J. Anat. (1991), 175, pp. 237-249 237With 7 figures

Printed in Great Britain

Lymphoid follicle formation in the bursa of Fabricius of thechick embryo

NOBUYOSHI SHIOJIRI AND MASATOSHI TAKAHASHI

Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422,Japan

(Accepted 24 October 1990)

INTRODUCTION

The bursa of Fabricius is one of the primary lymphoid organs in birds, and providesa milieu for the development of both myeloid and lymphoid cells. It has been shown,by the use of quail-chick chimaeras, that haematopoietic precursor cells first enter thebursa of Fabricius and migrate in the mesenchyme towards the bursal epithelium toform the lymphoid follicle, responding to the chemo-attractants produced by bursalepithelial cells (Le Douarin, Houssaint, Jotereau & Belo, 1975; Houssaint, Belo & LeDouarin, 1976; Le Douarin, Jotereau, Houssaint & Belo, 1976; Le Douarin,Dieterlen-Lievre & Oliver, 1984). Protease activity in haematopoietic precursor cellsmight be involved in this migration (Valinski, Reich & Le Douarin, 1981).Granulopoiesis in the bursa of Fabricius is transient and occurs only in itsmesenchyme in embryonic life (Houssaint et al. 1976).

Haematopoietic stem cells are generated in the wall of the aorta in the 3 to 4 daysavian embryo, in the dorsal mesentery and then in dense foci at the level of branchingof the anterior and posterior cardinal veins (Dieterlen-Lievre & Martin, 1981;Dieterlen-Li&vre, 1984; Cormier, De Paz & Dieterlen-Lievre, 1986; Cormier &Dieterlen-Lievre, 1988). It is not known how they reach the bursa from their site oforigin although Le Douarin (1986) stated that haematopoietic stem cells colonise thebursa via the venous channels. It also remains to be clarified how haematopoietic stemcells migrate in the bursal mesenchyme to reach the bursal epithelium and howepithelial cells, mesenchymal cells and haematopoietic precursor cells interact with oneanother during lymphoid follicle formation. It has been demonstrated in manysystems that the extracellular matrix is very important for cell migration, especially inits function as an anchor (Nakatsuji, 1984; Newgreen & Erickson, 1986; Perris &Bronner-Fraser, 1989; Perris & Johansson, 1990) and that migrating free cells developcontractile proteins in their cytoplasm that respond to the chemo-attractants (Wallace,Westo, Packman & Lichtman, 1984; Southwick, Dabiri, Paschetto & Zigmond, 1989).Lectin probes that bind to the sugar moieties in the basal lamina and the extracellularmatrix have been reported by Watanabe, Muramatsu, Shirane & Ugai (1981) andGallagher (1986). Identification of free cells containing abundant F-actin in thecytoplasm with rhodamine-phalloidin, histochemical studies of extracellular matrixwith fluorescent lectins and morphological studies of lymphoid follicle formation withscanning electron microscopy might be helpful in the elucidation of these questions.We have analysed the development of the bursa of Fabricius in the chick embryo

with special attention to the homing of haematopoietic precursors in the bursa andsubsequent lymphoid follicle formation, by morphological and histochemicalmethods. We report here that haematopoietic stem cells might colonise the bursa of

N. SHIOJIRI AND M. TAKAHASHI

Table 1. Lectins and haptenic sugars used in the present study

AbbreviationLectins* of lectins Haptenic sugar

Concanavalin A Con A a.-Methyl-D-mannoside (0-2 M)Ricinus communis agglutinin120 RCA120 Galactose (0-2 M)Wheat germ agglutinin WGA N-acetyl-D-glucosamine (0-2 M)Peanut agglutinin PNA Lactose (01 M)Soybean agglutinin SBA N-acetyl-D-galactosamine (0-1 M)

* All lectins except WGA were used at the concentration of 50,ug/ml. The concentration of WGA was3 ,ug/ml (Gallagher, 1986).

Fabricius exogenously via special routes, and that they migrate in the mesenchymetowards the epithelium, becoming attached to the mesenchymal cells and extracellularfibres to form the lymphoid follicle. The importance of tissue interactions in lymphoidfollicle formation is discussed.

