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HEMATOPOIESIS
From the Instituto de Biología Molecular y
Celular del Cáncer, Centro de Investigación del
Cáncer, Servicio de Citometría, and Servicio de
Anatomía Patológica, Universidad de Salamanca, Spain.
The stem cell factor c-kit signaling pathway (SCF/c-kit)
has been previously implicated in normal hematopoiesis, melanogenesis, and gametogenesis through the formation and migration of
c-kit+ cells. These biologic functions are also
determinants in epithelial-mesenchymal transitions during embryonic
development governed by the Snail family of transcription factors. Here
we show that the activation of c-kit by SCF specifically induces the
expression of Slug, a Snail family member. Slug mutant mice have a
cell-intrinsic defect with pigment deficiency, gonadal defect, and
impairment of hematopoiesis. Kit+ cells derived from Slug
mutant mice exhibit migratory defects similar to those of
c-kit+ cells derived from SCF and c-kit mutant mice.
Endogenous Slug is expressed in migratory c-kit+ cells
purified from control mice but is not present in c-kit+
cells derived from SCF mutant mice or in bone marrow cells from W/Wv mice, though Slug is present in spleen
c-kit+ cells of W/Wv (mutants expressing c-kit
with reduced surface expression and activity). SCF-induced migration
was affected in primary c-kit+ cells purified from
Slug Hematopoiesis is a lifelong process
responsible for replenishing hematopoietic progenitor cells and mature
blood cells from a pool of pluripotent, long-term reconstituting stem
cells.1 The daily turnover of blood cells in a normal
adult is tightly regulated, involving, in part, a complex interaction
between soluble and membrane-bound stimulatory and inhibitory cytokines
and their corresponding receptors.2 The molecular cloning
of these hematopoietic growth factors and their receptors has
been instrumental in delineating the pathways that lead from a
single hematopoietic stem cell to the various terminally differentiated
cells in the hematopoietic system.
Although a number of cytokines have effects on progenitor and stem
cells in vitro or in vivo, one cytokine discovered in the early 1990s,
c-kit ligand, appears to have unique and nonredundant activities on
primitive/progenitor cells.3 The in vivo roles of c-kit
are well understood because of the existence of mutant mice in which
genes encoding the receptor and its respective ligand are defective.
Mutations in the c-kit receptor and its ligand are well represented by
numerous white-spotting (W) and Steel (Sl) mutant alleles,
respectively. Mice afflicted with mutations at the W locus were
originally identified, as the name implies, by the presence of white
spots on pigmented mice.4 Detailed examination of the mice
showed that the mutation was pleiotropic. The mice also had defects in
germ cell development and in hematopoiesis (characterized by macrocytic
anemia). In 1988 it was shown that the W locus encoded a tyrosine
kinase receptor known as c-kit.5,6
Many years after the discovery of the W locus, a mutation in mice that
had a phenotype virtually identical to W mice was
identified.7 Because mutations on 2 different chromosomes
had the same complex phenotype that affects pigmentation, germ cells,
and hematopoiesis, researchers hypothesized that there would be some
relationship between the proteins encoded at these 2 loci.8 In 1990 the protein encoded at the Sl locus was
identified and named as mast cell growth factor, stem cell factor
(SCF), and c-kit ligand.9-13
Although the primary function of SCF in early hematopoiesis might
be to induce the growth of quiescent progenitor/stem cells through
synergistic interactions with other early-acting cytokines, ample
evidence indicates that SCF, in the absence of other cytokines, selectively promotes viability rather than proliferation of primitive murine progenitor cells.14 Although SCF/c-kit migratory
pathways and developmental fates are well documented, less is known
about the molecular mechanisms that provide biologic specificity to the
SCF/c-kit signaling pathway in the formation and migration of
c-kit+ cells.
These biologic events controlled by the SCF/c-kit signaling
pathway are reminiscent of those that take place in
epithelial-mesenchymal transitions during mammalian development.
