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Cell renewal in adjoining intestinal and tracheal epithelia of Manduca
Journal of Insect Physiology 57 (2011) 487–493
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Cell renewal in adjoining intestinal and tracheal epithelia of Manduca James B. Nardi a,*, Charles Mark Bee b, Lou Ann Miller c, Divya Mathur d, Benjamin Ohlstein d,1 a
Department of Entomology, University of Illinois, 320 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States Imaging Technology Group, Beckman Institute for Advanced Science and Technology, University of Illinois, 405 N. Mathews Avenue, Urbana, IL 61801, United States c Center for Microscopic Imaging, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Avenue, Urbana, IL 61801, United States d Columbia University Medical Center, Department of Genetics and Development, 701 W. 168th Street, HHSC 1130, New York, NY 10032, United States b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2010 Received in revised form 16 January 2011 Accepted 19 January 2011 Available online 28 January 2011
Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects’ responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells. Published by Elsevier Ltd.
Keywords: Regenerative cells Intestinal cells Midgut Tracheae Stem cells Fasciclin II
1. Introduction During normal development, the number of cells in the intestinal or midgut epithelium of the moth Manduca sexta increases approximately 200-fold during the larval stages – from egg hatching to pupation – over a period of about two weeks. This increase in cell number is accompanied by rapid division of midgut regenerative cells, with minimal death of existing intestinal cells (Baldwin and Hakim, 1991). As the site of entry for viral, bacterial, and eukaryotic vectors as well as chemical toxins, the insect midgut epithelium is a crossroads for foreign traffic and is an epithelial monolayer without a cuticular barrier separating its apical cell membrane from foreign surfaces. To compensate for the vulnerability of the midgut epithelium and any loss of cells that accompanies encounters of its membranes with foreign entities, its regenerative cells can also be rapidly deployed to replace any sudden loss of the two differentiated cell types in the midguts of larval moths and butterflies: columnar cells (enterocytes) and goblet cells (Hakim et al., 2010). Regenerative cells concurrently generate such differentiated midgut cells while retaining their proliferative state. Regenerative
cells have recently been described in adult midgut epithelium of Drosophila, and their genetic lineages have been tracked (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). The specification of these populations of regenerative cells during larval and pupal midgut organogenesis has now been elucidated in detail (Mathur et al., 2010). Like other insect tissues, the midgut epithelium is associated with tracheal, neural and muscle cells. While cells of most larval tissues increase in size without cell divisions, regenerative tracheal cells divide throughout larval life to keep pace with growth of the tissues, such as midgut epithelium, to which they supply oxygen (Wigglesworth, 1954). Humoral factors that affect proliferation and differentiation of Manduca midgut cells have been studied in cultures of midgut regenerative cells (Hakim et al., 2010), but how different cell types associated with the midgut regenerative cells function in situ to coordinate their growth with the growth of midgut epithelium is not known. These neighboring cells that surround the midgut epithelium on its basal surface could constitute a regenerative niche that orchestrates proliferation and differentiation among regenerative midgut cells. 2. Materials and methods 2.1. Staging of embryonic development and larval development
* Corresponding author. Tel.: +1 217 333 6590; fax: +1 217 244 3499. E-mail addresses: [email protected], [email protected] (J.B. Nardi), [email protected] (B. Ohlstein). 1 Tel.: +1 212 305 0558. 0022-1910/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.jinsphys.2011.01.008
Eggs were collected from tobacco leaves that had been placed in the moth-breeding cage 1 h earlier. All eggs and larvae were maintained in an incubator at constant temperature (26 8C) and
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photoperiod (18L:6D). At this temperature, embryonic development, from egg laying to egg hatching, lasts 100 h. By designating embryonic development time (DT) as a percentage of time between egg laying (0% DT) and egg hatching (100% DT), the number of hours (N) after collection of eggs closely approximates (N % DT). After hatching, all larvae were fed a standard artificial diet. 2.2. Preparation of larval midgut epithelia for fixation and imaging Two developmental landmarks facilitate staging of larvae during each larval stadium: (1) retraction of the new head capsule cuticle from the overlying old cuticle marks the end of one stadium and (2) the molting of the head capsule and body cuticle of stadium (n) marks the beginning of the next stadium (n + 1). All embryos and larvae were dissected in Petri dishes with black Sylgard lining their bottoms. Anesthetized animals were submerged in Grace’s insect culture medium and pinned at their anterior and posterior ends to the Sylgard surface. They were opened along the dorsal midline with iridectomy scissors and edges of the integument were impaled with stainless steel minutien pins (0.1 mm diameter) to expose the entire gut. Following addition of primary fixative, the gut was opened along its dorsal midline with iridectomy scissors; the tissue retained the configuration in which it had been pinned. After fixation, the entire gut was separated from the integument. Tissues used for electron microscopy and staining of sections with toluidine blue were prepared as described in Section 2.2.1. Tissues used for immunolabeling were fixed according to the procedure presented in Section 2.2.2. 2.2.1. Preparation of tissues for sectioning and scanning electron microscopy Sections of specimens for transmission electron microscopy and light microscopy and whole mounts for scanning electron microscopy were fixed in at 4 8C in a primary fixative of 2.5% glutaraldehyde and 0.5% paraformaldehyde dissolved in a rinse buffer of 0.1 M cacodylate (pH 7.4) containing 0.18 mM CaCl2 and 0.58 mM sucrose. After 3 h in this fixative, tissues were washed three times with rinse buffer before being transferred to the secondary fixative (2% osmium tetroxide in rinse buffer). Tissues remained in this solution for 4 h in the cold and were then washed three more times with rinse buffer. To enhance membrane contrast, rinsed tissues were placed in filtered, saturated uranyl acetate for 10 min immediately before being gradually dehydrated in a graded ethanol series (10–100%). At this stage in the preparation, tissues for scanning electron microscopy were processed separately from the tissues for sectioning. From absolute ethanol, tissues for sectioning were transferred to propylene oxide and infiltrated with mixtures of propylene oxide and resin before being embedded in pure LX112 resin. Resin was polymerized at 60 8C for three days followed by 15 repetitions of 1-min microwave treatments using a 400-ml water bath in the microwave. Embedded tissues were sectioned with a diamond knife either at 1.0 mm for light microscopy or at 0.09 mm for electron microscopy. Thick sections for light microscopy were mounted on glass slides and stained with a solution of 0.5% toluidine blue in 1% borax. Thin sections of those regions of alimentary canal chosen for ultrastructural examination were mounted on copper grids and stained briefly with saturated aqueous uranyl acetate and Luft’s lead citrate to enhance contrast. Images were taken with a Hitachi 600 transmission electron microscope operating at 75 kV. Specimens for scanning electron microscopy were processed in parallel with specimens for resin embedding until they had been dehydrated in 100% ethanol. From absolute ethanol, specimens