MATERIALS AND METHODS

Embryos of White Leghorn chicks (Gallus gallus domesticus) at 6, 7, 8, 9, 10, 11, 12,13, 15, 17 and 19 days of incubation were used. At least three embryos at each stagewere used for each of the following staining methods.

Histological methodsThe posterior regions of embryos taken before 11 days of incubation, and bursae

alone after 12 days, were fixed in Bouin's fluid or Zenker's solution and embedded inparaffin. Sections were stained with haematoxylin-eosin-alcian blue (HX-E-AB) orthe azan method. Sections fixed in Zenker's solution were stained by the Pappenheimmethod.

HistochemistryEmbryos and bursae for F-actin, keratin and lectin histochemistry were fixed in cold

95 % ethanol overnight and embedded in paraffin. Dewaxed sections at 5 ,tm wereincubated with rhodamine-labelled phalloidin (Molecular Probes Inc, 5 units/ml inphosphate-buffered saline (PBS)) (Murakami, Suzuki, Fujii & Yamaoka, 1989) orwith fluorescein isothiocyanate (FITC)-labelled lectin probes listed, with theirabbreviations, in Table 1 (Vector Lab) at room temperature for 30 minutes in thedark. After thorough washing in PBS, sections were mounted in PBS-glycerol (1:1,v/v) and observed with an Olympus fluorescent microscope (model BHS-RF). Controlslides for phalloidin staining were incubated in PBS in place of fluorescent phalloidin.Control slides for lectin staining were incubated with lectins and their haptenic sugarsshown in Table 1. Sections for keratin immunohistochemistry were incubated withrabbit polyclonal anti-keratin, wide spectrum at 1/200 dilution (Daco Corp,Carpinteria) at room temperature for one hour. FITC- or horseradish peroxidase-labelled goat anti-rabbit IgG antibodies (Miles Lab) were used as the secondaryantibody. Immunoreaction was observed with a fluorescent microscope or visualisedby the diaminobenzidine reaction. Control slides for keratin immunostaining wereincubated with normal rabbit serum (1/200 dilution) as the primary antibody.

Scanning electron microscopy (SEM)Tissues were fixed in 2% glutaraldehyde buffered with 0-1 M sodium cacodylate

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Morphogenesis of bursal lymphoid follicles

(pH 7 4) on ice for two hours, washed with cacodylate buffer and then fixed in 1 %osmium tetroxide buffered with 0-1 M sodium cacodylate. After dehydration in analcohol series, tissues were dried by the critical-point method, coated with gold andexamined with a JEOL scanning electron microscope (JSM 35CF1 lA).

RESULTS

Seeding of the bursa by haematopoietic precursor cellsAt 7 days of incubation, the bursa of Fabricius was a sac consisting of the

endodermal epithelium and the alcian blue-positive mesenchyme as the major tissue(Fig. 1 A). At 7 and 8 days, a few basophilic cells appeared in the distal part of thebursa. Most of them were located in the mesenchyme (Fig. 1 B). Some basophilic cellsadhered to the outer and inner walls of the blood vessels (Fig. 1 C) and of vessels notcontaining erythrocytes, possibly lymphatic vessels. These blood vessels and lymphaticvessels were located dorsal to the distal bursa. There were no basophilic cells in themesenchyme or in the blood vessels of the central and proximal bursa at early stagesof development. In addition, in the loose connective tissue near the distal bursa,basophilic cells were present. They were positively stained with rhodamine-phalloidin(Fig. 1 D). With further development, basophilic haematopoietic stem cells containingabundant F-actin in their cytoplasm increased in the mesenchyme (Fig. 1 E).

Migration of haematopoietic precursor cells in the mesenchyme to the bursalepithelium

Haematopoietic stem cells in the mesenchyme, which exhibited various morphologyand possessed long and short cell processes, were in close association with extracellularmatrices and mesenchymal cells (Fig. 2). They had the appearance of migrating in themesenchyme towards the epithelium, anchoring to the mesenchymal cells and thealcian blue-positive fine extracellular matrices. The mesenchyme subjacent to theepithelium was condensed, while the loose mesenchyme containing the blood vesselswas located in the central part of the plicae.The bursal mesenchyme was also fluorescent for PNA and SBA staining, from 8 to

12 days and from 9 to 13 days, respectively (Fig. 3). The PNA staining pattern wasdifferent from that in the anterior and central part of the gastrointestinal tract wherethe PNA binding sites were localised mainly on the basal lamina or on the luminalside, not in the mesenchyme.

