Contribution of microscopy to the study of proliferating blood cells in ...

Contribution of microscopy to the study of proliferating blood cells in
Ciona intestinalis immune response
M. A. Di Bella and G. De Leo
Department of Biopatologia e Biotecnologie Mediche e Forensi, University of Palermo, Via Divisi 83, 90133, Palermo,
Italy
Following an inflammatory stimulus such as the injection of erythrocytes into the tunic of Ciona intestinalis, hemocytes
respond to events occurring in the tunic within a short time, and infiltrate the inflamed tissue being massively involved in
the acute inflammatory response and capsule formation.
The proliferative response of circulating hemocytes and pharynx assume particular interest as it is significantly enhanced
during these defence reactions. Microscopy clearly may contribute to extend our understanding of the phenomenon
showing interesting mitotic figures and hematogenic nodules with proliferative characteristics.
Keywords: Ciona intestinalis; ascidians; cell proliferation; ultrastructure
1. Introduction
Ascidians, known as sea squirt, are cosmopolitan invertebrates that occupy a key phylogenetic position because they are
considered a sister group of vertebrates being classified in the phylum Chordata, subphylum Urochordata [1]. Owing to
their position they have acquired importance in immunity evolution studies, and Ciona intestinalis, a reference species
of the solitary ascidians, is a valuable model organism for the study of a variety of biological processes.
In the presence of pathogens or foreign material injected into the body wall, C. intestinalis displays acute
inflammatory responses consisting in humoral and cellular reactions performed by blood cells. Hemocytes are essential
for recognition of self and non-self, for phagocytosis, encapsulation and lysis of foreign agents, and repair of damaged
tissue. In a few hours the inflamed tissue appears densely populated with hemocytes that, coming from the blood
lacunae, insinuate themselves into the intercellular spaces and cross the epidermis contributing to the capsule formation
[2-7]. In this sense, circulating hemocytes, coming from the hemolymph, pharinx, and hematogenic sites, assume
particular interest.
In C. intestinalis several hemocyte types have been described [8], and among them, hemoblasts or stem cells are
considered able to proliferate and/or differentiate diverse hemocyte types also in other ascidian species [9].
The ultrastructural observations reported in the present study reinforce the view that after the inflammatory stimulus,
the hemoblasts in blood lacunae and hematogenic nodules may undergo an increased proliferative activity.
2. Material and Methods
C. intestinalis specimens about 10-12 cm long were collected from Palermo harbour. Animals free of encrusting marine
matter were maintained at 15-18°C in aerated sea water. Sheep erythrocytes (1 x 10 7 suspended in 0.2 ml phosphatebuffered
saline solution, pH 7.4) were injected into the tunic [3]. Four days later, the specimens showing tunic reaction
were isolated. Ciona specimens injected with 0.2 ml phosphate buffered saline solution (PBS) served as controls. Cubes
of the injected region, 1 to 3 mm long, were fixed using the following procedure.
They were placed for 1 hr at 4 °C in a solution containing 1.5% glutaraldehyde, buffered with 0.05 M sodium
cacodylate (pH 7.3 plus 1.7% sodium chloride). After brief rinsing, they were postfixed for 1hr at 4 °C with 1% osmium
tetroxide in 0.05 M sodium cacodylate at pH 7.3
All specimens were rinsed briefly and dehydrated in a graded series of ethanol solutions, cleared in propylene oxide
and embedded in Epon resin. Thin sections were stained with uranyl acetate and lead citrate and examined under a
transmission electron microscope (Philips CM 10), at 80 kV.
3. Results
Microscopy: Science, Technology, Applications and Education
______________________________________________
A. Méndez-Vilas and J. Díaz (Eds.)
After an experimental injury, the pharyngeal areas of C. intestinalis were studied in selected thin sections at EM. The
body wall of a tunicate consists of two epithelial sheets; the outer is the epidermis covered with the tunic, and the inner
is the atrial epithelium that encircles the pharinx: between these two sheets there are mesenchymal cells that move
freely.
©FORMATEX 2010 111

Microscopy: Science, Technology, Applications and Education
A. ______________________________________________
Méndez-Vilas and J. Díaz (Eds.)