Indeed, the process of mesoderm formation involves the acquisition of
migratory properties and cell fate determination. These
epithelial-mesenchymal transitions are controlled by a conserved
family of zinc-finger proteins, the Snail family.15-17 The
Drosophila gene snail is critical for mesoderm
formation and cell fate determination.18 The related murine Snail and Slug genes have also been
proposed to participate in mesoderm formation and cell
migration.19-21
In this study we have investigated the relationship between the
SCF/c-kit signaling pathway and the Snail family of proteins. We have
found that the SCF/c-kit signaling pathway specifically induces the
expression of a member of the Snail gene family of zinc-finger
transcription factors, Slug gene, in natural and
artificially engineered c-kit+ cells. Analysis of a
targeted null mutation that deleted all Slug coding sequences revealed
that Slug mutant mice, like c-kit and SCF-defective
mice,22 have a complex phenotype including pigmentation,
gonadal defects, and hematopoietic defects. Long-term transplantation
experiments demonstrated that the defect in Slug mutant mice, in which
Slug Cell culture
Mice
Phenotypic analysis of the cells Cell morphology was analyzed according to standard criteria. Single-cell suspensions were prepared from individual tissues, including bone marrow, spleen, thymus, and peripheral blood, by standard procedures.24 Approximately 1 × 106 cells were used for most stainings. Cells were immunophenotyped with the following antibodies: phycoerythrin (PE)-conjugated TER119 (Ly-76, a monoclonal antibody recognizing an antigen expressed on erythroid cells from erythroblasts to erythrocyte; PE-CD4, PE-Gr-1, PE-CD117; PE-CD19; PE-B220; fluorescein isothiocyanate (FITC)-conjugated CD8, FITC-IgM, FITC-MacI (all from PharMingen, San Diego, CA). Cells, suspended in Ca++-/Mg++-free phosphate-buffered saline (PBS) supplemented with 1% (vol/vol) FBS, were labeled with each antibody (approximately 1 µg/106 cells) for 30 minutes on ice. Cell fluorescence was analyzed with the FACScan flow cytometer (Becton Dickinson, Bedford, MA). Cells incubated with appropriately labeled isotype controls (PharMingen) were used to gate the nonspecific fluorescence signal. Before analysis, mature red cells were depleted by hypotonic lysis (0.38% ammonium chloride for 15 minutes on ice). Background controls were treated identically except that primary antibodies were omitted. Cells were initially gated by size and by scatter to identify live cells. In some experiments, cell viability was assessed by propidium iodide (5 µg/mL; Sigma) (in flow cytometry) exclusion.Cell purification Mononuclear spleen suspensions were prepared by cutting the spleens into small fragments in 5 mL Ca++-/Mg++-free PBS containing 10% (vol/vol) FBS and by passing the cell suspension through progressively smaller needles. Marrow cells were flushed from the femurs with a syringe containing 2 mL PBS-10% FBS. Marrow and spleen light-density mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (P = 1.077 g/mL) at 800g for 20 minutes at room temperature. For cell sorter separation, cells were incubated with c-kit-PE and c-kit+ cells sorted by fluorescence-activated cell sorting (FACS) (FACstar; Becton Dickinson). Sorted cells were then re-analyzed for purity with the cytometer.Reverse transcription-polymerase chain reaction To analyze the expression of Slug and Snail in cell lines and in purified c-kit+ cells, reverse transcription (RT) was performed according to the manufacturer's protocol in a 20-µL reaction containing 50 ng random hexamers, 3 µg total RNA, and 200 U Superscript II RNase H. reverse transcriptase (Gibco/BRL, Paisley, United Kingdom). Thermocycling parameters for polymerase chain reaction (PCR) and the sequences of the specific primers were as follows: mSlug, 30 cycles at 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, sense primer 5'-GCCTCCAAAAAGCCAAACTA-3', antisense primer 5'-CACAGTGATGGGGCTGTATG-3'; mSnail, 30 cycles at 95°C for 2 minutes, 60°C for 2 minutes, and 72°C for 2 minutes, sense primer 5'-CAGCTGGCCAGGCTCTCGGT-3', antisense primer 5'-GCGAGGGCCTCCGGAGCA-3'. Amplification of -actin RNA served
as a control to assess the quality of each RNA sample. Sequences of the
internal probes were as follows: mSlug, 5'-GACACACATACAGTGATTATTTCC-3'; mSnail, 5'-TGCAACCGTGCTTTTGCTGACCGCTCCAAC-3'.
