Connective Tissue Sheath Research Articles

Skin and hair follicle fibroblasts differentially express POMC peptides, receptors and associated processing convertases during the hair growth cycle and in vitro– implications for fibroblast topographic differentiation in skin

Mesenchymal cells are involved in reciprocal mesenchymal–epithelial interactions during development and growth of skin and its appendages. Fibroblasts exhibit topographic differentiation and so constitute a highly diverse family of cells with distinct and characteristic traits. This heterogeneity is also seen in the skin where hair growth inductive fibroblasts called follicular papilla (FP) fibroblasts are distinct from fibroblasts of the connective tissue sheath (CTS), and both, in turn, are distinct from peripheral interfollicular dermal fibroblasts (DFs). POMC peptides and their cognate receptors are expressed variably by several skin cells types, including fibroblasts. However, it is not clear how the POMC system is regulated in different skin fibroblasts populations. We characterized the expression of the POMC peptide family and their receptors and pro‐hormone convertases (PCs) in human haired scalp during the hair growth cycle and in matched sets of DF, FP and CTS fibroblasts cultured from normal adult male scalp. Expression of POMC peptides, PC 2 and µ‐OR in FP fibroblasts was highest during anagen. By contrast, β‐end, 7B2 and PC1 were broadly undetectable during anagen but expression levels increased considerably during the apoptosis‐driven catagen phase. Matched sets of cultured FP and DF fibroblasts showed similar protein expression levels of α‐MSH, ACTH, PC2, 7B2, µ‐OR and MC1‐R. However, FP cells expressed higher levels of PC1 and β‐end peptide and higher µ‐OR mRNA levels than DF cells. Thus, follicular and interfollicular fibroblasts represent heterogeneous subpopulations that are likely to respond variably to POMC peptides.

Melanocortin receptors in fibroblastic cell types of the skin –in vitro and in vivo expression and functional relevance

In contrast to the well‐established role of melanocortins on melanocyte function, the expression and relevance of melanocortin receptors (MC‐Rs) in skin fibroblasts is incompletely understood. We recently showed that human dermal fibroblasts derived from foreskin express functional MC‐1Rs which mediate an inhibitory action of α‐melanocyte‐stimulating hormone (α‐MSH) on collagen metabolism (Böhm et al., J Biol Chem 2004, in press). Here, we show by RT‐PCR, immunofluorescence, immunohistochemistry and immune electron microscopy that MC‐1R expression is conserved in vitro and in vivo in additional human fibroblastic cell types of the skin including adult dermal fibroblasts, fibrosarcoma cells, connective tissue sheath fibroblasts and dermal papilla cells of the hair follicle. In vitro expression of MC‐1R declines as a matter of cellular senescence in dermal fibroblasts. Interestingly, α‐MSH inhibits the inductive effect of interferon‐γ, an important proinflammatory mediator in inflammatory and fibrotic skin diseases, on expression of adhesion molecules such as intercellular adhesion molecule‐1 in human dermal fibroblasts. These data point towards an additional biological role of α‐MSH as a modulator of inflammatory reactions in the connective tissue compartment of the skin.

Fine Structure of the Hemopoietic Tissues in the Mealworm Beetle, Tenebrio molitor

ABSTRACTThe fine structure of the hemopoietic tissue and its detailed reticular organization in the mealworm beetle, T. molitor were examined using light and scanning electron microscopes. The major hemopoietic tissues in the abdomen were located on the upper surface of the dorsal diaphragm which continuous over the ventral wall of the heart. Histologic characteristics of this hemopoietic tissues are dense clusters of cells. They are irregular in outline and are not surrounded by any connective tissue sheath. The hemopoietic tissue of this insect is consisted of three cellular components which are the reticular cells, hemocytic stem cells and several kinds of mature hemocytes. The reticular cells had numerous cytoplasmic processes and forming a complex network. The stem cells give rise to differentiating hemocytes of the different cell lineages. Mature hemocytes within this hemopoietic tissue are originated from the stem cells and differentiated into several types of hemocytes including prohemocytes, plasmatocytes, and granulocytes.