Fig. 1. Electron micrograph at low magnification of vessel
endothelium and a nodular portion of it. Cells of the single
layer are flat, a free blood cell in the lumen can be observed.
Cells in the nodule are closely packed, some of them are
columnar in shape; nuclei are large and sometimes contain
two nucleoli (box). L= lumen; en= endothelium.
Magnification: 2 600x.
In the pharyngeal wall of C. intestinalis hemolymphatic lacunae and hematogenic tissue or lymph nodules occur.
Lymph nodules that lie below the atrial epithelium and along the blood channel have been observed as small ansae or
projections with a nodular appearance. The usual arrangement of the atrial epithelium and vessel endothelium is that of
a single layer of flattened cells with a regular profile on the side of the lumen on the basal lamina. In Figure 1 we
observe the cells of the nodule that are arranged in layers; they often thicken and assume a polygonal shape; cell
boundaries interdigited with each other; some laterally placed cells are columnar and present elongated cilia projecting
into the atrial cavity. These cells in the nodule contain a nucleus with one or two nucleoli, and some mitochondria; they
are rich in free ribosomes, all features that reflect an undifferentiated state.
Fig. 2. Group of cells with the feature of hemoblasts are seen adjacent to the atrial epithelium (ae). Cells border directly one on
another, giving angular outlines. Nucleus is spherical, containing nucleolus often adjacent to the nuclear membrane (Fig. 2c). The
cytoplasm essentially granular contains round or oval mitochondrial profiles. Note the atrial epithelium with a basement membrane
facing the cellular aggregates. Some epithelial cells protrude into the lumen and seem to surround the blood cells. In the peripheral
margin of the nodule some cells still show points of close membrane apposition while other cells are free in the lumen. Maturing
cells have dense granules in the cytoplasm some of which are very large (Fig. 2b). In the Fig. 2d a hemoblast cell with large nucleus
and tubular cisternae of RER in the cytoplasm. L= lumen. Magnification: (a) 3 200x; (b) 2 100x; (c) 5 250x; (d) 5 250x.
In Figure 2 patches of cells, most of which resemble hemoblasts as they do not show any specialized features, can be
observed; they occur more often than usual, and immediately adjacent to the atrial epithelium. Cells present in each
112 ©FORMATEX 2010

Microscopy: Science, Technology, Applications and Education
______________________________________________
A. Méndez-Vilas and J. Díaz (Eds.)
group show a relatively simple structure, being characterized by a spherical to oval nucleus which occupies a large
portion of the cytoplasm and may contain prominent nucleoli adjacent to the nuclear membrane. The cytoplasm is rich
in free ribosomes, and sometimes contains some elongated or tubular cisternae of RER, and mitochondria. A lot of cells
are often closely packed so that cell boundaries are difficult to distinguish as membranes of adjacent cells may be
completely adherent or with a thin intercellular space, and their shape often appears irregular (Fig. 2c). On the
peripheral margin of the clusters, the cells are less packed because the intercellular spaces become larger and more
irregular until the cells detach from the group and become free in the lacunae. In these areas maturing blood cells can be
observed. Fig. 2b show some of them lacking the nucleus and characterized by the presence of electron dense granules.
The granules may become very large and vesicles can also be seen in the cytoplasm.
Fig 3. Micrograph of free hemocytes in blood lacunae. (a) Group of hemocytes where a hemoblast-like cell is surrounded by
differentiated blood cells. The hemoblast (arrow) present a large nucleus with evident nuclear membrane and pores; cytoplasm lacks
organelles. The differentiated cells are granulocyte types, they present a unique granule that nearly occupies the whole cell except at
the edge where numerous vesicles can be seen. (b) A group of cells interconnected with two intercellular bridges. Cells are spherical
with a large round nucleus and nucleolus. Cytoplasm contains numerous polyribosomes and rare long cisternae of RER; few
mitochondria and a Golgi complex are observed. Note the smallest cell where the nuclear membrane is not clearly distinguishable.
