeratins [30]. Since embryonic quail ectodenn contains such cytokeratins and neural crest cells do not [18], CAM-5.2 can be used as a marker for contaminating ectoderm. Such contamination was usually found to be either absent or negligible; any cultures with significant ectodermal contamination were rejected. Proportions of positively and negatively stained cells in the monolayered outgrowths from mesencephalic and trunk crest cultures were assessed by cell counts of randomly sampled fields using an ocular counting graticule and X40 objectives [lo, 151. In order to confirm that the cells in the positively and negatively staining populations were not significantly different in size (which would invalidate the sampling method) cell sizes were measured using a Kontron IBAS TI image analysis programme to measure the area of individual cells ; no significant differences in cell area were detectable. Assessment of the rate of' cell division in neural crest cultures. Cell division within the neural crest is well documented both in vivo and in vitro [31, 521 and it was possible that the antibodies raised in this study were detecting molecules associated with different phases of the cell cycle and not necessarily heritable differences between subpopulations of crest cells. This possibility can be assessed by looking for correlation between mitosis and epitope expression. In order to estimate the number of cells in S-phase at any one time, neural crest cultures were incubated with c(-MEM FCS supplemented with 10 pM-brorno-2'-deoxyuridine (BrdU) for 30 min before fixing in 95% ethanol and staining with the antiBrdU antibody, BU-1 [20]. 30 min pulse-labelling experiments were carried out at intervals from 12 h to 72 h. At least 400 cells taken from four explants were counted for each time interval using the random sampling technique described above and the proportion of immunopositive cells calculated. Since staining with BU-1 required ethanol fixation, which denatures the epitopes detected by LH2D4, LH5D3 and LH6C2, double immunofluorescence with any one of these and BU-1 was not possible. Therefore, in order to compare mitosis data and epitope expression, a longitudinal profile of LH2D4, LH5D3 and LH6C2 staining over the same time period was obtained by fixing cultures at 15, 24, 36, 48 and 72 h after explantation, counting immunopositive positive cells using identical sampling methods to those described above and carrying out regression analysis on the two sets of data.

+

Photomicrography. Photographic records were made using an Olympus BH2 microscope, in phase contrast and epifluorescent modes, and fitted with a PMA-2 automatic camera unit. Kodak Tri-X film was used for black and white photography, with standard exposures of 1.25 min for fluorescence photography and automatic exposure for the corresponding phase contrast image. Film was developed in Kodak HCllO at a dilution of 1 :31 in tap water for 7.5 min at 25" C and printed on Ilford Multigrade paper.

Results Immunization and screening

The efficiency of the immunization procedure was tested by examining serum from immunized and non-immunized mice for the presence of antibodies directed against

quail neural crest cells. In an indirect immunofluorescence assay, serum from non-immunized mice showed no reactivity on 24 h primary explant cultures of mesencephalic crest whereas serum obtained five days after the first intrasplenic injection of crest cells gave bright staining on all the cells present in such cultures. From two fusions, a total of 350 culture supernatants were screened on primary mesencephalic crest cultures and three hybridoma lines were selected, taken through three limiting dilutions to ensure monoclonality and then bulked up. The antibodies secreted by these three lines are referred to as LH2D4, LH5D3 and LH6C2. Immunoglobulin subclass determination showed them to be IgM, IgM and IgG2b respectively. All three showed characteristic patterns of immunoreactivity on cultured neural crest cells. Although quail tissue is used almost exclusively in this work, immunoreactivity on equivalent chick tissue was tested and found to be indistinguishable in terms of cellular staining patterns. Patterns of immunoreactivity