In situ hybridization Digoxigenin-labeled sense and antisense RNA probes for Slug were synthesized from the BR1.4 plasmid.19 In situ hybridization was carried out using the protocol described by Latham et al.25RNA analysis Total cytoplasmic RNA (10 µg) was glyoxylated and fractionated in 1.4% agarose gels in 10 mM Na2HPO4 buffer (pH 7.0). After electrophoresis, the gel was blotted onto Hybond-N (Amersham), UV cross-linked, and hybridized to 32P-labeled probes. Loading was monitored by reprobing the filters with a mouse-actin cDNA. The
Slug probe comprised the coding sequence of mouse Slugh cDNA.
Bone marrow transplantation and sample collection Recipient female C57 BL/6J mice (8-12 weeks old) were irradiated with 2 split doses of 600 cGy 2 hours apart. This dose is sufficient to eliminate endogenous hematopoiesis completely. Bone marrow (BM) cells were injected into the tail vein of the irradiated mice at 2-4 × 106 cells per mouse for long-term reconstitution. All recipients were maintained in microisolator cages on sterilized food and acidified sterile water. Animals, 5 per group, were killed, and hematopoietic tissues were collected for FACS analysis.Hematopoietic colony assays Bone marrow cells (0.25-1.0 × 105 cells/plate) and spleen cells (104-105 cells/plate) isolated from normal and Slug mutant mice were seeded into FBS-free semisolid culture plates (Stem Cell Technologies, Vancouver, BC, Canada). Colony growth was stimulated with the following combinations of recombinant growth factors: rat stem cell factor (100 ng/mL; Sigma), mouse IL-3 (10 ng/mL; Sigma), and human erythropoietin (2 U/mL; Roche, Barcelona, Spain) for burst-forming unit erythroid (BFU-E) growth. The growth of erythroid colony-forming unit (CFU-E)-derived colonies was stimulated with erythropoietin alone (2 U/mL). The growth of myeloid colonies (CFU-GM) was stimulated with recombinant murine granulocyte macrophage-colony-stimulating factor (GM-CSF) (10 ng/mL; Sigma) in the presence or in the absence of SCF (100 ng/mL; Sigma). Cultures were incubated at 37°C in a humidified incubator containing 5% CO2 in air and were scored either 3 days (for CFU-E-derived colonies) or 7 days (for GM-CSF- and BFU-E-derived colonies) following initiation of the culture. The frequency of the colonies was determined in triplicate cultures.Isolation of primary bone marrow-derived mast cells, immunoprecipitation, and Western blotting Bone marrow cells were collected by flushing the marrow cavity of femurs, and mast cells were derived by selective growth for 6 weeks in IL-3-containing medium (Opti-Mem I, Gibco-BRL; 10% FBS, 0.5 ng/mL recombinant murine IL-3; R&D Systems, Madrid, Spain). Medium was replaced daily and cells were transferred to new dishes to remove adherent cells, including macrophages and megakaryocytes. Immunoprecipitation and Western blot assays were made using extracts from 1 × 107 mast cells per lane. Briefly, cells were starved for 12 hours in Opti-Mem I medium without IL-3 and containing only 0.5% serum, before stimulation with 100 ng/mL murine SCF (R&D Systems) for 10 minutes at 37°C, where indicated. Kit was detected using affinity-purified goat antiserum against the C-terminus of mouse kit, M-14 (Santa Cruz Biotechnology, Quimigranel, Madrid, Spain). Monoclonal antibody 4G10 (UBI) was used to detect phosphotyrosine.Histologic analysis Tissue specimens were fixed with 10% formalin overnight, processed, and embedded in paraffin, and 6-µm sections were stained with hematoxylin and eosin, examined histologically, and photographed. All sections were taken from homogenous and viable portions of the resected tissues. Mast cells were stained with Giemsa. The number of mast cells per square millimeter was determined.TUNEL assay Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) was performed by using the in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany), essentially following manufacturer's instructions with minor modifications, depending on the specimen preparation. In brief, sections were postfixed for 15 minutes in 4% paraformaldehyde, washed twice with PBS, and incubated in ethanol-acetic acid (2:1) for 5 minutes at 20°C. After 2 washes in PBS, sections were subjected to proteinase K digestion (10 µg/mL in 10 mM Tris HCl, pH 8.0, and 1 mM EDTA), washed twice with PBS, and counterstained with methyl green.