(c) Detail of the intercellular bridges. The bridges are cylindrically shaped, and consist of a thickening of the plasma membrane. The
region of cytoplasmic continuity between cells contains ribosomes and near it RER elongated cisternae appear (arrow head). en:
endothelium. Magnification: (a) 4 400x; (b) 5 500x; (c) 18 200x.
Figure 3 shows cells characterized by ultrastructural features and nucleus/cytoplasm ratio of undifferentiated
elements found in the circulating hemolimph. These cells previously morphologically described as hemoblast or
hemoblast-like cells [8] are frequently surrounded by other differentiated blood cells (Fig3a). This finding agrees with
the role proposed for hemoblast as a source of other blood cell types.
Cells show a round contour, their surface is smooth and they measure approximately 5µm in diameter. They show a
large roundish nucleus with an essentially granular nucleolus and nuclear membrane with pores. The cytoplasm is
©FORMATEX 2010 113

Microscopy: Science, Technology, Applications and Education
A. ______________________________________________
Méndez-Vilas and J. Díaz (Eds.)
homogenously grainy, containing few roundish mitochondria and a Golgi complex. Most of the cytoplasm is filled with
numerous polyribosomes, but some RER cisternae can also be observed; occasionally RER narrow profiles run through
the cytoplasm.
Surprisingly the most prominent feature of these circulating blood cells is the presence of intercellular bridges. Three
or more cells are in fact linked by bridges, thus there is a cytoplasmic continuity among them. In Figures 3b and c the
connecting intercellular bridges are shown; they have a cylindrical shape and a diameter of 0.3 µm. A local thickening
of the membrane surrounding the bridge can be evidenced so that the rim appears more electron-dense than the rest of
the surface membrane. Near the bridges some RER cisternae occur.
4. Discussion
In the absence of vertebrate-like adaptive immunity, the defence responses of invertebrates are mainly based on
humoral factors and cellular activities. In tunicates inflammatory responses as well as inflammatory events linked to
allorecognition responses both share the involvement of hemocytes [3, 10, 11]. Hemocytes often migrate from the
hemocoel or tunic vessel into the tunic in response to injury, or other events, likely mediated by chemotactic cues [12,
13] and they are likely provided with a complex of surface receptors and effector molecules enabling them to be active
in different immune responses.
In C. intestinalis several hemocyte types have been described including stem cells [8]. Stem cells are generally
defined as self-renewing, unspecialized, and capable of multilineage differentiation [14-17]. Ascidian hemocytes that
have been reported to function in stem cells or regeneration related activities include hemoblasts. They are thought to
divide and differentiate to form the different blood cells present in the haemolymph. Few hemoblasts have previously
been found in the circulating hemolymph [18-20, 8], although several authors report a distinction between hemoblasts
or hemoblast-like cells characterized by the presence of a nucleolus, and lymphocyte-like cells, similar cells without
nucleoli [21], both of the two cell types are put on a pair with stem cells capable of hemopoietic function.
In colonial tunicates the hemoblasts are also involved in tissue renewal during asexual reproduction [18, 22, 23] and
are found in many developing tissues where they sometimes proliferate so rapidly that they differentiate simultaneously
in different tissues [24].
In adult C. intestinalis, as we observed, after an inflammatory wound, the frequency of these cells is significantly
increased both in the circulatory system and in hemopoietic sites, suggesting that they can increase proliferation and
then, undergoing differentiation, they infiltrate through the epidermis reaching the inflamed tissue where an intense
heightened population density is maintained until the tissue is repaired [7].
Hemopoietic tissue in ascidians is organized into clusters called lymph nodules scattered in the pharyngeal wall, in
transverse vessels of the brachial sac, [25, 26], and also in the endostyle region [27]. The atrial epithelium, the
specialized tissue that underlies the epidermis and encircles the pharinx, is also able to undergo transformation as it
becomes multilayered. The cells assume a squamous shape, the nuclei are in general ellipsoidal.
The blood cells of Styela clava were shown by autoradiography with tritiated thymidine to be engaged in premeiotic
DNA synthesis occurring both in the haematopoietic nodules and in blood channels [28]; most of these cells are
presumed to be hemoblasts.