From their immunofluorescent staining patterns, LH2D4 and LH5D3 appeared to recognize cytoplasmic epitopes whereas LH6C2 recognized a cell surface epitope (as judged by immunostaining of permeabilized cultured cells, of non-permeabilized cultured cells where the pre-treatment with Triton X-100 was omitted, and by the location of immunostaining in sectioned tissue). A significant feature is that each of the three recognized only a subpopulation of cells within neural crest cultures. Positive and negative cells were interspersed throughout the cultures and were morphologically indistinguishable from one another (see Fig. 1). For each antibody used, including PA1, HNK-1 and anti-PGP9.5, specific patterns of immunoreactivity were identical at the level of the individual cell (not illustrated) in cultures of both mesencephalic and trunk crest. Quantitative assessments of the sizes of positively and negatively-staining populations within premigratory mesencephalic, cultured mesencephalic and cultured trunk crest, is shown in histogram form for all six antibodies in Fig. 2. Examination of the cytospin data shows that almost 100% of the cells of the premigratory mesencephalic neural crest of st. 9 embryos were recognized by LH2D4 and LH5D3, whereas LH6C2, PA1 and antiPGP9.5 revealed a total absence of staining (see Fig. 3). A small proportion of cells (4%) gave a positive result with HNK-1. Student's t-tests revealed that the proportion of cells giving a positive reaction in premigratory mesencephalic crest is significantly different to that in both mesencephalic and trunk crest cultures at 24 h for all epitopes. The proportion of positive cells in trunk cultures was consistently and significantly smaller than in the equivalent mesencephalic crest cultures, with the exception of HNK-1 staining. Statistical analysis confirmed that there was no significant difference (at the 0.05 probability level) between LH2D4 and LH5D3 in incidence of staining across all three types of preparation used. More detailed information on the changes in posi-

155

Fig. 1. Phase contrast (A, C and E) and immunofluorescence (B, D and F) micrographs of cells in 24 h mesencephalic crest cultures stained with LH2D4 (A and B), LH5D3 (C and D) and LH6C2 (E and F; focussed on the upper surface of the cells). The epitopes of both LH2D4 and LH5D3 are localized in discrete patches within

the cytoplasm while the LH6C2 epitope has a punctate distribution on the cell surface (arrowheads in F). Note that in all three cases positively staining cells are morphologically indistinguishable from negatively staining cells (see for example, arrows in A, C, and E). Bur equals 50 pm

tive population size was obtained by examining mesencephalic crest cultures at 15, 24, 36, 48 and 72 h after explantation. Over this period the proportion of positive cells fell significantly: LH2D4 from 80% to 26%, LH5D3 from 82% to 26%, LH6C2 from 14% to 6% and HNK-1 from 64% to 18% (Fig. 4).

BrdU) at any one sampling time dropped from 14.2% (s.d. 1.6%) at 12 h to 1.42% (s.d. 0.4%) at 72 h. These results are consistent with a rapid proliferation rate immediately after explantation followed by either a general lengthening of the cell cycle throughout the culture, or with some cells withdrawing from the mitotic pool while the remaining cells continue to divide at a constant rate [21, 311. The data obtained in this study does not allow us to distinguish between these two alternatives. Comparison of the lines of regression for the proportions of cells labelled with LH2D4, LH5D3 and LH6C2

Epitope expression in relation to the cell c-vcle

Pulse-labelling of neural crest cultures with BrdU shows that the proportion of cells in S-phase (i.e. incorporating

156

Percentage cells poslllve

Percentage cells posltlve

fl

Exclusivity of epitope expression

LH5D3:

LH2D4: 0.8

0.8

100

LOO

a0

au

60

60

40

40

20

............. ............. ............. .............

20

............. .............

n

0 c/spln

24hr mesenc

24hr trunk

c/spln

24hr mesenc

LH6C2:

24hr trunk

pA1: ~~~

Percentage cell posltlve

Percentage cells posltlve 1-l1 0f

80

IL

Ii

80

60 40 -

6.4

200

c/sDln

24hr mesenc

24hr trunk

c/sDln

24hr trunk

HNK 1:

anti-PGP9.5: Percentage cells posltlve

Percentage cells posltlve

60

8oiI

40

40

20

20

80

24hr mesenc

60

n

The application of double immunofluorescence to examine mesencephalic crest explants (Fig. 5 ) revealed a greater degree of heterogeneity within the cultures than was apparent following examination with any single antibody. When using any two primary antibodies the maximum number of subpopulations which can be identified within an explant is increased to four; one staining with each of the two primary antibodies, one staining with both primaries and one staining with neither. In this study, for all antibody combinations examined, the majority of cells were recognized by either one or other of the antibodies alone. However, as can be seen in Fig. 6, for every combination of antibodies used there were a few cells in each explant which showed reactivity with both antibodies. In every case cells were also seen which were negative for both antibodies used. Despite the small size of the LH6C2-positive population, some cells were identified which were exclusively LH6C2-positive when LH6C2 was used in combination with LH2D4, HNK-1 or anti-PGP9.5. However, when explants were stained with LH6C2 and LHSD3 simultaneously, there were no cells which were positive with LH6C2 without also being recognized by LH5D3. A similar situation was seen in explants stained with antiPGP9.5 and LH2D4 where few cells could be identified which were positive for LH2D4 only. The proportion of cells in each culture which co-expressed the epitopes recognized by LH2D4 and anti-PGP9.5, and those coexpressing the HNK-1 and anti-PGP9.5 epitopes, was high relative to the number which shared the LH5D3 and anti-PGP9.5 epitopes. Although there is no evidence from these results that the antibodies used here necessarily define specific cell lineages, the double immunofluorescent technique gives some insight into the likely complexity of differentiation pathways within the neural crest.

n

c/spln

24hr mesenc

24hr trunk

c/spln

24hr mesenc

24hr trunk

Fig. 2. Histograms representing the proportion of antibody-positive cells in cytospin preparations of premigratory mesencephalic neural crest (‘cispin’), 24 h cultures of mesencephalic neural crest (‘24 hr mesenc’) and of trunk neural crest (‘24 hr trunk’), following indirect immunofluorescence staining with various antibodies. (standard errors indicated above each column)

over time in culture, with the line of regression for the degree of BrdU incorporation over the same period (as judged by BU-1 staining), showed that the two groups of data are significantly different. The rate of decline in the number of cells recognized by each of the antibodies is not statistically related to the decline in the number of cells synthesizing DNA. Therefore, there is (apparently) no relationship between epitope expression and the cell cycle for HNK-1 or any of the three antibodies raised against neural crest.

Temporospatial patterns of LH2D4, LH5D3 and LH6C2 immunoreactivity during embryogenesis

The results of indirect immunofluorescence using LH2D4, LH5D3 and LH6C2 on sectioned embryonic material reveal variations in both the degree of developmental regulation and tissue specificity of their respective antigens. In general, LH2D4 and LH5D3 had similar widespread patterns of tissue reactivity on embryos from st. 7-22. This reactivity was not confined to neural crest (Figs. 7a, b) or to crest-derived cells over this period. At st. 7, LH2D4 displayed a diffuse cytoplasmic staining pattern in all cells of the developing embryo, with a variation in staining intensity amongst mesenchyme cells. As development proceeded the range in staining intensity throughout the embryo increased so that by st. 12 mesenchyme in the head was largely negative and differentiating tissues such as the neural tube, somites, gut, otic placodes, optic vesicles and heart were

157

Fig. 3. Phase contrast (A and C) and immunofluorescence (Cand D) micrographs of cells from cytospin preparations of premigratory, mesencephalic neural crest cell suspensions. A and B stained with LH2D4; almost all cells (98.7*0.8%) in this type of preparation were immunopositive. C and D stained with LH6C2; none

100

Percentaue Cells Positive

80

60

40

20

0 12

18 24 30 36 42 48 5 4 60 66 72

Hours After Explant Fig. 4. Diagram illustrating the relationship between the expression of various antigens by neural crest cells and the number of crest cells in S-phase over time in culture. For any one time point the number of cells in S-phase was calculated following a 30 min pulse label with 5-bromo-2'-deoxyuridine (BrdU) and immunofluorescence staining with the antiBrdU-antibody (BU-I). Antibody staining was carried out on cultures sampled at various intervals after explantation (time points shown in Figure). The relationship between expression of LH2D4, LH5D3, LH6C2 and HNK-1 and the cell cycle was examined by comparing the lines of regression for the data which appears in this diagram (see text). ( 0 = BU-1; = LH2D4; A = LH5D3 ; o = LH6C2; 4 = HNK-1)