were critical-point dried and coated with gold–palladium. Images were taken with a Philips XL30 ESEM-FEG microscope operated by the Imaging Technology Group at the Beckman Institute, University of Illinois. 2.2.2. Preparation of whole mounts and sections for immunolabeling 2.2.2.1. Fluorescent labeling of tracheal stem cells and mitotic cells. Whole mounts of guts used for fluorescent labeling were always fixed in 4% paraformaldehyde (w/v) dissolved in phosphate-buffered saline (PBS, pH 7.4). After several rinses with PBS to remove residual fixative, tissues were permeabilized for at least 30 min by addition of blocking buffer (PBS + 10% normal goat serum + 0.1% Triton X-100). The epithelia relaxed into a planar configuration following addition of blocking buffer. Intestinal tissue was pre-incubated for at least 30 min in blocking buffer before addition of the two primary antibodies – one prepared in rabbit, one prepared in mouse. Tracheal stem cells were labeled with a monoclonal antibody to M. sexta fasciclin II (MAb 2F5, 1:5000). The origin of this antibody is described in Nardi (1990). Only nuclei of mitotic cells labeled with the rabbit mitotic marker anti-phosphohistone H3 (1 mg/10 ml, Upstate). This primary rabbit antibody was diluted to 10 mg/ml in blocking buffer. After an overnight incubation with both primary antibodies at 4 8C, tissues were rinsed at least three times with blocking buffer and then incubated overnight at 4 8C room with 7.5 mg/ml fluorescein isothiocyanate (FITC)-coupled goat antirabbit antibody (Vector). Following three more rinses with blocking buffer, tissues were next incubated with 1:100 rhodamine (TRITC)-coupled goat anti-mouse antibody (Zymed, 1.5 mg/ ml) followed by several rinses with blocking buffer. After thoroughly rinsing the anti-mouse secondary antibody with blocking buffer, tissues were mounted in 70% glycerin (v/v) in 0.1 M Tris (pH 9.0). Specimens were imaged with a Nikon E600. 2.2.2.2. Labeling of tissues with antibody coupled to horseradish peroxidase (HRP). The same fixative, blocking buffer and mouse primary antibody MAb 2F5 described above for preparation of fluorescently labeled intestinal epithelia were used in this procedure. After an overnight incubation with this primary antibody, tissue was rinsed with blocking buffer (PBS + 3% normal horse serum + 0.1% Triton X-100) before an overnight exposure to biotinylated horse anti-mouse secondary antibody (1:200). After rinsing excess secondary antibody from the tissues with PBS + 0.05% Tween-20 (PBT), avidin-HRP diluted in PBT was added as an enzyme marker for 3–5 h at room temperature. Excess of this last reagent was thoroughly rinsed from tissue with PBT. The biotinylated secondary antibody and avidin-HRP were supplied in a kit from Vector Laboratories. To visualize the cells marked with HRP, the addition of diaminobenzidine (DAB) and H2O2 in a metalenhanced DAB substrate kit (Pierce) produced a dark precipitate. Tissues were mounted on slides as described in the previous section. 3. Results Shortly after dorsal closure of the midgut during embryogenesis (55% DT), the nerve, muscle and tracheal cells associated with the developing midgut epithelium occupy predictable positions relative to each other. Images of these cells have been prepared from whole mounts of the midguts as well as from sections cut perpendicular to the plane of these whole mounts. In preparing whole mounts, tubular midguts were first cut along their longitudinal axes and then flattened to lie within a single plane. As shown schematically in Fig. 1, tracheal cells at the termini of tracheal branches lie between longitudinal muscles and are
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Fig. 1. In the two schematic diagrams shown in this figure, the relative positions of enteric muscles (longitudinal and circular), tracheal epithelial cells and midgut epithelial cells are indicated between molts (a) and at the time of a molt (b). These cells occupy a plane that lies perpendicular to the basal surface of the midgut and that passes from this basal midgut surface to its luminal surface (at bottom of image). Each cell type (tracheal, muscle and midgut) is delimited by a well-defined basal lamina. The positions of muscle cells remain relatively constant on the basal surface of the midgut, but the undifferentiated, regenerative tracheal cells shift positions as they divide and differentiate. As tracheal cells differentiate at each molt, each develops a cuticular lumen that is reinforced by distinctive taenidial ridges. The differentiating tracheal cells leave the basal surface of the midgut to move into the plane of the midgut monolayer, interdigitating with its endodermal cells (arrowheads). TR, tracheal regenerative cell; T, differentiated trachea or tracheole; TBL, tracheal basal lamina; R, midgut regenerative cell; MBL, midgut basal lamina; E, enterocyte or columnar cell; G, goblet cell; LM, longitudinal muscles; CM, circular muscles. Differentiating midgut epithelial cells are indicated with arrows.
Fig. 2. These scanning electron micrographs of the basal surface of a first instar midgut, immediately after hatching from the egg, show a global view of the arrangement of tracheal, muscle and nerve cells associated with underlying midgut epithelium. Longitudinal muscles extend from right to left of the image; representative longitudinal muscles are marked with arrowheads. Circular muscles run perpendicular to the longitudinal muscles. t, differentiated tracheoles; e, enteric neurons and glia. Regenerative cells lie hidden beneath the surface’s basal lamina. Scale bars = 200 mm in 2a and 20 mm in 2b.
covered with a basal lamina. Circular muscles that are oriented perpendicular to the longitudinal muscles of the gut lie between these tracheal cells and the midgut epithelial cells. To supply oxygen to the differentiated midgut cells, differentiated tracheal cells extend tracheoles from the basal surface of the midgut to positions within the midgut monolayer. Undifferentiated tracheal progenitors (tracheal regenerative cells) are found at the tips of these differentiated tracheal branches (Fig. 1a); a tracheal progenitor cell will eventually differentiate and form a cuticlelined lumen that will transport oxygen to newly differentiated midgut cells (Fig. 1b). To facilitate interpretation of the scanning electron microscope image of the midgut basal surface in Fig. 2 and the immunolabeled whole mounts of the insect midguts shown in Figs. 3–7, these schematic views of the cells in Fig. 1a and b are presented as sections cut perpendicular to the surfaces shown in these figures of midgut whole mounts. The membrane protein neuroglian is expressed on all epithelial cells of the tracheae and tracheoles (Nardi, 1994; immunolabeling not shown). Another membrane protein in the immunoglobulin superfamily, however, is expressed only on cells at the growing tips and nodes of tracheal branches (Nardi, 1990). This protein, fasciclin II, is found on the undifferentiated cells at the termini of the tracheal branches (tracheal regenerative cells or tracheal
progenitors) as well as the enteric neurons and glia that supply the developing midgut epithelium. The fasciclin II-positive cells are contiguous with differentiated, tracheal epithelial cells that are readily identified as cells ensheathing fine cuticular tubes. Distinctive tracheal taenidial ridges line these cuticular tubes and lie perpendicular to the long axis of each tracheal tube. Fasciclin II-positive cells first appear on the basal surface of the midgut epithelium soon after dorsal closure of the midgut (Fig. 3) and are observed throughout larval development. The form assumed by each of these regenerative cells is a function of its stage in the molt cycle (Figs. 4–7). Immediately after a molt, the fasciclin II-positive cells extend long processes along the transverse axis of the midgut (Fig. 4). Halfway between larval molts, tracheal regenerative cells divide and form coherent aggregates (Figs. 5 and 6). Cell divisions within the tracheal and midgut epithelia are scored by labeling mitotic cells with an antibody to phosphohistone H3. A cross section of the larval midgut epithelium labeled with anti-fasciclin II-HRP (Fig. 6, inset) shows the relative positions of tracheal regenerative cells and the longitudinal muscles that also faintly label with anti-fasciclin II. Tracheal regenerative cells are also clearly associated with circular muscles of the Manduca midgut epithelium (Figs. 6 inset, 8 and 9).