Matrigel assay Cell migration was determined in the BioCoat Matrigel Invasion Chamber assay (Becton Dickinson). Purified hematopoietic c-kit+ cells from wild-type mice (Slug+/+), Slug heterozygous mice (Slug+/), and Slug homozygous mice (/) were
suspended in DMEM-0.1% BSA at a concentration of
5 × 104 cells/mL, placed in the upper compartment, and
incubated for 24 hours at 37°C in 5% CO2 in the absence
or presence of SCF (100 ng/mL). After incubation, nonmigrating cells
were removed from the upper surface of the membrane by scrubbing. Cells
on the reverse side were stained with 0.1% crystal violet and were
counted under a microscope at × 100 magnification. Percentage cell
migration was calculated from the ratio of the number of cells
recovered from the lower compartment to the total number of cells
loaded in the upper compartment. Each experiment was performed using at
least 4 chambers for each c-kit+ cell sample and was
repeated at least twice.
Induction of Slug expression by the activation of kit receptor for SCF The ability of c-kit to stimulate the expression of the Snail family members was first assayed in naturally expressing c-Kit+ cells using the LAMA 84 cell line (Figure 1A). As shown in Figure 1B, the expression of Slug increased rapidly in SCF-treated LAMA-84 cells. However, the level of Snail expression was not modified in the presence of SCF. To extend these previous data indicating the capacity of c-kit in SCF-treated LAMA-84 cells to specifically activate Slug gene expression, Ba/F3 cells lacking endogenous c-kit26 were engineered to express a wild-type, full-length c-kit receptor (Ba/F3+ c-kit) (Figure 1C). Ba/F3 c-kit-transfected cells specifically expressed Slug on SCF stimulation (Figure 1D) and in a time-dependent-manner (Figure 1E). However, the Snail gene was expressed at similar levels in SCF-unstimulated and SCF-stimulated Ba/F3+ c-kit cells. These experiments demonstrated that activation of c-kit specifically induces expression of a member of the Snail gene family of zinc-finger transcription factors, indicating a clear relationship between c-Kit/SCF activation and Slug expression.
An important aspect of establishing that Slug acts downstream of kit is that the 2 gene products be expressed in the same cell type and at the same time in vivo. For this reason we analyzed Slug mRNA expression in primary c-kit+ cells purified from the bone marrow of wild-type mice by in situ hybridization. As shown in Figure 1F, Slug expression is observed in primary c-kit+ cells, and Slug expression is induced in these cells on SCF stimulation (Figure 1G). Because mutations on 2 different genes, the c-Kit receptor and its ligand (SCF), have the same complex phenotype that affects pigmentation, germ cells, and hematopoiesis, we carefully analyzed mice lacking a Slug gene to determination in vivo which functions of the c-kit/SCF pathway are mediated by Slug. Pigmentation, gonadal defects, and hematopoietic defects in Slug mutant mice The most obvious phenotype of Sl and W mutants in vivo is the presence of severe runting, which is observed shortly after birth. This characteristic is also observed in mice carrying a null mutation of the Slugh gene (Slugh 1
homozygous mutant mice), which appeared significantly smaller than
their littermates.19 As in c-kit- and SCF-defective mice, the growth retardation of Slugh1 homozygous mutant mice
occurred in the first 3 weeks of life. Accordingly, we next studied
whether Slug, like c-kit receptor and its ligand, is also important for
dermal, gonadal, and hematopoietic development.
Pigmentation deficiencies. Melanoblasts originate in the pluripotent neural crest and migrate along characteristic pathways. For survival and migration, they depend on numerous signaling systems.27 Heterozygous mutant mice (W/+ or Sl/+) have a characteristic white forehead blaze and additional areas of depigmentation on the ventral body, tail, and feet. Homozygous mutant mice (W/Wv or Sl/Sld) are more affected and completely lack pigmentation of skin and hair, whose melanocytes are derived from the neural crest.22 Heterozygous Slug mice did not present alterations in pigmentation. However, Slug homozygous mutant mice had a diluted coat with additional areas of depigmentation on the tails and feet and the characteristic white forehead blaze (Figure 2A). These dermal defects in Slug/ mice consisted of various degrees of depigmentation. However, the retina and inner layer of the iris, whose
melanocytes are derived from the optic cup and are independent of
SCF/c-kit signaling pathway, are systematically pigmented in Slug/
mice. These dermal defects observed in Slug/ mice are similar to
the dermal phenotype observed in W/+ and Sl/+ mice (Figure 2A) and
suggest a role for Slug in the development of melanocytes derived from
the neural crest.