brightly fluorescent. At st. 18 a shift in the pattern of staining was apparent which became more pronounced until at least st. 22 (the latest stage examined). The staining intensity of differentiating tissues in the wall of the gut, CNS, heart, eye, ear, mesenchyme and notochord

of the cells in this type of preparation were immunopositive for LH6C2. Due to the spherical morphology adopted by these cells in suspension immediately prior to preparation of the cytospins, the characteristic reactivity patterns of the antibodies (see Fig. 1 j are lost. Bur equals 50 Fm

decreased gradually so that by the later stages the amnion and apical surface of the ectoderm were the only tissues expressing the epitope recognized by LH2D4 (Fig. 7e, 0. The intensity of LH5D3 immunoreactivity was stronger than that of LH2D4. LH5D3 gave bright cytoplasmic staining in all cells of st. 7 embryos although the most intense reactivity was seen at the apical surfaces of the ectoderm and the neuroepithelium. Premigratory and migratory neural crest cells, mesenchyme, notochord, neural tube, somites, pharynx, lens, heart optic vesicles and otic placodes all stained with LH5D3 at various stages, although the intensity of staining was less than that on the apical surfaces of the ectoderm, pharynx and amnion. From st. 18-22, the expression of the LH5D3 epitope became restricted to the amnion and the apical surface of much of the ectoderm, although it did not appear to be present on the surface of the invaginating olfactory pit. The distribution of the LH6C2 epitope shows a welldefined tissue specificity, although it appears not to be restricted to tissues of neural crest origin. The developing CNS at 26 h of incubation was the first structure to be recognized by LH6C2 ; the initial widespread reactivity of LH6C2 in the walls of the neural tube subsequently became restricted to the floor plate (Fig. 8a, b). The LH6C2 epitope could be detected as a stippled pattern on almost all the cells in the wall of the developing pharynx from st. 9 and persisted until at least st. 22. A similar stippled pattern of the LH6C2 epitope was seen on the cells of the epimyocardium (Fig. 8c, d). The area of staining in the epimyocardium was clearly delineated by the boundary of the heart wall with the splanchnic mesenchyme; this activity persisted until st. 22. From the time of their formation (st. 14) until at least st. 22, the epithelium of the branchial arches and clefts

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Fig. 5. Immunofluorescence micrographs of neural crest cells within the confluent monolayer of 24 h mesencephalic culture following double indirect immunofluorescence staining with LH6C2 + TRITC and LH2D4SFITC. A and B illustrate the same field, with FITC and TRITC fluorescence respectively; note that the same group of cells are immunopositive for LH2D4 and LH6C2 epitopes amongst a field of negative cells. C shows a confluent field in which three populations of cells can be identified: those positive for the LH2D4 epitope, those positive for LH6C2 ( a

single cell identified by the large arrowhead) and those negative for both epitopes (the FITC green fluorescence differs from that in A due to the double exposure used to incorporate the TRITC fluorescence). In C, focus is on the punctate staining of the cell surface epitope recognized by LH6C2, and consequently the immunopositive staining of the other cells for the cytoplasmic epitope recognized by LH2D4, is at a different focal depth. Bars equal 25 pm

LH6C2

LH2D4

a

wards and in the nodose ganglion from st. 18 onwards. At st. 20 the presence of the LH6C2 epitope was exI pressed as a fine punctate staining pattern at the border between the neural and pigmented retinas and within the neural retina throughout the eye. By St. 22, the latest stage examined, the epitope had become localized to the region of the retina closest to the lens (these latter find0 0 ings are not illustrated). anti-PGP9.5 LH2D4

""0"