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Figs. 3–8. Tracheal regenerative cells are found on the basal surface of the intestinal (midgut) epithelium. In whole mounts of the midgut, the tracheal regenerative cells label with a primary mouse antibody to the surface protein fasciclin II and either (A) an anti-mouse secondary antibody coupled to horseradish peroxidase (HRP) in Figs. 3, 4, 6 and 7 or (B) an anti-mouse secondary antibody coupled to rhodamine (Fig. 5). Longitudinal muscles (representative muscles marked with arrowheads) faintly label with antifasciclin II and extend from top to bottom of each image. Circular muscles run perpendicular to the longitudinal muscles. t, differentiated tracheoles; e, enteric neurons and glia also label with anti-fasciclin II. In Fig. 8, the cells observed in this section of the basal surface of the midgut epithelium have been stained with toluidine blue. (Fig. 3) In Manduca embryos the first tracheal regenerative cells (representative regenerative cells marked with arrows) are evident around 65% development time (DT) on the basal surface of the midgut epithelium. (Fig. 4) Long processes extend from tracheal regenerative cells immediately after hatching of the embryo, at the inception of the first larval stadium. Representative tracheal regenerative cells are marked with arrows. (Fig. 5) In this whole mount of the midgut epithelium fixed halfway through the first larval stadium, the antibody to phosphohistone H3 is marked with fluorescein (green) and anti-fasciclin II is marked with rhodamine (red). Most cell divisions are localized to tracheal regenerative cells. Some cell divisions also occur concurrently within another plane of focus – the plane of the midgut monolayer occupied by midgut regenerative cells (arrows). e, enteric neurons and glia. (Fig. 6) When tracheal regenerative cells halfway through the first larval stadium are labeled with HRP, the aggregates of regenerative cells that lie between the longitudinal muscles (arrowheads) are even more evident. (Inset) This cross section of the larval midgut epithelium also fixed halfway through the first larval stadium and labeled with anti-fasciclin II-HRP shows the positions of tracheal regenerative cells (arrowhead) and longitudinal muscles (lm) and circular muscles (cm) relative to the endodermal monolayer whose luminal surface is indicated with arrows. (Fig. 7) Immediately prior to the molt from first to second instar, tracheal regenerative cells (representative cells marked with arrows) cease dividing and occur as single, dispersed cells. (Fig. 8) At the molt from the second larval instar to the third larval instar, a differentiating tracheole cell generated by tracheal regenerative cells (trc) on the basal surface of the midgut epithelium is shown crossing the circular muscle layer that separates tracheal regenerative cells from midgut cells. Arrow points to growing tip of tracheole cell. One tracheal regenerative cell is dividing (arrowhead). Midgut regenerative cells (double arrow) lie among differentiated goblet cells (gc). lm, longitudinal muscles; cm, circular muscles. Scale bars = 50 mm (Figs. 3–7). Scale bars = 5 mm (Fig. 8 and inset in Fig. 6).
Within these aggregates of regenerative tracheal cells, while some cells differentiate into cuticle-lined tracheoles, others remain undifferentiated on the basal periphery of the midgut epithelium (Figs. 7 and 8). Prior to each larval–larval molt, progeny of tracheal regenerative cells leave the basal surface of the midgut epithelium, extend long processes, differentiating as
they enter the plane of the midgut monolayer and no longer express fasciclin II (Figs. 7 and 8). After the differentiating tracheal cells cross into the plane of the midgut epithelium, the fasciclin II-positive regenerative cells remaining on the basal periphery of the midgut epithelium cease dividing and extend lengthy lateral processes, remaining dispersed
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Fig. 9. Halfway through the third larval stadium, clusters of tracheal regenerative cells (trc) lie beneath a thin basal lamina (bl), adherent to circular muscles (cm), and between longitudinal muscles (lm) of the gut. Differentiated tracheoles (t) extend into the plane of the midgut epithelial cells. Note the highly convoluted basal surfaces of differentiated midgut or intestinal epithelial cells (i.e.) surrounding a midgut or intestinal regenerative cell (irc). Scale = 2 mm.
on the basal surface of the newly expanded midgut monolayer (Fig. 7). Ultrastructural examination of cells on the basal surface of the midgut epithelium reveals the associations of basal laminae with different cell types (Figs. 9 and 10). A relatively thick basal lamina segregates tracheal cells from midgut cells; and a relatively thin basal lamina covers the hemocoel surface of the tracheal regenerative cells and the enteric muscles. In the two-dimensional section of cells shown in Fig. 9, the aggregate of tracheal regenerative cells that lies between two longitudinal muscles and in contact with circular muscles measures about 7 mm. Although obscured by basal laminae that drape over the enteric muscles and tracheal regenerative cells, outlines of some underlying tracheal regenerative cells presumably are represented by the rounded contours of the basal lamina (Fig. 10, arrows). This figure also illustrates the three-dimensional relationship among tracheal regenerative cells and differentiated tracheal branches. The approximate spacing between the longitudinal muscles is 10 mm. The underlying circular muscles (arrowheads) are all oriented perpendicular to the longitudinal muscles. Regenerative cells of tracheal epithelia are destined to supply the respiratory needs of new midgut cells in the rapidly growing endodermal epithelium. Tracheal regenerative cell proliferation begins anew with the start of a new molt cycle, peaking in concert with proliferation of the midgut cells that occurs during each cycle (Fig. 11). A series of fine structural images of differentiating tracheal cells and differentiating midgut cells that intercalate with differentiated midgut cells reveals that tracheal cells occupy inpocketings of the basal surface of the midgut epithelium (Figs. 13,14). As developing midgut cells stretch toward the gut lumen, they are followed in their wakes by tracheal cells that have extended long processes and that have developed cuticle-lined lumens in these processes. A basal lamina occupies the interface between each differentiating tracheal process each regenerative midgut cell that is undergoing
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Fig. 10. In this view of the basal surface of the midgut epithelium from the middle of the third larval stadium, the regenerative cells that occupy the termini of these differentiated tracheal branches (t) lie hidden between the longitudinal muscles (lm) and beneath the thin basal lamina. Two representative longitudinal muscles are labeled. Outlines of tracheal regenerative cells are suggested in several places by rounded contours of the basal lamina (arrows). Outlines of some circular muscles can also be faintly discerned beneath this basal lamina (arrowheads). Scale = 10 mm.
differentiation. Each of these differentiating tracheal processes is embedded in an extracellular matrix that is contiguous with the midgut’s basal lamina. Differentiating tracheal processes associate with differentiating regenerative midgut cells and intercalate with the differentiated midgut cells.