Lymphoid follicle formationLymphoid follicle formation started with the invasion of the bursal epithelium by

haematopoietic stem cells as cell groups at 10 and 11 days. Lymphoid folliclesdeveloped in those parts of the epithelium that had been invaded by thehaematopoietic stem cells. We also reconfirmed the time when the haematopoieticstem cells invaded the bursal epithelium by staining the embryonic bursa with anti-keratin antiserum and fluorescent lectins. Anti-keratin antiserum reacted only tobursal epithelial cells, not to the lymphoid stem cells. The patchy pattern of keratin-negative staining in the bursal epithelium started to be visible at 10 to 11 days ofincubation (Fig. 4). The negative staining was first observed in the distal part of thebursa, and then in the central and proximal bursa during development.The basal lamina of the bursal epithelium was stained with all fluorescent lectins

examined and possessed no breaks before 9-10 days. At 10 and 11 days, the basallaminar staining with the lectins became weak or negative in some regions of the distal

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N. SHIOJIRI AND M. TAKAHASHI

a A > _Fig. 1 (A-E). (A) The bursa of Fabricius at 7 days of incubation; it consists of the endodermalepithelium and mesenchyme. HX-E-AB staining. x 250. (B) Basophilic haematopoietic cells(arrowheads) in the mesenchyme of the bursa of Fabricius at 9 days. Pappenheim staining. x 500. (C)Basophilic haematopoietic cells in the blood vessel near the 9 days distal bursa (arrow). Pappenheimstaining. x 500. (D) F-actin-positive free cells in the mesenchyme of the distal part of the 8 daysbursa. Rhodamine-phalloidin staining. x 500. (E) F-actin-positive free cells in the mesenchyme inthe 12 days bursa. Rhodamine-phalloidin staining. x 250.

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241Morphogenesis of bursal lymphoid folliclesW.f^ A.. m *_r

Fig. 2. A haematopoietic stem cell Qarge arrow) which was in close association with extracellularfibres (small arrows) in the mesenchyme at 12 days. Scanning electron micrograph. x 5300.Bar, I /sm.

Fig. 3 (A-B). PNA binding sites in the embryonic bursa. (A) The continuous basal laminar stainingof a 10 days bursa with PNA. Bursal mesenchyme was also strongly stained. x 250. (B) The basallaminar staining of an 11 days bursa with PNA. The basal lamina was diffusely or weakly stained inthe region where the invasion of haematopoietic precursor cells takes place (arrows). x 250.

bursa due to the invasion of the epithelium by lymphoid precursor cells (Fig. 3).Haematopoietic precursor cells often appeared to push up the epithelium into thelumen (Figs. 4A, 5C). The basal lamina in the interfollicular region was positivelystained.

Epithelial cells did not extend long cell processes towards the invading haemato-poietic precursor cells (Fig. 5 A). Even after surrounding the haematopoietic precursorcells in epithelial buds, epithelial cells did not extend such cell processes (Fig. 5 B). Atlymphoid follicle formation, the overall staining intensity of F-actin in the bursalepithelium became strong and epithelial cells surrounding basophilic lymphoid

N. SHIOJIRI AND M. TAKAHASHI

Fig. 4(A-B). (A) Basophihc haematopoietic stem cells invading the bursal epithelium as cell groupsat 12 days. Pappenheim staining. x 500. (B) Cytokeratin staining in an I I days bursa with polyclonalantibodies against calf keratin. Haematopoietic precursor cells in the bursal epithelium were negative(arrow). Immunoperoxidase method, counterstained with haematoxylin. x 500.