LH6C2

LH5D3

159

*

anti-PGP9.5 LH5D3

Discussion

Epitope expression in the mesencephalic neural crest

LH6C2

HNK-1

anti-PGP9.5 HNK-1

m LH6C2 anti-PGP9.5

I

I

2

0 0

4

00

Fig. 6 . Venn diagrams illustrating the approximate relative sizes of the subpopulations observed in 24 h mesencephalic neural crest cultures following double immunofluorescence using various antibody combinations. The data represented in this figure were obtained from a relatively small number of cultures (8 for each antibody combination) which were not subjected to the extensive random sampling techniques used to obtain the data represented in Fig. 2. Consequently there is an apparent increase in the number of cells labelled with LH6C2 within double-labelled cultures. This is attributable to a necessary bias in sampling methods towards fields of view containing LH6C2-positive cells (due to their normally very low incidence of 7.27 f2.3%). The non-random sampling methods do not affect the general conclusion, which is that double immunofluorescence using combinations of these antibodies reveals a greater diversity within early crest cultures than is apparent when examination is restricted to the use of a single antibody

were strongly immunoreactive with LH6C2 (Fig. 8e, 0. The distribution of the epitope was similar to that seen in the developing epimyocardium in that there was a distinct boundary, in this case with negative epithelium. The intensity of fluorescence was distinctly brighter in the epithelium lining the branchial pouches than in the epithelium covering the arches. Faint signs of LH6C2 staining were seen at the border of the dermamyotome and sclerotome from st. 14 on-

The objective of this study was to analyse the population composition of the avian mesencephalic neural crest at stages before, and equivalent to, migration. Differences in epitope expression were assumed to reflect different subpopulations (i.e. population heterogeneity), with the implication that this in turn reflected differences in developmental fate. Our results clearly support the view that cellular heterogeneity is relatively widespread during the early stages of neural crest ontogeny [2, 3, 4, 6, 13, 42, 551. However, in contrast to previous reports we provide evidence that, at least in terms of the reactivity patterns of the antibodies used here, the premigratory neural crest is an homogeneous population of cells, in so far as there is virtual uniformity throughout the population with regard to presence or absence of any particular epitope in question. Examination of cytospin preparations of premigratory neural crest with LH2D4 and LH5D3 demonstrated that virtually all cells expressed these cytoplasmic epitopes, with 98.7% and 99.1 YOincidence of positive staining respectively. In contrast, the cell surface epitopes recognized by LH6C2 and PA1 were totally absent from the premigratory population. The epitope recognized by the fifth mAb employed, HNK-1, was expressed on less than 4% of the cells in the premigratory crest. Given the known recognition by HNK-I of an epitope shared between a number of adhesive molecules, we have interpreted this to reflect the small proportion of the neural crest population which have already undergone those cell surface changes associated with de-epithelialization and migration. Similar analysis of primary cultures of neural crest revealed that as early as 15 h after explantation the crest had begun to diversify into distinct subpopulations. Thus, at 24 h we see a down-regulation of the LH2D4 and LH5D3 epitopes (66.7% and 68.3% incidence of cells positive) and an up-regulation of the LH6C2, PAI, and HNK-1 epitopes (to 7.27%, 54.6%, and 54.3% incidence of immunopositive cells respectively). These observations reconcile the contradictory conclusions reached in earlier work using different experimental strategies. Bronner-Fraser and Fraser's [9] description of the crest as a largely homogeneous, multipotential population was based on the introduction of lineage markers into presumptive trunk neural crest at a stage before crest cells had emigrated from the neural tube. Identical conclusions have been reached from la-