Noninvasive Assessment of Subcutaneous Fibrous Tunnel Patency of Fractured Ventriculoperitoneal Shunt by Ultrasonography

Shunt malfunction caused by fracture of the distal tube is a common complication of ventriculoperitoneal shunts. Functionality of the fractured distal shunt tube must be evaluated before its removal, because a connective tissue sheath or subcutaneous fibrous channel around the peritoneal catheter may be patent. Although an accurate assessment of shunt function is made by invasive tests such as radioisotope or radiopaque injection via tapping the reservoir, these methods have risks. In our case, patency of a subcutaneous fibrous tunnel that had developed around the distal catheter before the fracture was demonstrated noninvasively by real-time ultrasonography.

Impact of inflammatory disease on the nerve microenvironment

The peripheral nervous system (PNS) has a distinctive anatomic organization that may contribute to its unique physiology as well as vulnerability to certain types of disease. The enclosure of bundles of myelinated and unmyelinated fibers by a semi-rigid and semi-permeable connective tissue sheath creates physiological compartments that are served by transperineurial and intrafascicular vessels but are devoid of lymphatic channels. Unlike tissues drained by lymphatics, interstitial fluid in the PNS flows from proximal to distal regions, propelled by positive endoneurial fluid pressure (EFP). There is a proximo-distal gradient from the dorsal root ganglia and roots to the extraganglionic nerves where the pressure is significantly lower [1]. EFP in the ganglia and roots is nearly twice as high as in the extraganglionic nerves. From the higher pressure in spinal fluid to the relatively low pressure in the sciatic fluid, a downward gradient continues until the nerve ending. At that point, the perineurium terminates as an ensheathing sleeve, and the normal slightly negative interstitial fluid pressure of the extraneural interstitium prevails. This gradient continues into the lymphatic channels where negative fluid pressure is greatest. The endoneurial microenvironment is sensitive to

Microscopic anatomy of the infraorbital canal, nerve, and foramen

Objective We performed this study to investigate the morphologic and topographic anatomy, and variations of the infraorbital canal (IOC), infraorbital nerve (ION), and infraorbital foramen (IOF). Study design and setting This study was performed at the Anatomy Department of Ankara University School of Medicine between 1999 and 2000. The morphometry of these anatomic areas on cadaver heads were studied microscopically. The morphologic and topograpic anatomies of IOC, ION, and IOF were carried out microscopically with ×25 to ×40 magnification on 10 cadaver heads (20 sides). Results We found the shape of the IOF as oval in 30% (6 sides), round in 40% (8 sides), and semilunar in 30% (6 sides) of the cadaver heads. We detected the IOF as single in 90% (18 sides), double in 5% (1 side), and triple in 5% (1 side) of the cases. We determined a complete roof (we called it as “canal”) in 50% of cases and groove plus canal in 50% of the heads. The mediolateral relationship of the vessels to the nerve while emerging from the IOF was investigated, and the artery was always found in a mediosuperior position and the vein was inferior to it. In all specimens, the ION was found to be consisting of 3 to 8 fascicles interwoven with the artery and the vein in the loose connective tissue sheath. Conclusion A detailed knowledge of the anatomic morphometry of this area is necessary for a surgeon while performing maxillofacial surgery and regional block anesthesia. Anatomic variations on this area may take place and a surgeon must take this into consideration so as to increase the surgical success.

Microscopic anatomy of the infraorbital canal, nerve, and foramen.

We performed this study to investigate the morphologic and topographic anatomy, and variations of the infraorbital canal (IOC), infraorbital nerve (ION), and infraorbital foramen (IOF). This study was performed at the Anatomy Department of Ankara University School of Medicine between 1999 and 2000. The morphometry of these anatomic areas on cadaver heads were studied microscopically. The morphologic and topograpic anatomies of IOC, ION, and IOF were carried out microscopically with x 25 to x 40 magnification on 10 cadaver heads (20 sides). We found the shape of the IOF as oval in 30% (6 sides), round in 40% (8 sides), and semilunar in 30% (6 sides) of the cadaver heads. We detected the IOF as single in 90% (18 sides), double in 5% (1 side), and triple in 5% (1 side) of the cases. We determined a complete roof (we called it as "canal") in 50% of cases and groove plus canal in 50% of the heads. The mediolateral relationship of the vessels to the nerve while emerging from the IOF was investigated, and the artery was always found in a mediosuperior position and the vein was inferior to it. In all specimens, the ION was found to be consisting of 3 to 8 fascicles interwoven with the artery and the vein in the loose connective tissue sheath. A detailed knowledge of the anatomic morphometry of this area is necessary for a surgeon while performing maxillofacial surgery and regional block anesthesia. Anatomic variations on this area may take place and a surgeon must take this into consideration so as to increase the surgical success.