precursors also contained more F-actin than those in the interfollicular region (Fig.5 C). The reactivity of haematopoietic precursors in the epithelium to the rhodamine-phalloidin and lectins decreased.As a result of the invasion of haematopoietic precursors into the epithelium and

their inclusion in the epithelium, epithelial buds developed and became invaginatedinto the mesenchyme. There was often a space between the epithelial buds andsurrounding mesenchyme (Fig. 5 D). Epithelial cells and free cells often projected intothe lumen around 12 to 15 days.With progress of development, epithelial buds developed into large lymphoid

follicles and increased in number by further invasion of haematopoietic precursorsand their proliferation in the epithelium. Presumptive reticular cells were recognisedin the central part of a 12 days large epithelial bud with the aid of keratin staining andthey became intermingled with lymphoid cells in the immature lymphoid follicles (Fig.6A). The tuft epithelium, with strong rhodamine-phalloidin staining on the luminalside and with strong reactivity to the anti-keratin antiserum, differentiated adjacent tothe lumen in large follicles at 14 days. The basal laminar staining in the follicularepithelium, when stained with the lectins, was more or less continuous after 15 days,but haematopoietic precursor cells crossing the epithelial-mesenchymal interface weresometimes seen. The basal lamina in the tuft epithelial cells was not stained with thelectin probes. Scanning electron microscopy also revealed the smooth basal surface ofthe lymphoid follicles with some perforations at 15 days (Fig. 6B). At 17 days, largefollicles with many lymphoid cells became differentiated (Fig. 6C), but, even at 19days, small follicles with much fewer lymphoid cells were sometimes seen.The mesenchyme surrounding the lymphoid follicles differentiated into the

Fig. 5(A-D). (A) A haematopoietic cell (large arrow) invading the bursal epithelium (E) at 12 days.Small arrows, fibres in the mesenchyme. Scanning electron micrograph. x 7200. (B) A primordiumof the lymphoid follicle at 12 days. E, bursal epithelium. Arrow, a haematopoietic cell in theepithelium. x 2900. (C) The 12 days bursal epithelium surrounding haematopoietic precursor cells(arrow). Rhodamine-phalloidin staining. x 500. (D) The space between an epithelial bud andsurrounding mesenchyme at 12 days. Arrowhead, granulocytes in the mesenchyme. HX-E-ABstaining. x 500.

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Morphogenesis of bursal lymphoid follicles

Fig. 5.

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Fig. 6(A-D). (A) Cytokeratin staining of a 15 days bursa. Reticular cells were dispersed in themedulla of a lymphoid follicle. E, bursal epithelium; L, lymphoid follicle. Arrow indicates the tuftregion. x 500. (B) A basal view ofa 14 days lymphoid follicle. Arrowhead indicates a gap in the basallamina. Scanning electron micrograph. x 1800. (C) Lymphoid follicles in a 17 days bursa. HX-E-ABstaining. x 250. (D) Granulocytes in the bursal epithelium (arrow) at 13 days. HX-E-AB staining.x 500.

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Morphogenesis of bursal lymphoid follicles

Fig. 7(A-D). Scanning electron micrographs to show: (A) The rough surface morphology of the 11days bursal epithelium. x 800. (B) The relatively flat surface of the 12 days bursal epitheliumn,compared to the 11 days epithelium (A). Some indentations were seen. x 460. (C) Indentations in the12 days bursal epithelium. x 1500. (D) The epithelial surface of 14 days bursa. x 450.

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supporting connective tissue. The clear cortical zone of the lymphoid follicles in thebursa developed after hatching. The mesenchyme in the centre of the plicae becamevery loose.

Granulopoiesis started in the mesenchyme at 9-10 days and granulocytes exhibiteda more rounded morphology, with many short cell processes, in comparison with thehaematopoietic stem cells. The major site for granulopoiesis was in the mesenchyme,but some granulocytes were seen in the interfollicular epithelium around 13 days (Fig.6D). Granulopoiesis ceased to develop in the bursa after hatching.

Presence of the haptenic sugar in the lectin solution abolished the lectin stainingcompletely. Control slides used in the keratin immunohistochemistry and rhodamine-labelled phalloidin staining were invariably negative. Rhodamine-labelled phalloidinalso stained the apical microfilaments in the epithelium and the smooth muscle of thesmall intestine of the chick embryo. Staining pattern of F-actin in paraffin sections wassimilar to that in frozen section.