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Fig. 7. Phase contrast (A and C) and imniunofluorescence (B and D) micrographs of a transverse section through the mesencephalon of a st. 9 quail embryo staining with LH2D4. Note the intensity of the staining in the ectoderm, walls o f the neural tube, notochord and premigratory neural crest. The low power micrographs A and B clearly illustrate the heterogeneity of staining between cells amongst the cells of the mesenchyme. C and D are high power micrographs of A and B. Judging by their location and morpholo-

gy, a t least some of the positively staining cells ( a r r o w 4 within the mesenchyme are migrating neural crest cells. Phase contrast (E)and immunofluorescent (F) micrographs of a section through the head o f a st. 22 quail embryo showing ectoderm (e) and amnion (a)stained with LHSD3. The expression o f the LHSD3 epitope in the amnion is discontinuous and LH5D3 reactivity is confined to the apical aspect of the ectoderm. Bar equals 200 pm (A) and SO pm (C and E)

belling of cells during early migration itself [ 191. In contrast, the phenotypically heterogeneous clones described by Baroffio et al. [6], originated from mesencephalic crest at a stage (stage 10-11) when, at that axial level, cell migration was already quite advanced. Perhaps not surprisingly, more recent clonal analysis reveals that only 3.3% of the inesencephalic clones are derived from a progenitor cell common to both ectomesenchymal and neurogenic lineages [7]. Our data, using a third and dif-

ferent strategy, links these premigratory and migratory (or equivalent) stages and demonstrates, within the neural crest at one axial level, the early emergence of subsets of crest cells from an apparently homogeneous starting population. Subsequent experiments based on sub-cloning of crest cells will reveal to what extent expression of a particular epitope is a stable trait of an individual clone. During the later period ( 2 4 7 2 h post-explantation),

Fig. 8. Phasc contrast (A, C, E and C ) and immunofluorescent (B, D, F and H) micrographs of sections through quail embryos stained with LH6C2. A and B illustrate the immunopositive staining of the ventral floor plate of the rhombencephalon in a st. 22 embryo; note the clearly regional staining which does not extend dorsolaterally into the walls of the neural tube. C and D show the chamber of the developing heart in a stage 16 embryo; there is a sharp boundary of LH6C2 epitope expression as the heart wall joins the splanchnic mesoderm (arrowhead in D). E and F illustrate the developing branchial arches (ba) and pouches (hp) of a st. 16 embryo in coronal section. The intensity of LH6C2

fluorescence is distinctly brighter in the branchial pouchcs than over the arches, which may be a reflection of tighter cell packing in the clefts when compared to the arches. The distribution of the antigen is similar to that seen in the developing epimycoardium (A and B): there is a very distinct boundary (arrowhead in F) between cells which express the epitope (in this case epithelium of the branchial arches and pouches) and those which do not (adjacent embryonic cctoderm). C and H show the invaginating pharyngeal pouch of a st. 22 embryo in transverse section, the epithelium of which is stringly immunopositive. Bars equal 50 pm (A and C ) and 200 Fm (C and E)

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for each of the antibodies employed, the size of the epitope-negative populations of cells increased in relation to the epitope-positive populations. There are two possible mechanisms which may account for this. The first is that these negative cells are the progeny of epitopenegative parents, whose proliferation rate is higher than that of epitope-positive cells. The second is down-regulation of the epitope to the point where cells cease to be positive and join the epitope-negative population. Analysis of the cell cycle parameters of the two populations would be necessary to distinguish between these two possibilities. The application of double immunofluorescence techniques to examine population composition within explants of mesencephalic neural crest confirms the existence of at least four cellular subsets. It should be noted that this figure represents an absolute minimum since it is extremely unlikely that subpopulations defined by one antibody combination map exactly onto the subpopulations defined by a different antibody combination. This data therefore implies considerable complexity and overlap of subpopulations. Precisely how these subpopulations arise in relationship to each other and the extent to which cells may enter and leave a given cellular subset is yet to be determined. The fact that the LH6C2 epitope is not expressed on the premigratory crest, as judged by the cytospin data, yet equivalent cells used as an immunogen elicited an immune response to produce the LH6C2 antibody, requires comment. There are three possible explanations for this phenomenon. One is that, following their injection into the mouse spleen the viable neural crest cells continued their development to the point where the LH6C2 epitope is expressed before the cells die. The second possibility is that LH6C2 may have been produced during a naturally-induced immune response and which fortuitously cross-reacts with some migrating crest cells. The third explanation is that the LH6C2 epitope is normally masked or exists in a different configuration during early crest ontogeny ; degradative events following immunization may reveal the hidden epitope which would otherwise only be displayed as the result of normal developmental events. However, a precise understanding of the underlying mechanism is not necessary for an appreciation of LH6C2 as a tool to probe population composition. Differences in epitope expression between cephalic and trunk crest