The dinosaurian origin of feathers: perspectives from dolphin (Cetacea) collagen fibers.

The early origin of birds is a hotly disputed debate and may be broadly framed as a conflict between paleontologists and ornithologists. The paleontological emphasis has shifted from Archaeopteryx and its origins to recent finds of Cretaceous birds and "feathered" dinosaurs from China. The identification of alleged feathers has, however, relied principally on the visual image. Some workers have interpreted these integumentary structures as collagen fibers. To test the latter hypothesis, using light microscopy, collagen from the hypodermis (blubber) and subdermal connective tissue sheath was examined from a dolphin that had been buried for a year as part of an experiment. Within the blubber, toward the central thicker parts of the material, the collagen fibers had compacted and the three-dimensional latticework of normal blubber had more or less collapsed. Chromatographic analysis of the blubber revealed pronounced oxidation of the unsaturated lipids, probably accounting for the collapse of the latticework. Fibers normally bound together in bundles became separated into individual fibers or smaller bundles by degradation of the glue-like substance binding them together. These degraded collagen fibers show, in many instances, feather-like patterns, strikingly reminiscent of many of those identified as either "protofeathers" or "modern" feathers in dromaeosaurid dinosaurs. The findings throw serious doubt on the virtually complete reliance on visual image by supporters of the feathered dinosaur thesis and emphasize the need for more rigorous methods of identification using modern feathers as a frame of reference. Since collagen is the main fiber type found in most supporting tissues, the results have wide implications regarding the degradation and fossilization of vertebrate integument, such as that of the ichthyosaurs, dinosaurs and birds.

Protease-Activated Receptor-1 (Thrombin Receptor) Is Expressed in Mesenchymal Portions of Human Hair Follicle

Protease nexin-1, a serine protease inhibitor, is expressed specifically in the dermal papilla (DP) of anagen hair follicles and is suggested to be one of the modulators of the cyclic growth of hair follicles. Accumulating evidence has shown that protease nexin-1 plays its biologic role by inhibiting thrombin action in various systems other than the hair follicle. Thrombin has various physiologic functions including blood coagulation cascade, mostly via activation of protease-activated receptors (PAR). In this study, we investigated the expression of PAR mRNA using RT-PCR in dissected human hair follicles. We showed that PAR-1 mRNA was expressed specifically in the mesenchymal portions, including DP and connective tissue sheath, of anagen hair follicles. Furthermore, immunoreactivity for PAR-1 was detected in the DP and lower portion of connective tissue sheath in the anagen and catagen phases and in the DP of telogen hair follicles. Because only a pharmacologic level (100 nM) of thrombin significantly stimulated cell proliferation and DNA synthesis of the cultured dermal papilla cells, thrombin does not seem to have a mitogenic effect on dermal papilla cells physiologically. These results raise the possibility that thrombin is involved in the cyclic hair growth through its receptor of PAR-1.

Communication network in the follicular papilla and connective tissue sheath through gap junctions in human hair follicles.

Epithelial-mesenchymal interactions play a crucial role in the induction of life-long cyclic transformations of hair follicles. Many studies have already demonstrated several candidates for the soluble factors secreted from the mesenchymal components of the hair follicle, i.e. the follicular papilla (FP) and connective tissue sheath (CTS), which may be responsible for hair cycling. In this paper, we focused on cell-cell contact between FP cells (FPCs), between CTS cells (CTSCs), and between FPCs and CTSCs that may allow these mesenchymal components to function as a syncytium during hair cycling. Electron microscopic examination of the FP and the CTS obtained from human scalp revealed a tri-lamellar structure of the plasma membranes, which is a characteristic of gap junctions at the cell-cell contacting area. The immunohistochemical study with anticonnexin 43 Ab using a confocal laser scanning microscope demonstrated numerous spotted positive signals scattered throughout the FP. In the CTS, spotted positive signals were arranged linearly along the basement membrane of the hair follicle. In particular, these positive spots were aggregated in the transitional region between the FP and the CTS. By Western blot analysis of total protein extracts from the cultured FPCs and neonatal human dermal fibroblasts using anticonnexin 43 antibody, a positive band corresponding to connexin 43 was detected at 43 kDa on both the FPC lane and fibroblast lane. These findings suggest that the FP and the CTS form a communicating network through gap junctions, which may play a role in controlling the dynamic structural changes of hair follicles during hair cycling.