Change of the surface morphology of the bursal epithelium with developmentBefore 11 days, the bursal epithelial surface was rough (Fig. 7A). Cells with well-

developed microvilli and with poor microvilli on the luminal surface were mixed in theepithelial sheet at 11 days. At 12 and 13 days of incubation, when invasion ofhaematopoietic precursors into the epithelium culminated, the epithelial surfacebecame relatively flat (Fig. 7B). Near the presumptive follicle region, the epitheliumwas sometimes indented due to the projection of the follicular epithelium in the closelyadjoining plicae at these stages (Fig. 7C). At 14 days, the epithelial surface becamerough again and the follicular primordia could be clearly recognised in a regular latticepattern, but there were no indentations in the epithelial layer (Fig. 7D).

DISCUSSION

Colonisation of haematopoietic precursor cells in the bursa of FabriciusIn the present study, rhodamine-phalloidin-positive free cells and basophilic cells in

Pappenheim-stained preparations were first detected in the mesenchyme of the distalbursa in 7 and 8 days chick embryos, suggesting that they might correspond tohaematopoietic precursor cells and migrate into the bursa at very early stages ofdevelopment. The results are consistent with the experimental study by Le Douarinand her collaborators (Le Douarin et al. 1975; Houssaint et al. 1976; Le Douarinet al. 1976). It is well-known that the actin-based cytoskeleton and microtubulesnormally act in concert when a cell migrates (Vasiliev et al. 1970; Malawista &Chevance, 1982; Marsh & Letourneau, 1984; Bray & Hollenbeck, 1988). The presenceof abundant F-actin in the cytoplasm of haematopoietic precursor cells suggests theiractive migration to the bursa of Fabricius. We also noted that haematopoietic cellsactually migrating in the bursal mesenchyme contained much F-actin in the cytoplasm.Most haematopoietic precursor cells at early stages of development were distributed

in the mesenchyme and, sometimes, in the walls of the blood vessels and probablylymphatic vessels, which were located dorsal to the distal bursa. Basophilic cells werealso present in the loose connective tissue adjacent to the distal bursa. These resultssuggest that haematopoietic precursor cells seed the bursa via the blood vessels, orlymphatic vessels, or the connective tissue. Localisation of haematopoietic precursorcells in the distal bursa at early stages implies the presence of special mechanisms fortheir seeding in the bursa. Moore & Owen (1966) have shown experimentally thathaematopoietic stem cells could seed the bursa through the blood vessels. In contrast,

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Morphogenesis of bursal lymphoid follicles 247Martin, Beaupain & Dieterlen-Lievre (1980) and Dieterlen-Lievre & Martin (1981)suggested a different pathway of colonisation of haematopoietic stem cells into thebursa rather than by the blood stream, i.e. through lymphoid channels.

Invasion of haematopoietic precursor cells into the bursal epithelium in thetransverse plane of the plicae appeared to occur at about the same time in many placesand thus produced many lymphoid follicle primordia at constant intervals in thebursal epithelium. The haematopoietic precursor cells migrated in the mesenchyme fora long distance before invading into the epithelium. Can the migration and invasionpatterns of haematopoietic precursor cells be explained simply by chemotaxis? Othermechanisms permitting haematopoietic precursor cells to migrate extensively towardsthe epithelium might be also operative in the bursa. One possibility is the extracellularmatrix as a cell guidance agent. The extracellular matrix can serve as a substrate forthe migration of cells and may provide contact guidance for the direction of cellmovement. This has been suggested by the presence of orientated matrix structurescontaining specialised cell-matrix linkages in vivo along neural crest migratorypathways (Lofberg, Ahlfors & Fallstr6m, 1980; Brauer & Markwald, 1988; Newgreen,1989). The cytoskeletal proteins could be coupled indirectly with the extracellularmatrix proteins via the receptor for such proteins (Duband, Dufour & Thiery, 1988).Our observations of the embryonic bursa with scanning electron microscopydemonstrated that haematopoietic precursor cells, with various forms, were in closeassociation with the extracellular fibres embedded in the mesenchyme. The amount ofextracellular fibres in the bursal mesenchyme was much greater than that in theintestinal mesenchyme at the same stage. The stage of positive staining of the bursalmesenchyme (especially the extracellular matrix) with PNA and SBA coincided withthat of the colonisation and migration of haematopoietic precursor cells into thebursal epithelium. The fibres in the bursal mesenchyme might play important roles formigration of haematopoietic precursor cells.