Gonadal deficiencies.
Slug-deficient females were fertile, and the ovaries appeared
normal. Most Slug Hematopoietic deficiencies. SCF and c-kit null mutant mice have severe hematopoietic deficiencies. SCF acts on hematopoietic progenitor cells, where it is reported to increase survival rather than recruitment into the cell cycle.14 Accordingly, we analyzed the role of Slug in normal hematopoiesis. Macrocytic anemia in Slug / mutant mice. The hematologic parameters examined, in particular hemoglobin, mean cell volume, and mean cell
hemoglobin concentration, define macrocytic anemia with normal peripheral blood cell counts (Table 1),
one aspect of the mouse Sl and W, both of which are due to naturally
occurring loss-of-function mutations in either SCF or c-kit receptor,
respectively.
We next analyzed the capacity of expansion of erythropoiesis in Slug
mutant mice on hematopoietic stress. The vast expansion of
erythropoiesis that occurs in the murine spleen in response to
hemolytic anemia or other hematopoietic stress (during pregnancy) is
caused by the migration of BFU-E from the marrow to the
spleen.28 Thus, we first examined the effects on
erythropoiesis in the splenic red pulp of Slug mutant mice during
pregnancy. Murine pregnancy is characterized by transient splenomegaly
at mid-gestation by a dramatic increase in numbers of erythroblasts.
This pregnancy-associated anemia is the major reason for the gross
change in size and cell content of maternal spleen (Table
2). On the contrary spleens of 12-day
pregnant Slug mutant mice are smaller than spleens of control mice
(Table 2). Histologic examination of spleens demonstrated that
increases in splenic red pulp were less evident in Slug+/
Next we quantitated the BFU-E and CFU-E numbers in Slug mutant mice in
which we had previously induced hemolytic anemia with PHZ. Injection of
PHZ causes acute red cell destruction followed by expansion of
erythropoiesis.29 Accordingly, age-matched mice were
injected with PHZ, and its effect was systematically monitored by day 3 in mice given PHZ by a prompt decrease in Hct and an increase in the
reticulocyte count (data not shown). In Slug+/ T-cell compartment in Slug mutant mice In mice lacking functional Slug expression, T-cell numbers in peripheral blood are normal, though analysis of thymus composition from 4-week-old mice shows reduced cell production and differentiation toward CD4+CD8+ cells similar to that in Sl and W mutant mice (Figure 4). This specific T-cell differentiation block was also eventually observed in Slug+/
mice. The thymus of Slug/ mice was small and was examined on
histologic sections. Morphologic differences between the thymi of /
and +/+ animals of the same litter could be detected because the
histologic appearance of the thymus of Slug/ mice was similar to
the thymus of Sl and W mutant mice (Figure 4). In thymus sections from
Slug-deficient mice, we also observed many cells at the cortical level
that appeared to correspond to apoptotic bodies that were not seen
frequently in sections from wild-type mice (Figure 4). Consistent with
this interpretation, we detected a significant increase in
TUNEL-positive cells in thymus sections from Slug-deficient mice.
Increased apoptosis in Slug-deficient animals correlated with thymus
atrophy. These results are congruent with the idea that SCF promotes
the growth of primitive mouse CD4 CD8
thymocytes, but not CD4+CD8+ cells or single
CD4+ and CD8+ cells.31,32
B-cell, myeloid, and mast cell development appear normal in Slug mutant mice Extensive expression analysis by flow cytometry of the cell surface differentiation markers was performed on cells from spleen and from bone marrow of 5-week-old wild-type, Sl and W mutant mice, and Slug mutant mice. No reduction in cells of the myeloid and B-cell lineages was observed in the Slug mutant mice (Figure 5A-B). Thus, unlike the critical role of Slug such as c-Kit/SCF interaction in the generation of erythroid and T-cell lineages, Slug does not seem to be required for normal B-cell and myeloid development in adult mice.