Plasticity and Cytokinetic Dynamics of the Hair Follicle Mesenchyme: Implications for Hair Growth Control

The continuously remodeled hair follicle is a uniquely exploitable epithelial-mesenchymal interaction system. In contrast to the cyclical fate of the hair follicle epithelium, the dynamics of the supposedly stable hair follicle mesenchyme remains enigmatic. Here we address this issue using the C57BL/6 hair research model. During hair growth, increase in total follicular papilla size was associated with doubling of papilla cell numbers, much of which occurred before intra-follicular papilla cell proliferation, and subsequent to mitosis in the proximal connective tissue sheath. This indicates that some papilla cells originate in, and migrate from, the proliferating pool of connective tissue sheath fibroblasts. Follicular papilla cell number and total papilla size were maximal by anagen VI, but intriguingly, decreased by 25% during this period of sustained hair production. This cell loss, which continued during catagen, was not associated with intra-follicular papilla apoptosis, strongly indicating that fibroblasts migrate out of the late anagen/early catagen papilla and re-enter the proximal connective tissue sheath. Low-level apoptosis occurred only here, along with the "detachment" of cells from the regressing connective tissue sheath. Thus, the hair follicle mesenchyme exhibits significant hair cycle-associated plasticity. Modulation of these cell interchanges is likely to be important during clinically important hair follicle transformations, e.g. vellus-to-terminal and terminal-to-vellus during androgenetic alopecia.

Electron Microscopy Study of Anterior Neuropituitary of the Sterlet Acipenser ruthenus

Structural organization of sterlet anterior neuropituitary (ANP), a homologue of the median tubercle of Tetrapoda, was studied by method of electron microscopy. The ANP was conventionally divided in three zones in dorsoventral direction. Neurosecretory fibers (NsF) were concentrated mainly in the central and in the external zones. Terminals of NsF are directly adjacent to the basal membrane (BM) of the brain vascular sheath to form a morphological substrate for release of neurohormones into the pericapillary space. The presence of invaginations of the brain connective tissue sheath, which are in a close contact with NsF, indicates an increase of the area of neurosecretory contacts. Neurosecretory granules (NsG) of various shape and size are revealed in NsF and their terminals. Comparison of light-microscopy immunohistochemical data with localization of NsF terminals containing NsG of different diameters allows reconstructing distribution of neurosecretory elements of various ergicity in the ANP. Patterns of co-localization of various biologically active substances were revealed in the same varicose dilatations or terminals. Detection of typical synaptic contacts in the sterlet ANP indicates possibility of the cholinergic pituitary regulation, alongside with the peptide- and monoaminergic regulation.

Using Activa™ TG-RM to bind beef muscles after removal of excessive seam fat between the m. longissimus thoracis and m. spinalis dorsi and heavy connective tissue from within the m. infraspinatus

Binding beef muscles together after removing excess seam fat and heavy connective tissue was studied. Ribeye rolls and top blade roasts were fabricated from corresponding sides of 24 beef carcasses with cuts from one side serving as treated (bonded) and cuts from the other side serving as controls. For the ribeye rolls, the bonded treatment consisted of removing the kernel fat that lies between the m. spinalis dorsi and the m. longissimus thoracis. For the top blade roasts, the bonded treatment consisted of removing the heavy connective tissue sheath after the m. infraspinatus was cut open lengthwise. To bind muscles together after removal of fat or connective tissue, a dry-sprinkle mixture of Activa™ TG-RM (featuring the enzyme, transglutaminase) along with 5% sodium caseinate by weight was applied to the surfaces to be bonded (approximately 0.25% by weight was used). Steaks were cut for raw and cooked proximate analysis and energy assessment and for in-home and simulated retail case consumers studies. Bonded steaks were higher in dissectible lean, lower in energy from fat and total energy for all cuts, and higher in protein for the raw top blade, raw ribeye, and cooked ribeye steaks. Bonded ribeye steaks were lower in chemical and dissectible fat and higher in moisture. Consumers rated bonded ribeye steaks higher than control steaks for leanness and bonded top blade steaks higher for all raw appearance and cooked palatability traits. When asked to purchase bonded or control steaks from a simulated retail case, consumers purchased bonded ribeye steaks because of leanness and bonded top blade steaks because of perceived greater marbling compared to control steaks. Modifying subprimals to remove excessive seam fat and connective tissue resulted in improved physical and chemical composition of steaks and improved many visual and eating quality characteristics.