Fig. 11. During each molt cycle, proliferation of tracheal regenerative cells (circles) closely tracks proliferation of midgut regenerative cells (triangles). A labeled nucleus represents one that labels with anti-phosphohistone H3. Each point represents the average standard error of 10 counts (n = three larvae, 3 or 4 counts from each) within a field measuring 0.66 mm2.
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Figs. 12–14. This series of images depict the association of midgut regenerative cells, tracheoles (t), and basal laminae during addition of tracheoles to the differentiated midgut epithelium. The basal surface of the midgut epithelium is shown halfway through the second larval stadium (Fig. 12), shortly before the molt from the second to the third larval instar (Fig. 13), and immediately after (Fig. 14) the molt from the second to third instar. In each of the three images, two intestinal regenerative cells (irc) lie adjacent to goblet cells whose central cavities (cc) are lined with microvilli. Goblet cells have distinctive nuclei (nu) and highly convoluted basal membranes (bm). Tracheoles (t) and basal laminae occupy positions (arrows) adjacent to the midgut regenerative cells (irc). A basal lamina (*) and circular muscles (cm) lie over the midgut epithelium. Midgut lumen is at top of figures. Scale = 5 mm.
4. Discussion 4.1. Distribution of tracheal regenerative cells With an antibody marker that recognizes the membrane protein fasciclin II, a covert spacing pattern of tracheal regenerative cells has been revealed on the basal surface of the midgut. The extension of long processes from these tracheal regenerative cells (Figs. 3, 4, 6 and 7) and their interactions with adjacent tracheal cells as well as with tracheal cells that are two or more cell diameters away could account for the regular distribution of these regenerative cells over the basal surface of the insect midgut. 4.2. Distribution of differentiated tracheole cells The regular spacing pattern of differentiated tracheole cells that associate with differentiating and differentiated midgut epithelial cells (Fig. 2) is mediated by a communication among differentiating tracheal and midgut cells that does not require direct cell–cell contact. As tracheole cells intercalate with midgut cells, the two cell types do not come in direct contact but meet at an extracellular matrix (basal lamina) interface (Figs. 13 and 14). In epidermal epithelia that have been deprived of their tracheoles, basal processes extend in the direction of the existing tracheole cells and recruit these cells as an oxygen supply. The movement and distribution of differentiated tracheole cells within these developing insect epidermal epithelia apparently are not determined intrinsically but seem to be imposed by the epidermal cells with which they supply oxygen (Nardi, 1984; Wigglesworth, 1959). 4.3. Coordination of growth of different cells during organogenesis The proliferation of tracheal regenerative cells of Manduca is closely coupled with proliferation of nearby regenerative cells of midgut epithelia. These regenerative cells respectively reside within an ectodermal epithelium and an endodermal epithelium that divide, grow, and differentiate in concert (Figs. 8, 11–14). During organogenesis of salivary gland epithelium, interactions with other surrounding tissues such as blood vessels, nerves and mesenchymal cells maintain the populations of epithelial regenerative cells that are required for development and regeneration of mouse salivary epithelium. The peripheral parasympathetic innervation of salivary epithelia maintains the progenitor cell population of salivary epithelia in its undifferentiated state (Knox
et al., 2010). Coordinated growth of tracheal-midgut in Manduca may represent another example of a branching epithelium (tracheal) maintaining the undifferentiated state of midgut regenerative cells. In the literature on insect regenerative cells, the terms stem cells, regenerative cells and progenitor cells have often been used interchangeably. Stem cells are cells that can concurrently generate such differentiated midgut cells while retaining their proliferative state. Although regenerative cells of insect midgut epithelia have conventionally been referred to as ‘‘stem’’ cells and compared with stem cells that have been described in adult midgut epithelia of Drosophila (Hakim et al., 2010; Nardi et al., 2010), the rigorous genetic lineage labeling of epithelial cells that has definitively identified a subset of Drosophila regenerative cells as stem cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006) has not been possible with insects other than Drosophila. To adjust to the constantly changing requirements of a rapidly growing epithelium, stem cells of different tissues must be able to differentiate at the right time and right place. The cues directing their proliferation and differentiation have been postulated to reside not only in the stem cells themselves with minimal input from neighboring cells (Wilson and Kotton, 2008) but also with neighboring cells referred to as stem cell ‘‘niches’’ that supply the cues regulating the behavior of the stem cells (Mathur et al., 2010). The coordinated development of tracheal regenerative cells and midgut regenerative cells described in this manuscript provides evidence that tracheal cells that adhere to the basal lamina surface of the midgut monolayer not only occupy the appropriate location for a presumed midgut stem cell niche but also clearly proliferate and differentiate in concert with putative midgut stem cells. Acknowledgments The authors gratefully acknowledge the helpful suggestions of two anonymous reviewers. References Baldwin, K.M., Hakim, R.S., 1991. Growth and differentiation of the larval midgut epithelium during molting in the moth, Manduca sexta. Tissue and Cell 23, 411– 422. Hakim, R.S., Baldwin, K., Smagghe, G., 2010. Regulation of midgut growth, development, and metamorphosis. Annual Review of Entomology 55, 593–608. Knox, S.M., Lombaert, I.M.A., Reed, X., Vitale-Cross, L., Gutkind, J.S., Hoffman, M.P., 2010. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science 329, 1645–1647.
J.B. Nardi et al. / Journal of Insect Physiology 57 (2011) 487–493 Mathur, D., Bost, A., Driver, I., Ohlstein, B., 2010. A transient niche regulates the specification of Drosophila intestinal stem cells. Science 327, 210–213. Micchelli, C.A., Perrimon, N., 2006. Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature 439, 475–479. Nardi, J.B., 1994. Rearrangement of epithelial cell types in an insect wing monolayer is accompanied by differential expression of a cell surface protein. Developmental Dynamics 199, 315–325. Nardi, J.B., 1990. Expression of a surface epitope on cells that link branches in the tracheal network of Manduca sexta. Development 110, 681–688. Nardi, J.B., 1984. Tracheole migration in an insect wing. Evidence for guidance by epithelial processes. Roux’s Archives 194, 1–8.