The SCF/c-kit signaling pathway is required for mast cell
development.22 Thus, mast cells from 4- and 8-week-old
Slug mutant mice were examined on histologic sections of different
tissues.33 No morphologic differences between the mast
cells of Defect in Slug mutant mice is intrinsic to the stem cell Because receptor signaling depends on ligand interaction, it is not surprising that mutant forms of the c-kit receptor and its ligand produce almost identical developmental defects. However, transplantation experiments reveal a critical difference between the 2 mutations: the hematopoietic stem cells of Sl mice function normally in wild-type recipients, whereas those of W mutants do not (reviewed in Fleischman22). Accordingly, we first analyzed whether Slug mutant mice have a normal SCF/c-kit signaling pathway. To ensure that we had a normal c-kit-encoded transmembrane tyrosine kinase receptor for stem cell factor (Kit/SCF-R), we examined primary mast cells from bone marrow of +/+, +/, and / age-matched mice.
Kit/SCF-R from /, +/, and control mice was of the same size and
was expressed at comparable levels (Figure
6A). The Kit/SCF-R was also kinase active
and autophosphorylated on tyrosine residues on stimulation with SCF
(Figure 6A).
To define whether the nature of the defect was either extrinsic or
intrinsic to the stem cell, we analyzed the ability of Slug mutant
hematopoietic stem cells to reconstitute permanent hematopoiesis in
irradiated hosts. Engraftment of bone marrow cells from a healthy donor
cures the hematopoietic phenotype seen in Slug Primary BM c-kit+ cells do not express Slug in W and Sl mutant mice Slug subserves pivotal functions in promoting the development, survival, and proliferation of hematopoietic progenitor cells, neural crest-derived cells, and germ cells, a role well illustrated by the depletion of erythroid precursors and associated macrocytic anemia, gonadal defects, and hypopigmentation manifested by Slug-deficient mice. The findings that activation of c-kit specifically induces the expression of Slug and that Slug-deficient mice have phenotypes similar to those of Sl and W mutant mice prompted us to test whether Slug expression levels are up-regulated as a consequence of SCF/c-kit activation in control versus Sl and W primary c-kit+ cells. Accordingly, we induced hemolytic anemia with PHZ in control and Sl and W mutant mice. By day 3 c-kit+ cells from bone marrow and spleen were purified by sorting in control and Sl and W mutant mice (Figure 7A). Then we tested whether Slug was also present in c-kit+ cells purified from these mice. Examination of Slug expression by RT-PCR revealed that Slug was present in primary c-kit+ cells derived from bone marrow and spleen of control mice (Figure 7B).-Actin expression was used
to assess the integrity and loading of each RT-PCR reaction (Figure 7B,
bottom section). The expression of Slug was higher within migratory
cells seen in the spleen than in the c-kit+ cells that
remained in the BM. In contrast and using the same experimental
conditions, we could not detect the expression of Slug in primary
c-kit+ cells purified from the bone marrow of W and Sl
mutant mice. Only we observed Slug expression in primary
c-kit+ cells derived from the spleen (migratory cells) of W
mutant mice.
Effects of SCF of costimulation and migration of c-kit+
cells in Slug / mice, SCF did not
increase the numbers of BFU-E and CFU-GM in combination with
erythropoietin and GM-CSF, respectively (Table
5). Moreover, to functionally test the
effect of Slug on the migration of c-kit+ cells on SCF
stimulation, we performed Matrigel assays with purified hematopoietic
c-kit+ cells from BM of wild-type mice (Slug+/+), Slug
heterozygous mice (Slug+/), and Slug homozygous mice (Slug/)
(Table 6). Control Slug+/+ c-kit cells
traversed the reconstituted basement membrane at high cell frequency
(28%) on SCF stimulation. In contrast, Slug/ c-kit cells migrated
through Matrigel at significantly lower rates on SCF stimulation (3%).
These findings, together with the discovery that the activation of
c-kit specifically induces the expression of Slug and Slug-deficient
mice have a phenotype similar to that of Sl and W mutant mice,
indicate that Slug is a molecular target that contributes to the
biologic specificity of the SCF/c-kit signaling pathway.
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Abstract
Introduction