Moreover on response to mitogens or allogeneic cells, in vitro proliferation of circulating blood cells have been
evidenced by autoradiography using populations enriched by density gradient centrifugation and in vitro culture
medium [29].
Using TEM we observed the circulating dividing cells and the high presence of precursor cells in hemopoietic tissue.
The rapid rate of proliferation is suggested by the presence of undifferentiated blood cells increased conspicuously in
number near the vessel epithelium and the presence of intercellular bridges between hemoblasts circulating in blood
lacunae. Some cells, in fact, result from incomplete cytoplasmic separation that leaves them connected by sizeable
intercellular bridges.
An intercellular bridge is a way for a nucleated cell to share its cytoplasm with its mitotically derived sister cells. The
formation of structurally intercellular bridges occurs during development of a wide range of cell types and species
ranging from insects to plants and mammals. There are a number of examples in development of intercellular bridges
that result from the arrest of cleavage furrows to convert them into stable intercellular bridges [30]. There are also a
number of examples of intercellular bridges that are a consequence of improper cytokinesis.
The disappearance of midbody and spindle remnants in the intercellular bridges we observed, suggest that these
could be transient intercellular bridges seen before the completion of cytokinesis. Such intercellular bridges might be in
the process of breaking down resulting in the final separation of the cells. Alternatively these intercellular bridges,
holding the cell together, may probably facilitate the synchronization of one of the following processes as cell division,
migration or differentiation. The connection might also allow the free passage of signals and the sharing of regulatory
molecules that initiate sister cells to be active in immune response.
These data first support with TEM observations of sample obtained after an in vivo response, that after an inflamed
stimulus there is an increase in frequency of circulating hemocytes with stem-cell specific features, undifferentiated
114 ©FORMATEX 2010

appearance and probably greater proliferative activities; thus hemoblasts as renewing cells provide a constant supply of
blood cells drawn toward the site of the injury, and replace lost cells.
This work should contribute as input for more detailed studies to link electron microscopic observations and
molecular, immunocytochemical approaches that could lead to our understanding of the functional significance of some
cellular aspects during immune responses.
Acknowledgements The authors are grateful to Dr. Sheila Mc Intyre for polishing the English. This work has been supported by
grants from the Italian Ministero della Università e della Ricerca and the University of Palermo research grant to M.A.D. and G.DL.
References
Microscopy: Science, Technology, Applications and Education
______________________________________________
A. Méndez-Vilas and J. Díaz (Eds.)
[1] Delsuc F, Brinkmann H, Chorrot D, Hervé P. Tunicate and not cephalochordates are the closest living relatives of vertebrates Nat.
Lett. 2006;439:965-968.
[2] Parrinello N, Patricolo E, Canicattì C. Tunicate immunobiology. I. Tunic reaction of Ciona intestinalis to erythrocyte injection.
Boll. Zool. 1977;44:373-381.
[3] Parrinello N, Patricolo E, Canicattì C. Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). I. Encapsulation
and tissue injury. Biol. Bull. 1984; 167:229-237.
[4] Parrinello N, Patricolo E. Inflammatory-like reaction in the tunic of Ciona intestinalis (Tunicata). II. Capsule components. Biol.
Bull. 1984; 167:238-250.
[5] De Leo G, Parrinello N, Parrinello D, Cassarà G, Di Bella MA. Encapsulation response of Ciona intestinalis (Ascidiacea) to
intratunical erythrocyte injection. I. The inner capsular architecture. J. Invert. Pathol., 1996; 67:205-212.
[6] De Leo G, Parrinello N, Parrinello D, Cassarà G, Russo D, Di Bella MA. Encapsulation response of Ciona intestinalis
(Ascidiacea) to intratunical erythrocyte injection. II. The outermost inflamed area. J. Invert. Pathol., 1997; 69:14-23.
[7] Di Bella MA, De Leo G. Hemocyte migration during inflammatory-like reaction of Ciona intestinalis (Tunicata, Ascidiacea). J.
Invert. Pathol. 2000;76:105-111.