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Nardi, J.B., Bee, C.M., Miller, L.A., 2010. Stem cells of the beetle midgut epithelium. Journal of Insect Physiology 56, 296–303. Ohlstein, B., Spradling, A., 2006. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470–474. Wigglesworth, V.B., 1954. Growth and regeneration in the tracheal system of an insect, Rhodnius prolixus (Hemiptera). Quarterly Journal of the Microscopical Society 95, 115–137. Wigglesworth, V.B., 1959. The role of the epidermal cells in the ‘migration’ of tracheoles in Rhodnius prolixus (Hemiptera). Journal of Experimental Biology 36, 632–640. Wilson, A.A., Kotton, D.N., 2008. Another notch in stem cell biology: Drosophila intestinal stem cells and the specification of cell fates. BioEssays 30, 107–109.
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Cell renewal in adjoining intestinal and tracheal epithelia of Manduca James B. Nardi a,*, Charles Mark Bee b, Lou Ann Miller c, Divya Mathur d, Benjamin Ohlstein d,1 a
Department of Entomology, University of Illinois, 320 Morrill Hall, 505 S. Goodwin Avenue, Urbana, IL 61801, United States Imaging Technology Group, Beckman Institute for Advanced Science and Technology, University of Illinois, 405 N. Mathews Avenue, Urbana, IL 61801, United States c Center for Microscopic Imaging, College of Veterinary Medicine, University of Illinois, 2001 S. Lincoln Avenue, Urbana, IL 61801, United States d Columbia University Medical Center, Department of Genetics and Development, 701 W. 168th Street, HHSC 1130, New York, NY 10032, United States b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2010 Received in revised form 16 January 2011 Accepted 19 January 2011 Available online 28 January 2011
Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects’ responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells. Published by Elsevier Ltd.
Keywords: Regenerative cells Intestinal cells Midgut Tracheae Stem cells Fasciclin II
1. Introduction During normal development, the number of cells in the intestinal or midgut epithelium of the moth Manduca sexta increases approximately 200-fold during the larval stages – from egg hatching to pupation – over a period of about two weeks. This increase in cell number is accompanied by rapid division of midgut regenerative cells, with minimal death of existing intestinal cells (Baldwin and Hakim, 1991). As the site of entry for viral, bacterial, and eukaryotic vectors as well as chemical toxins, the insect midgut epithelium is a crossroads for foreign traffic and is an epithelial monolayer without a cuticular barrier separating its apical cell membrane from foreign surfaces. To compensate for the vulnerability of the midgut epithelium and any loss of cells that accompanies encounters of its membranes with foreign entities, its regenerative cells can also be rapidly deployed to replace any sudden loss of the two differentiated cell types in the midguts of larval moths and butterflies: columnar cells (enterocytes) and goblet cells (Hakim et al., 2010). Regenerative cells concurrently generate such differentiated midgut cells while retaining their proliferative state. Regenerative
cells have recently been described in adult midgut epithelium of Drosophila, and their genetic lineages have been tracked (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). The specification of these populations of regenerative cells during larval and pupal midgut organogenesis has now been elucidated in detail (Mathur et al., 2010). Like other insect tissues, the midgut epithelium is associated with tracheal, neural and muscle cells. While cells of most larval tissues increase in size without cell divisions, regenerative tracheal cells divide throughout larval life to keep pace with growth of the tissues, such as midgut epithelium, to which they supply oxygen (Wigglesworth, 1954). Humoral factors that affect proliferation and differentiation of Manduca midgut cells have been studied in cultures of midgut regenerative cells (Hakim et al., 2010), but how different cell types associated with the midgut regenerative cells function in situ to coordinate their growth with the growth of midgut epithelium is not known. These neighboring cells that surround the midgut epithelium on its basal surface could constitute a regenerative niche that orchestrates proliferation and differentiation among regenerative midgut cells. 2. Materials and methods 2.1. Staging of embryonic development and larval development
* Corresponding author. Tel.: +1 217 333 6590; fax: +1 217 244 3499. E-mail addresses: [email protected], [email protected] (J.B. Nardi), [email protected] (B. Ohlstein). 1 Tel.: +1 212 305 0558. 0022-1910/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.jinsphys.2011.01.008
Eggs were collected from tobacco leaves that had been placed in the moth-breeding cage 1 h earlier. All eggs and larvae were maintained in an incubator at constant temperature (26 8C) and
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photoperiod (18L:6D). At this temperature, embryonic development, from egg laying to egg hatching, lasts 100 h. By designating embryonic development time (DT) as a percentage of time between egg laying (0% DT) and egg hatching (100% DT), the number of hours (N) after collection of eggs closely approximates (N % DT). After hatching, all larvae were fed a standard artificial diet. 2.2. Preparation of larval midgut epithelia for fixation and imaging Two developmental landmarks facilitate staging of larvae during each larval stadium: (1) retraction of the new head capsule cuticle from the overlying old cuticle marks the end of one stadium and (2) the molting of the head capsule and body cuticle of stadium (n) marks the beginning of the next stadium (n + 1). All embryos and larvae were dissected in Petri dishes with black Sylgard lining their bottoms. Anesthetized animals were submerged in Grace’s insect culture medium and pinned at their anterior and posterior ends to the Sylgard surface. They were opened along the dorsal midline with iridectomy scissors and edges of the integument were impaled with stainless steel minutien pins (0.1 mm diameter) to expose the entire gut. Following addition of primary fixative, the gut was opened along its dorsal midline with iridectomy scissors; the tissue retained the configuration in which it had been pinned. After fixation, the entire gut was separated from the integument. Tissues used for electron microscopy and staining of sections with toluidine blue were prepared as described in Section 2.2.1. Tissues used for immunolabeling were fixed according to the procedure presented in Section 2.2.2. 