Relationship between the descending branch of the inferior gluteal artery and the posterior femoral cutaneous nerve applicable to flap surgery.

Flap surgery in the distal part of the gluteal region has to deal with a lack of detailed descriptions of the inferior gluteal artery and the posterior femoral cutaneous nerve. The existing papers are mainly clinical studies, based on low numbers of observations. Our study includes 118 cadaveric gluteal regions. The descending branch was present in 91% and gave rise to a cutaneous branch. When the descending branch was absent, this cutaneous branch came from the medial or lateral femoral circumflex artery or as a perforator of the deep artery of the thigh. The posterior femoral cutaneous nerve was found in a common sheath of connective tissue with the descending branch of the inferior gluteal artery in 72% of cases. Nerve loops around the vessel are present in 29%. Our results show that a cutaneous or fasciocutaneous flap, either local or free, in this region can be reliably lifted on a cutaneous branch of the descending branch of the inferior gluteal artery without loss of sensitivity. However, the close relationship of the artery and nerve limits the arc of rotation in the case of a local flap.

Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice.

Airway hyperresponsiveness, airway inflammation, and reversible airway obstruction are physiological hallmarks of asthma. These responses are increasingly being studied in murine models of antigen exposure and challenge, using whole body plethysmography to noninvasively assess airway hyperresponsiveness. This approach infrequently has been correlated with indexes of airway hyperresponsiveness measured by invasive means. Furthermore, correlation with quantitative histological data for tissue infiltration by inflammatory and immune cells, particularly in the wall of airways, during daily airway challenge is lacking. To address these uncertainties, we used C57BL/6 mice that were immunized with ovalbumin or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle 8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14-22. We assessed airway hyperresponsiveness to methacholine noninvasively on days 14, 15, 18, or 22; we studied the same mice 24 h later while they were anesthetized for invasive analyses of airway hyperresponsiveness. Plasma total IgE concentration was significantly higher in the ovalbumin-treated mice compared with the vehicle-treated mice, but this did not correlate with eosinophil number. Peak airway hyperresponsiveness measured by either approach correlated early during daily antigen challenge (days 14 and 15), but this correlation was lost later during subsequent daily antigen challenges (days 18 and 22). On days 14 and 15, peak airway hyperresponsiveness correlated with transmigration of neutrophils and macrophages, but not lymphocytes, in the peribronchovascular connective tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between noninvasive and invasive measures of peak airway hyperresponsiveness was inconsistent.

Involvement of Transforming Growth Factor-β2 in Catagen Induction During the Human Hair Cycle

The involvement of transforming growth factor-beta isoforms in the induction of the regressing phase (catagen) of human hair follicles were examined in vivo. In the growing phase (anagen), transforming growth factor-beta1 was detected at the hair cuticle and connective tissue sheath. Transforming growth factor-beta2 was restricted to the outermost cell layer of the outer root sheath. Transforming growth factor-beta3 was observed in the precortical hair matrix of anagen hair follicles. During the anagen-catagen transition phase, strong transforming growth factor-beta2 immunoreactivity appeared in the lower bulb matrix cells adjacent to the dermal papilla. In addition, transforming growth factor-beta2 and transforming growth factor-beta type II receptor were colocalized in the regressing epithelial strands, where terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive apoptotic cells were also found. Transforming growth factor-beta1 and transforming growth factor-beta3 were mostly negative in the strand. Using an organ culture system, we investigated whether transforming growth factor-beta2 and its antagonists affected the transition process. Elongation of hair was significantly suppressed by transforming growth factor-beta2. Next, a neutralizing antibody and fetuin, a potent transforming growth factor-beta antagonist was tested. In the presence of the antibody as well as fetuin, hair follicles were markedly elongated in a concentration-dependent manner. These results strongly suggest that transforming growth factor-beta2 plays an essential part in the induction of the catagen phase of the human hair cycle.