In addition to the dynamic changes of epitope expression during cephalic neural crest ontogeny, key differences between mesencephalic and trunk crest populations were detected. Although the cellular pattern of immunocytochemical staining for any single antibody was identical in cells from the two axial levels, the study revealed quantitative differences in the incidence of epitope expression between the two populations (for example, 68.3% of mesencephalic crest cells expressed the LH5D3 epitope at 24 h in vitro as compared with only 21.3% of trunk crest cells after an equivalent time in culture).

For all the antibodies used the size of the epitope-positive populations were consistently and significantly smaller in trunk crest than in mesencephalic crest cultures. Whether this phenomenon observed in vitro exists in a strictly demarcated fashion in vivo, with a clear boundary between cephalic and trunk crest, or if anterioposterior gradients of epitope expression exist, remains to be determined. Certainly the results obtained are consistent with other work demonstrating differences between trunk and cephalic neural crest [ I l , 26, 34, 35, 371. Of the very few studies comparable to this, it is interesting to note that Barald’s quantitative data [3] describes a similar trend to the one described here in that her mAbs CG1, CG4, CG14 recognize a smaller subpopulation (of putative cholinergic precursors) in the trunk than in the cephalic crest. However, in contrast to the proposals made by Barald, there is no evidence from the data presented here that our antibodies necessarily recognize specific cell lineages. Using a mAb recognizing choline acetyl transferase (ChAT), Leblanc et al. [27] also report that cephalic and trunk crest show differential ability to give rise to cholinergic neurones; interestingly, cephalic cultures consistently contained a higher proportion of ChAT immunoreactive cells. Mechanisms generating population heterogeneity

Using epitope expression as a criterion, during the transition from premigratory to migratory (or equivalent) stages during crest ontogeny, there is a shift from a homogeneous population to a set of subpopulations. Given that this heterogeneity can emerge within 15 h or less, in cultures of isolated neural crest, then clearly subpopulations can be generated, at least initially, independent of cues emanating from the migration environment. Specification of cell fate may take place while the cells are resident in the neural folds and a candidate class of signals may be the products of the homeobox-containing genes. Recently, experimental evidence has been presented which suggests that the homeotic genes play a significant role in the patterning of vertebrate embryos (e.g. [16]). Indeed, it is now well established that the universal feature of homeobox genes analysed so far is that the axial limits of their expression are very precisely defined and governed, not according to cell type but to position (e.g. [57]). Mapping of the regions of expression of these genes reveals a pattern of overlapping domains within the neural tube and may reflect a combinatorial molecular specification for anterio-posterior position, intrinsic to the cells of the CNS (reviewed [56]). Thus, homeobox genes may well be candidates for region-specific spatial programming in premigratory neural crest [24], should the existence of such programming be verified. An alternative mechanism, but one for which no evidence exists as yet, due largely to the cellular complexity of the vertebrate embryo, is that cell fate is lineage-determined. Consequently, progression of a lineage through a programme of cell division, in which each mitosis specifies the fate of successive daughter cells, as