2.2.1. Preparation of tissues for sectioning and scanning electron microscopy Sections of specimens for transmission electron microscopy and light microscopy and whole mounts for scanning electron microscopy were fixed in at 4 8C in a primary fixative of 2.5% glutaraldehyde and 0.5% paraformaldehyde dissolved in a rinse buffer of 0.1 M cacodylate (pH 7.4) containing 0.18 mM CaCl2 and 0.58 mM sucrose. After 3 h in this fixative, tissues were washed three times with rinse buffer before being transferred to the secondary fixative (2% osmium tetroxide in rinse buffer). Tissues remained in this solution for 4 h in the cold and were then washed three more times with rinse buffer. To enhance membrane contrast, rinsed tissues were placed in filtered, saturated uranyl acetate for 10 min immediately before being gradually dehydrated in a graded ethanol series (10–100%). At this stage in the preparation, tissues for scanning electron microscopy were processed separately from the tissues for sectioning. From absolute ethanol, tissues for sectioning were transferred to propylene oxide and infiltrated with mixtures of propylene oxide and resin before being embedded in pure LX112 resin. Resin was polymerized at 60 8C for three days followed by 15 repetitions of 1-min microwave treatments using a 400-ml water bath in the microwave. Embedded tissues were sectioned with a diamond knife either at 1.0 mm for light microscopy or at 0.09 mm for electron microscopy. Thick sections for light microscopy were mounted on glass slides and stained with a solution of 0.5% toluidine blue in 1% borax. Thin sections of those regions of alimentary canal chosen for ultrastructural examination were mounted on copper grids and stained briefly with saturated aqueous uranyl acetate and Luft’s lead citrate to enhance contrast. Images were taken with a Hitachi 600 transmission electron microscope operating at 75 kV. Specimens for scanning electron microscopy were processed in parallel with specimens for resin embedding until they had been dehydrated in 100% ethanol. From absolute ethanol, specimens
were critical-point dried and coated with gold–palladium. Images were taken with a Philips XL30 ESEM-FEG microscope operated by the Imaging Technology Group at the Beckman Institute, University of Illinois. 2.2.2. Preparation of whole mounts and sections for immunolabeling 2.2.2.1. Fluorescent labeling of tracheal stem cells and mitotic cells. Whole mounts of guts used for fluorescent labeling were always fixed in 4% paraformaldehyde (w/v) dissolved in phosphate-buffered saline (PBS, pH 7.4). After several rinses with PBS to remove residual fixative, tissues were permeabilized for at least 30 min by addition of blocking buffer (PBS + 10% normal goat serum + 0.1% Triton X-100). The epithelia relaxed into a planar configuration following addition of blocking buffer. Intestinal tissue was pre-incubated for at least 30 min in blocking buffer before addition of the two primary antibodies – one prepared in rabbit, one prepared in mouse. Tracheal stem cells were labeled with a monoclonal antibody to M. sexta fasciclin II (MAb 2F5, 1:5000). The origin of this antibody is described in Nardi (1990). Only nuclei of mitotic cells labeled with the rabbit mitotic marker anti-phosphohistone H3 (1 mg/10 ml, Upstate). This primary rabbit antibody was diluted to 10 mg/ml in blocking buffer. After an overnight incubation with both primary antibodies at 4 8C, tissues were rinsed at least three times with blocking buffer and then incubated overnight at 4 8C room with 7.5 mg/ml fluorescein isothiocyanate (FITC)-coupled goat antirabbit antibody (Vector). Following three more rinses with blocking buffer, tissues were next incubated with 1:100 rhodamine (TRITC)-coupled goat anti-mouse antibody (Zymed, 1.5 mg/ ml) followed by several rinses with blocking buffer. After thoroughly rinsing the anti-mouse secondary antibody with blocking buffer, tissues were mounted in 70% glycerin (v/v) in 0.1 M Tris (pH 9.0). Specimens were imaged with a Nikon E600. 2.2.2.2. Labeling of tissues with antibody coupled to horseradish peroxidase (HRP). The same fixative, blocking buffer and mouse primary antibody MAb 2F5 described above for preparation of fluorescently labeled intestinal epithelia were used in this procedure. After an overnight incubation with this primary antibody, tissue was rinsed with blocking buffer (PBS + 3% normal horse serum + 0.1% Triton X-100) before an overnight exposure to biotinylated horse anti-mouse secondary antibody (1:200). After rinsing excess secondary antibody from the tissues with PBS + 0.05% Tween-20 (PBT), avidin-HRP diluted in PBT was added as an enzyme marker for 3–5 h at room temperature. Excess of this last reagent was thoroughly rinsed from tissue with PBT. The biotinylated secondary antibody and avidin-HRP were supplied in a kit from Vector Laboratories. To visualize the cells marked with HRP, the addition of diaminobenzidine (DAB) and H2O2 in a metalenhanced DAB substrate kit (Pierce) produced a dark precipitate. Tissues were mounted on slides as described in the previous section. 3. Results Shortly after dorsal closure of the midgut during embryogenesis (55% DT), the nerve, muscle and tracheal cells associated with the developing midgut epithelium occupy predictable positions relative to each other. Images of these cells have been prepared from whole mounts of the midguts as well as from sections cut perpendicular to the plane of these whole mounts. In preparing whole mounts, tubular midguts were first cut along their longitudinal axes and then flattened to lie within a single plane. As shown schematically in Fig. 1, tracheal cells at the termini of tracheal branches lie between longitudinal muscles and are
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Fig. 1. In the two schematic diagrams shown in this figure, the relative positions of enteric muscles (longitudinal and circular), tracheal epithelial cells and midgut epithelial cells are indicated between molts (a) and at the time of a molt (b). These cells occupy a plane that lies perpendicular to the basal surface of the midgut and that passes from this basal midgut surface to its luminal surface (at bottom of image). Each cell type (tracheal, muscle and midgut) is delimited by a well-defined basal lamina. The positions of muscle cells remain relatively constant on the basal surface of the midgut, but the undifferentiated, regenerative tracheal cells shift positions as they divide and differentiate. As tracheal cells differentiate at each molt, each develops a cuticular lumen that is reinforced by distinctive taenidial ridges. The differentiating tracheal cells leave the basal surface of the midgut to move into the plane of the midgut monolayer, interdigitating with its endodermal cells (arrowheads). TR, tracheal regenerative cell; T, differentiated trachea or tracheole; TBL, tracheal basal lamina; R, midgut regenerative cell; MBL, midgut basal lamina; E, enterocyte or columnar cell; G, goblet cell; LM, longitudinal muscles; CM, circular muscles. Differentiating midgut epithelial cells are indicated with arrows.
Fig. 2. These scanning electron micrographs of the basal surface of a first instar midgut, immediately after hatching from the egg, show a global view of the arrangement of tracheal, muscle and nerve cells associated with underlying midgut epithelium. Longitudinal muscles extend from right to left of the image; representative longitudinal muscles are marked with arrowheads. Circular muscles run perpendicular to the longitudinal muscles. t, differentiated tracheoles; e, enteric neurons and glia. Regenerative cells lie hidden beneath the surface’s basal lamina. Scale bars = 200 mm in 2a and 20 mm in 2b.