Elucidation of the Mechanism of Acetylcholinesterase Staining of Vagus Nerve Tissue

An attenuated total reflectance Fourier transform infrared (ATR-FTIR) study was undertaken to determine the mechanism of a new staining technique on the structural organization of upper vagus nerve tissue. This new blue phosphate-buffered copper sulfate/acetylthiocholine iodide/glycine solution turned nerve tissue white in color. From the ATR-FTIR data, it became apparent that this was a stepwise process involving conformational changes of the nerve tissue components. Initially, the conformation of the saccharide components of the connective tissue sheath were altered by the copper/glycine complex, causing the sheath to swell. Significant quantities of copper were confirmed to be present in the stained tissue by inductively coupled plasma (ICP) analysis. This swelling then facilitated the diffusion of the lipotropic acetylthiocholine salts to and subsequent interaction with the lipid/protein molecules in the myelin sheath. It is hypothesized that this structural alteration of the myelin sheath produced a more dense protein layer, resulting in the white coloration of the nerve tissue. (The J Histotechnol 25:85, 2002)Submitted March 5, 2002; accepted April 5, 2002

Elucidation of the Mechanism of Acetylcholinesterase Staining of Vagus Nerve Tissue

An attenuated total reflectance Fourier transform infrared (ATR-FTIR) study was undertaken to determine the mechanism of a new staining technique on the structural organization of upper vagus nerve tissue. This new blue phosphate-buffered copper sulfate/acetylthiocholine iodide/glycine solution turned nerve tissue white in color. From the ATR-FTIR data, it became apparent that this was a stepwise process involving conformational changes of the nerve tissue components. Initially, the conformation of the saccharide components of the connective tissue sheath were altered by the copper/glycine complex, causing the sheath to swell. Significant quantities of copper were confirmed to be present in the stained tissue by inductively coupled plasma (ICP) analysis. This swelling then facilitated the diffusion of the lipotropic acetylthiocholine salts to and subsequent interaction with the lipid/protein molecules in the myelin sheath. It is hypothesized that this structural alteration of the myelin sheath produced a more dense protein layer, resulting in the white coloration of the nerve tissue. (The J Histotechnol 25:85, 2002)Submitted March 5, 2002; accepted April 5, 2002

Symposium on Peripheral Nerve and Neuropathies: a festschrift to celebrate the contribution of P. K. Thomas as Editor of the Journal of Anatomy, 1990–2001

Symposium on Peripheral Nerve and Neuropathies: a festschrift to celebrate the contribution of P. K. Thomas as Editor of the <i>Journal of Anatomy</i>, 1990–2001

Signals that determine Schwann cell identity.

While the signals that direct neural crest cells to choose the glial lineage and generate Schwann cell precursors are still obscure, studies both in vivo and in vitro indicate that the survival and differentiation of these cells to form Schwann cells is regulated by at least two signals, neuregulin-1 and endothelin. We know little about the signals that cause some immature Schwann cells to choose myelin differentiation, while other cells form non-myelinating cells. Three transcription factors, Sox-10, Oct-6 and Krox-20, have been shown to play key roles in the Schwann cell lineage. The transcription factor Krox-20 has been identified as a major target of the signals that induce myelin differentiation. Gene transfer experiments in vitro show that this protein has a remarkable ability to promote a large number of phenotypic changes in immature Schwann cells that characterize the transition of these cells to myelinating cells. Furthermore, Krox-20 shows important functional interactions with neuregulin and transforming growth factor beta (TGFbeta), two factors that have been implicated in the regulation of myelination in postnatal nerves. Another signal of importance in developing peripheral nerves, Desert Hedgehog, secreted by Schwann cells directs formation of the peripheral nerve connective tissue sheaths. Ongoing gene screening experiments are likely to reveal new genes of interest in this system.