Lymphoid follicle formation in the bursa of FabriciusIn the present study, we could determine the time when haematopoietic precursor

cells started to invade the bursal epithelium precisely by means of lectin histochemistryand keratin immunohistochemistry. The invasion ofhaematopoietic precursor cells intothe bursal epithelium occurred at 10 and 11 days of incubation. This result is similarto the report by Houssaint et al. (1976). Haematopoietic precursor cells have beenreported to possess protease activity during their migration in the mesenchymetowards the epithelium (Valinski et al. 1981). The basal lamina of epithelial cells, thatreacted to the lectins, disappeared or decreased in staining intensity in the regionwhere the haematopoietic precursor cells invaded. This suggests that the basal laminamight be digested during the invasion of haematopoietic precursor cells into theepithelium and that the cells might also have enzyme activity for degradating sugarmoieties of the basal lamina, but not of the mesenchyme, in addition to proteaseactivity. The protease activity in haematopoietic precursors might be involved also inthe degradation of the basal lamina of the bursal epithelium during the invasion.The epithelial cells which made contact with haematopoietic precursor cells did not

extend long cell processes towards the invading cells. Thus the epithelium seemed tobe passive to the invasion at first. In the next step, the bursal epithelium increased itsF-actin content in the cytoplasm, surrounded haematopoietic precursor cells and theninvaginated itself into the mesenchyme. Thus epithelial cells at this step seemed toparticipate actively in lymphoid follicle formation. A space was often seen between theepithelium and the mesenchyme in the immature lymphoid follicles, implying that the

248 N. SHIOJIRI AND M. TAKAHASHI

adhesion of the immature lymphoid follicular epithelium to the mesenchyme might beweak and suitable for further invagination of epithelial buds.

Before and after invasion of haematopoietic precursor cells into the epithelium, thesurface morphology of the epithelium was changed, as revealed by SEM. At 12 and13 days, when extensive invasion of haematopoietic precursor cells occurred, theepithelial surface became rather flat, suggesting that the epithelial layer is undertension. Moreover, the furrows which were thought to be a replica of the epithelialprojections facing towards the lumen in the adjoining plicae were generated in theepithelium near the epithelial buds. Their meaning is unclear. The phenomenon maybe related to epithelial remodelling accompanying lymphoid follicle formation and thegrowth of plicae.

Granulopoiesis in the bursaHoussaint et al. (1976) reported that granulopoiesis occurs only in the bursal

mesenchyme and that haematopoietic precursor cells, which invaded the bursalepithelium, developed into lymphocytes and similar cells remaining in the mesenchymeserved for granulopoiesis. In the present study, we have reconfirmed that the major sitefor granulopoiesis is in the bursal mesenchyme but we also found some granulocytesin the bursal epithelium during lymphoid follicle formation, especially in theinterfollicular epithelium. The haematopoietic precursor cells which generategranulocytes only might remain in the bursal mesenchyme and haematopoietic stemcells which differentiate into lymphocytes, or into both lymphocytes and granulocytes,could migrate in the mesenchyme and invade the bursal epithelium responding to thechemo-attractant from epithelial cells.

SUMMARY

The colonisation of lymphoid stem cells and the following lymphoid follicleformation in the bursa of Fabricius of the chick embryo was studied histochemicallyand morphologically. Most basophilic haematopoietic stem cells first appeared in themesenchyme of the distal bursa at 7 and 8 days. Basophilic cells were also seen in theloose connective tissue near the distal bursa, and in the vessels which were locateddorsal to the distal bursa. They began to invade the epithelium at 10 and 11 days ofincubation by digesting the basal laminar components which the fluorescent lectinprobes stained. Haematopoietic stem cells were in close association with theextracellular matrix in the mesenchyme and contained abundant F-actin. F-actin alsoincreased in the epithelial cells surrounding basophilic haematopoietic stem cellsduring lymphoid follicle formation. F-actin in migrating haematopoietic stem cellsand epithelial cells might be involved in the migration of haematopoietic cells and thehistogenesis of the lymphoid follicles. Granulopoiesis occurred mainly in the bursalmesenchyme, but some granulocytes were seen in the epithelium. The results supportthe extrinsic origin of the bursal lymphoid stem cells and their active migrationtowards the epithelium in the chick embryo. Cell-cell interactions and tissueinteractions in the lymphoid follicle formation are also discussed.

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