covered with a basal lamina. Circular muscles that are oriented perpendicular to the longitudinal muscles of the gut lie between these tracheal cells and the midgut epithelial cells. To supply oxygen to the differentiated midgut cells, differentiated tracheal cells extend tracheoles from the basal surface of the midgut to positions within the midgut monolayer. Undifferentiated tracheal progenitors (tracheal regenerative cells) are found at the tips of these differentiated tracheal branches (Fig. 1a); a tracheal progenitor cell will eventually differentiate and form a cuticlelined lumen that will transport oxygen to newly differentiated midgut cells (Fig. 1b). To facilitate interpretation of the scanning electron microscope image of the midgut basal surface in Fig. 2 and the immunolabeled whole mounts of the insect midguts shown in Figs. 3–7, these schematic views of the cells in Fig. 1a and b are presented as sections cut perpendicular to the surfaces shown in these figures of midgut whole mounts. The membrane protein neuroglian is expressed on all epithelial cells of the tracheae and tracheoles (Nardi, 1994; immunolabeling not shown). Another membrane protein in the immunoglobulin superfamily, however, is expressed only on cells at the growing tips and nodes of tracheal branches (Nardi, 1990). This protein, fasciclin II, is found on the undifferentiated cells at the termini of the tracheal branches (tracheal regenerative cells or tracheal
progenitors) as well as the enteric neurons and glia that supply the developing midgut epithelium. The fasciclin II-positive cells are contiguous with differentiated, tracheal epithelial cells that are readily identified as cells ensheathing fine cuticular tubes. Distinctive tracheal taenidial ridges line these cuticular tubes and lie perpendicular to the long axis of each tracheal tube. Fasciclin II-positive cells first appear on the basal surface of the midgut epithelium soon after dorsal closure of the midgut (Fig. 3) and are observed throughout larval development. The form assumed by each of these regenerative cells is a function of its stage in the molt cycle (Figs. 4–7). Immediately after a molt, the fasciclin II-positive cells extend long processes along the transverse axis of the midgut (Fig. 4). Halfway between larval molts, tracheal regenerative cells divide and form coherent aggregates (Figs. 5 and 6). Cell divisions within the tracheal and midgut epithelia are scored by labeling mitotic cells with an antibody to phosphohistone H3. A cross section of the larval midgut epithelium labeled with anti-fasciclin II-HRP (Fig. 6, inset) shows the relative positions of tracheal regenerative cells and the longitudinal muscles that also faintly label with anti-fasciclin II. Tracheal regenerative cells are also clearly associated with circular muscles of the Manduca midgut epithelium (Figs. 6 inset, 8 and 9).
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Figs. 3–8. Tracheal regenerative cells are found on the basal surface of the intestinal (midgut) epithelium. In whole mounts of the midgut, the tracheal regenerative cells label with a primary mouse antibody to the surface protein fasciclin II and either (A) an anti-mouse secondary antibody coupled to horseradish peroxidase (HRP) in Figs. 3, 4, 6 and 7 or (B) an anti-mouse secondary antibody coupled to rhodamine (Fig. 5). Longitudinal muscles (representative muscles marked with arrowheads) faintly label with antifasciclin II and extend from top to bottom of each image. Circular muscles run perpendicular to the longitudinal muscles. t, differentiated tracheoles; e, enteric neurons and glia also label with anti-fasciclin II. In Fig. 8, the cells observed in this section of the basal surface of the midgut epithelium have been stained with toluidine blue. (Fig. 3) In Manduca embryos the first tracheal regenerative cells (representative regenerative cells marked with arrows) are evident around 65% development time (DT) on the basal surface of the midgut epithelium. (Fig. 4) Long processes extend from tracheal regenerative cells immediately after hatching of the embryo, at the inception of the first larval stadium. Representative tracheal regenerative cells are marked with arrows. (Fig. 5) In this whole mount of the midgut epithelium fixed halfway through the first larval stadium, the antibody to phosphohistone H3 is marked with fluorescein (green) and anti-fasciclin II is marked with rhodamine (red). Most cell divisions are localized to tracheal regenerative cells. Some cell divisions also occur concurrently within another plane of focus – the plane of the midgut monolayer occupied by midgut regenerative cells (arrows). e, enteric neurons and glia. (Fig. 6) When tracheal regenerative cells halfway through the first larval stadium are labeled with HRP, the aggregates of regenerative cells that lie between the longitudinal muscles (arrowheads) are even more evident. (Inset) This cross section of the larval midgut epithelium also fixed halfway through the first larval stadium and labeled with anti-fasciclin II-HRP shows the positions of tracheal regenerative cells (arrowhead) and longitudinal muscles (lm) and circular muscles (cm) relative to the endodermal monolayer whose luminal surface is indicated with arrows. (Fig. 7) Immediately prior to the molt from first to second instar, tracheal regenerative cells (representative cells marked with arrows) cease dividing and occur as single, dispersed cells. (Fig. 8) At the molt from the second larval instar to the third larval instar, a differentiating tracheole cell generated by tracheal regenerative cells (trc) on the basal surface of the midgut epithelium is shown crossing the circular muscle layer that separates tracheal regenerative cells from midgut cells. Arrow points to growing tip of tracheole cell. One tracheal regenerative cell is dividing (arrowhead). Midgut regenerative cells (double arrow) lie among differentiated goblet cells (gc). lm, longitudinal muscles; cm, circular muscles. Scale bars = 50 mm (Figs. 3–7). Scale bars = 5 mm (Fig. 8 and inset in Fig. 6).
Within these aggregates of regenerative tracheal cells, while some cells differentiate into cuticle-lined tracheoles, others remain undifferentiated on the basal periphery of the midgut epithelium (Figs. 7 and 8). Prior to each larval–larval molt, progeny of tracheal regenerative cells leave the basal surface of the midgut epithelium, extend long processes, differentiating as
they enter the plane of the midgut monolayer and no longer express fasciclin II (Figs. 7 and 8). After the differentiating tracheal cells cross into the plane of the midgut epithelium, the fasciclin II-positive regenerative cells remaining on the basal periphery of the midgut epithelium cease dividing and extend lengthy lateral processes, remaining dispersed
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Fig. 9. Halfway through the third larval stadium, clusters of tracheal regenerative cells (trc) lie beneath a thin basal lamina (bl), adherent to circular muscles (cm), and between longitudinal muscles (lm) of the gut. Differentiated tracheoles (t) extend into the plane of the midgut epithelial cells. Note the highly convoluted basal surfaces of differentiated midgut or intestinal epithelial cells (i.e.) surrounding a midgut or intestinal regenerative cell (irc). Scale = 2 mm.