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seen in highly determinate systems (e.g. [45]) could generate limited subsets of crest cells in the absence of extrinsic cues and of direct interaction with non-crest populations. Limitations of HNK-I as a marker Results obtained from this study clearly demonstrate that the antibody HNK-1 does not recognize all neural crest cells and that HNK-1 negative cells form a significant proportion of both mesencephalic and trunk crest populations. Comparable data for trunk crest alone have been reported [32] in which only 42% of trunk crest cells were found to be HNK-1 -positive following two days in culture, a figure which compares favourably with the 37% positive cells which we found after one day in culture. Following the separation of HNK-1 -positive and HNK-1-negative cells by fluorescence-activated cell sorting, Maxwell et al. produced evidence for difference in developmental potential between the two populations. Thus, whereas both HNK-1 -positive and HNK-1-negative populations gave rise to melanocytes and unpigmented cells, only the HNK-I-positive progeny gave rise to catecholamine-reactive cells. Comparable conclusions regarding a lack of HNK-1 immunoreactivity in some trunk-derived cells emerges from experiments using an intercalating membrane vital dye to label crest cells in vivo [41]. A proportion of labelled trunk crest cells were found to be HNK-1 negative although the lack of correlation was not quantified. These two reports on trunk crest, together with the quantified mesencephalic and trunk crest results presented here, have profound implications for the generally accepted use of HNK-1 as a marker to identify neural crest cells in vivo (e.g. [8, 17,38,44,46]) and to eliminate neural crest cells from mixed cultures by complementmediated lysis [39]. Rather than identifying all or most neural crest cells early in their ontogeny, as generally assumed, it now appears that HNK-1 only detects approximately half of the population, in which case previous reports will have significantly underestimated numbers of neural crest cells and misinterpreted their overall distribution within the embryo. Epitope expression in tissues other than the neural crest The epitopes recognized by the three principal mAbs raised in this study against premigratory neural crest, LH2D4, LH5D3 and LH6C2, were all found to be expressed by certain other cell types. However, this does not in any sense invalidate our use of them to analyse the population composition of the neural crest; that they recognize subsets of crest cells has been sufficient for our present purposes. The fact that their expression in other tissues displays spatiotemporal patterns, indicating a developmental regulation of each epitope, or rather of the antigen bearing it, raises a number of interesting questions. LH2D4 and LH5D3 both have similar, widespread patterns of tissue reactivity on embryos from st 7-22.

This reactivity is not confined to the neural crest or even to crest-derived cells over this period. In embryos of st 18-22 h of development, immunoreactivity has become restricted specifically to the amnion and the apical surface of the ectoderm. However, the widespread, almost ubiquitous, presence of the LH2D4 and LH5D3 antigens during earlier development suggests a fundamental role for these molecules during early ontogeny. LH6C2 showed little immunoreactivity when applied to sections of early embryos but, as development proceeded, the tissue distribution of the epitope widened to include the floor of the neural plate, branchial arch epithelium, the wall of the heart and of the digestive tract. Although some of these tissues contain an important crest contribution, the spatiotemporal patterns observed clearly indicated that the LH6C2 epitope is not exclusive to neural crest cells. The developing CNS at 26 h of incubation was the first structure to be recognized by LH6C2. The initial widespread immunoreactivity within the wall of the neural tube became confined to the region overlying the notochord termed the floor plate and which consists of a specialized set of midline neuroepithelial cells determined by an earlier interaction between the notochord and the overlying neural plate. The floor plate is thought to play a role in the patterning of commissural outgrowth possibly by secretion of a chemotropic guidance factor [47, 511. Whether LH6C2 in this region reflects some aspect of the interaction between the notochord and neuroepithelial cells, is a manifestation of the differentiation of the specialized cells of the floor plate, or simply reflects the different origin of the floor plate cells from the rest of the neural tube [40] remains open to question. The premigratory neural crest is an homogeneous population with respect to the expression of the epitopes recognized by the antibodies utilized in this study. Subpopulations, definable by epitope expression, emerge progressively during early crest ontogeny and in the absence of the normal environmental cues which the crest cells might encounter during in vivo migration. An inherent constraint of this approach is that phenotypic differences revealed by differential immunoreactivity do not necessarily equate directly to differences in commitment state or developmental potential. Proof of such a relationship awaits biochemical characterization of the epitopes, a definition of the in vivo functional role of the molecules concerned and an in vitro assessment of epitope expression in relation to subsequent differentiated phenotype. Acknowledgements. We gratefully acknowledge a gift of the BU-1 antibody from Dr. N. Gonchoroff, of the anti-PGP9.5 from Professor R. Thompson, and of the HNK-1 hybridoma line from Dr. C. Stern. Lindsay Heath was supported by a postgraduate studentship from SERC.

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