[8] De Leo, G. Ascidian hemocytes and their involvement in defence reactions. Boll. Zool. 1992; 59:195-213.
[9] Raftos DA, Cooper EL. Proliferation of lymphocyte-like cells from the tunicate Styela clava, in response to allogeneic stimuli. J.
Exp. Zool. 1991; 260:391-400
[10] Sabbadin A. Formal genetics of ascidians. Amer. Zool. 1982; 22:765-777.
[11] Ballarin L. Immunobiology of compound ascidians Botryllus schlosseri: state of art. Inv. Surv.J. 2008;5:54-74
[12] Pinto MR, Chinnici C, Kimura Y, Melillo D, Marino R, Spruce LA, et al. CiC3-1amediated chemotaxis in the deuterostome
invertebrate Ciona intestinalis (Urochordata). J. Immunol. 2003; 171:5521-5528
[13] Cima F, Sabbadin A, Zaniolo G, Ballarin L. Colony specifity and chemotaxis in the compound ascidian Botryllus schlosseri.
Comp. Biochem. Physiol. A mol. Integr. Physiol. 2006;1145:376-382.
[14] Metcalf D, Moore MAS. Hematopoietic cells. Amsterdam: North-Holland; 1971
[15] Hall PA, Watt FM. Stem cells: the generation and maintenance of cellular diversity. Development. 1989; 106:619-633.
[16] Hyslop LA. Armstrong L, Stojkovic M, Lako M. Human embryonic stem cells: biology and clinical implications. Expert Rev.
Mol. Med. 2005; 7:1-21.
[17] Wong MD, Jin Z, Xie T. Molecular mechanisms of germline stem cell regulation. Annu. Rev. Genet. 2005; 39:173-195.
[18] Freeman G. The role of blood cells in the process of asexual reproduction in the tunicate Perophora viridis. J Exp Zool 1964;
156: 157-184
[19] Rowley AF. The blood cells of the sea squirt Ciona intestinalis: morphology, differential counts and in vitro phagocytic activity.
J. Invert. Pathol. 1981; 37:91-100.
[20] Wright RK. Urochordates. In: Ratcliffe NA, Rowley AF, eds. Invertebrate blood cells. Vol. 2. New York, N Y: Academic Press;
1981; 565-626.
[21] Peddie CM, Smith VJ. Lymphocyte-like cells in ascidians: Precursors for vertebrate lymphocytes? Fish Shellfish Immunol.
1995; 5:613-629.
[22] Nunzi MG, Burighel P, Schiaffino S. Muscle cell differentiation in the ascidian heart. Dev. Biol. 1979; 68:371-380.
[23] Sugino YM, Matsumura M, Kawamura K. Body muscle cell differentiation from coelomic stem cells in colonial tunicates. Zool.
Sci. 2007; 24:542-546.
[24] Kawamura K, Sugino YM. Cell proliferation dynamics of somatic and germ line tissues during zooidal life span in the colonial
tunicate Botryllus primigenus. Dev. Dyn. 2008; 237:1812-1825.
[25] George WC. A comparative study of the blood of the Tunicates. Quart. J. Micr. Sc. 1939; 81:391-428.
[26] Millar RH. Ciona. In: Colman JS, ed. L.M.B.C. Memoirs. Liverpool: The University Press; 1953;35:1-123.
[27] Voskoboynik A, Soen Y, Rinkevic Y, Rosner A, et al. Identification of the endostyele as a stem cell niche in a colonial chordate.
Cell Stem Cell. 2008; 3:456-464.
[28] Ermak TH. An autoradiographic demonstration of blood cell renewal in Styela clava (Urochordata: Ascidiacea). Experientia.
1975; 31:837-838.
[29] Peddie CM, Richesr AC, Smith VJ. Proliferation of undifferentiated blood cells from the solitary ascidian Ciona intestinalis in
vitro. Dev. Comp. Immunol. 1995;5:377-387.
[30] Robinson DN, Cooley L. Stable intercellular bridges in development: The cytoskeleton lining the tunnel. Trends Cell Biol.
1996;6:474-479.
©FORMATEX 2010 115