on the basal surface of the newly expanded midgut monolayer (Fig. 7). Ultrastructural examination of cells on the basal surface of the midgut epithelium reveals the associations of basal laminae with different cell types (Figs. 9 and 10). A relatively thick basal lamina segregates tracheal cells from midgut cells; and a relatively thin basal lamina covers the hemocoel surface of the tracheal regenerative cells and the enteric muscles. In the two-dimensional section of cells shown in Fig. 9, the aggregate of tracheal regenerative cells that lies between two longitudinal muscles and in contact with circular muscles measures about 7 mm. Although obscured by basal laminae that drape over the enteric muscles and tracheal regenerative cells, outlines of some underlying tracheal regenerative cells presumably are represented by the rounded contours of the basal lamina (Fig. 10, arrows). This figure also illustrates the three-dimensional relationship among tracheal regenerative cells and differentiated tracheal branches. The approximate spacing between the longitudinal muscles is 10 mm. The underlying circular muscles (arrowheads) are all oriented perpendicular to the longitudinal muscles. Regenerative cells of tracheal epithelia are destined to supply the respiratory needs of new midgut cells in the rapidly growing endodermal epithelium. Tracheal regenerative cell proliferation begins anew with the start of a new molt cycle, peaking in concert with proliferation of the midgut cells that occurs during each cycle (Fig. 11). A series of fine structural images of differentiating tracheal cells and differentiating midgut cells that intercalate with differentiated midgut cells reveals that tracheal cells occupy inpocketings of the basal surface of the midgut epithelium (Figs. 13,14). As developing midgut cells stretch toward the gut lumen, they are followed in their wakes by tracheal cells that have extended long processes and that have developed cuticle-lined lumens in these processes. A basal lamina occupies the interface between each differentiating tracheal process each regenerative midgut cell that is undergoing
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Fig. 10. In this view of the basal surface of the midgut epithelium from the middle of the third larval stadium, the regenerative cells that occupy the termini of these differentiated tracheal branches (t) lie hidden between the longitudinal muscles (lm) and beneath the thin basal lamina. Two representative longitudinal muscles are labeled. Outlines of tracheal regenerative cells are suggested in several places by rounded contours of the basal lamina (arrows). Outlines of some circular muscles can also be faintly discerned beneath this basal lamina (arrowheads). Scale = 10 mm.
differentiation. Each of these differentiating tracheal processes is embedded in an extracellular matrix that is contiguous with the midgut’s basal lamina. Differentiating tracheal processes associate with differentiating regenerative midgut cells and intercalate with the differentiated midgut cells.
Fig. 11. During each molt cycle, proliferation of tracheal regenerative cells (circles) closely tracks proliferation of midgut regenerative cells (triangles). A labeled nucleus represents one that labels with anti-phosphohistone H3. Each point represents the average standard error of 10 counts (n = three larvae, 3 or 4 counts from each) within a field measuring 0.66 mm2.
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Figs. 12–14. This series of images depict the association of midgut regenerative cells, tracheoles (t), and basal laminae during addition of tracheoles to the differentiated midgut epithelium. The basal surface of the midgut epithelium is shown halfway through the second larval stadium (Fig. 12), shortly before the molt from the second to the third larval instar (Fig. 13), and immediately after (Fig. 14) the molt from the second to third instar. In each of the three images, two intestinal regenerative cells (irc) lie adjacent to goblet cells whose central cavities (cc) are lined with microvilli. Goblet cells have distinctive nuclei (nu) and highly convoluted basal membranes (bm). Tracheoles (t) and basal laminae occupy positions (arrows) adjacent to the midgut regenerative cells (irc). A basal lamina (*) and circular muscles (cm) lie over the midgut epithelium. Midgut lumen is at top of figures. Scale = 5 mm.
4. Discussion 4.1. Distribution of tracheal regenerative cells With an antibody marker that recognizes the membrane protein fasciclin II, a covert spacing pattern of tracheal regenerative cells has been revealed on the basal surface of the midgut. The extension of long processes from these tracheal regenerative cells (Figs. 3, 4, 6 and 7) and their interactions with adjacent tracheal cells as well as with tracheal cells that are two or more cell diameters away could account for the regular distribution of these regenerative cells over the basal surface of the insect midgut. 4.2. Distribution of differentiated tracheole cells The regular spacing pattern of differentiated tracheole cells that associate with differentiating and differentiated midgut epithelial cells (Fig. 2) is mediated by a communication among differentiating tracheal and midgut cells that does not require direct cell–cell contact. As tracheole cells intercalate with midgut cells, the two cell types do not come in direct contact but meet at an extracellular matrix (basal lamina) interface (Figs. 13 and 14). In epidermal epithelia that have been deprived of their tracheoles, basal processes extend in the direction of the existing tracheole cells and recruit these cells as an oxygen supply. The movement and distribution of differentiated tracheole cells within these developing insect epidermal epithelia apparently are not determined intrinsically but seem to be imposed by the epidermal cells with which they supply oxygen (Nardi, 1984; Wigglesworth, 1959). 4.3. Coordination of growth of different cells during organogenesis The proliferation of tracheal regenerative cells of Manduca is closely coupled with proliferation of nearby regenerative cells of midgut epithelia. These regenerative cells respectively reside within an ectodermal epithelium and an endodermal epithelium that divide, grow, and differentiate in concert (Figs. 8, 11–14). During organogenesis of salivary gland epithelium, interactions with other surrounding tissues such as blood vessels, nerves and mesenchymal cells maintain the populations of epithelial regenerative cells that are required for development and regeneration of mouse salivary epithelium. The peripheral parasympathetic innervation of salivary epithelia maintains the progenitor cell population of salivary epithelia in its undifferentiated state (Knox
et al., 2010). Coordinated growth of tracheal-midgut in Manduca may represent another example of a branching epithelium (tracheal) maintaining the undifferentiated state of midgut regenerative cells. In the literature on insect regenerative cells, the terms stem cells, regenerative cells and progenitor cells have often been used interchangeably. Stem cells are cells that can concurrently generate such differentiated midgut cells while retaining their proliferative state. Although regenerative cells of insect midgut epithelia have conventionally been referred to as ‘‘stem’’ cells and compared with stem cells that have been described in adult midgut epithelia of Drosophila (Hakim et al., 2010; Nardi et al., 2010), the rigorous genetic lineage labeling of epithelial cells that has definitively identified a subset of Drosophila regenerative cells as stem cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006) has not been possible with insects other than Drosophila. To adjust to the constantly changing requirements of a rapidly growing epithelium, stem cells of different tissues must be able to differentiate at the right time and right place. The cues directing their proliferation and differentiation have been postulated to reside not only in the stem cells themselves with minimal input from neighboring cells (Wilson and Kotton, 2008) but also with neighboring cells referred to as stem cell ‘‘niches’’ that supply the cues regulating the behavior of the stem cells (Mathur et al., 2010). The coordinated development of tracheal regenerative cells and midgut regenerative cells described in this manuscript provides evidence that tracheal cells that adhere to the basal lamina surface of the midgut monolayer not only occupy the appropriate location for a presumed midgut stem cell niche but also clearly proliferate and differentiate in concert with putative midgut stem cells. Acknowledgments The authors gratefully acknowledge the helpful suggestions of two anonymous reviewers. References Baldwin, K.M., Hakim, R.S., 1991. Growth and differentiation of the larval midgut epithelium during molting in the moth, Manduca sexta. Tissue and Cell 23, 411– 422. Hakim, R.S., Baldwin, K., Smagghe, G., 2010. Regulation of midgut growth, development, and metamorphosis. Annual Review of Entomology 55, 593–608. Knox, S.M., Lombaert, I.M.A., Reed, X., Vitale-Cross, L., Gutkind, J.S., Hoffman, M.P., 2010. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science 329, 1645–1647.
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