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Juxtamembrane signaling via the membrane growth factor KitL is critical for Kit mediated functions. KitL has a conserved cytoplasmic domain, and has been shown to possess a monomeric leucine dependent basolateral targeting signal. To investigate the consequences in vivo of impaired basolateral KitL targeting in polarized epithelial cells, we have mutated this critical leucine to alanine using a knock-in strategy. KitLL263A/L263A mutant mice are pigmented normally and steady-state hematopoiesis is unaffected although peritoneal and skin mast cell numbers are significantly increased. KitL localization is affected in the Sertoli cells of the KitLL263A/L263A testis and testis size is reduced in these mice due to aberrant spermatogonial proliferation. Further, the effect of the KitL L263A mutation on the testicular phenotype is dosage dependent. The tubules of hemizygous KitLL263A/Sl mice completely lack germ cells in contrast to the weaker testicular phenotype of KitLL263A/L263A mice. The onset of the testis phenotype coincides with the formation of tight junctions between Sertoli cells during postnatal development. Thus, the altered sorting of KitL is dispensable for hematopoietic and melanogenic lineages, yet is crucial in the testicular environment, where the basal membranes of adjacent polarized Sertoli cells form a niche for the proliferating spermatogonia.
The receptor tyrosine kinase Kit and its cognate ligand KitL are encoded at the White spotting (W) and Steel (Sl) loci respectively (Chabot et al., 1988; Copeland et al., 1990; Geissler et al., 1988; Huang et al., 1990; Zsebo et al., 1990). Insight on the roles of Kit and KitL has been obtained from mice carrying mutations in either the W or Sl loci. Mutations at these loci affect various aspects of hematopoiesis, gametogenesis, melanogenesis and intestinal tract motility (Besmer et al., 1993; Russell, 1979; Silvers, 1979; Ward and Sanders, 2001). In hematopoiesis, Kit receptor signaling is critical in the stem cell hierarchy, in erythroid and mast cell lineages (Besmer, 1997; Galli et al., 1994). In addition, Kit also has a critical role in regulating age-dependent maintenance of pro T and pro B cell subsets during adult lymphopoiesis (Agosti et al., 2004). In embryonic gametogenesis, the KitL-Kit system control events in primordial germ cell (PGC) proliferation, survival and migration (Bachvarova et al., 1993; Besmer et al., 1993). Postnatally in the testis, Kit is important for the proliferation and survival of spermatogonia while in the ovary Kit has a role in follicle development (Manova et al., 1990; Yoshinaga et al., 1991). Concurrent with its function, Kit is expressed in the spermatogonia type A and B, primary spermatocytes and Leydig cells while in the ovary the receptor is abundant in primordial oocytes and growing oocytes (Manova et al., 1993). KitL is expressed in the microenvironment of Kit expressing cells such as the migratory path of PGCs, in Sertoli cells of the testis and granulosa cells of the ovary (Manova et al., 1993).
The diverse functions of Kit in vivo are governed by unique cell-type specific signaling networks. Two signaling Kit mutants KitY719F/Y719F and KitY567F/Y567F generated by us and others, blocking the PI3-kinase and Src kinase pathways respectively, provide insight in the roles of each signaling cascade in various cell types (Agosti et al., 2004; Blume-Jensen et al., 2000; Kimura et al., 2004; Kissel et al., 2000). In the KitY567F/Y567Fmutants, lymphopoiesis is affected while steady-state erythropoeisis remains unchanged (Agosti et al., 2004). This mutation also has little effect on gametogenesis. Interestingly, in KitY719F/Y719F and KitY567F/Y567F mice, mast cell numbers in dorsal skin sections are not reduced and both mutations affect peritoneal mast cell numbers to differing degrees (Agosti et al., 2004). In contrast to KitY567F/Y567F, there are severe deficiencies in spermatogenesis and oogenesis in the KitY719F/Y719F with little effect on hematopoiesis (Kissel et al., 2000). In parallel to the differentiating effects of the Kit signaling pathways in diverse cell types, the expression of either the soluble (KitL1) or membrane (KitL2) forms of KitL can also contribute to functional differences. KitLSld/Sld mice expressing a secreted soluble form of KitL alone have a severe phenotype (Nakayama et al., 1988) in contrast to mice that contain only the membrane form of KitL (Tajima et al., 1998a). A critical role for the KitL membrane form is underlined by the phenotypes observed in KitLSld/Sld and KitLSl17H/Sl17H mutant mice. KitLSld/Sld produces only the secreted soluble form of KitL as a result of an intragenic deletion while in KitLSl17H/Sl17H the cytoplasmic domain of KitL is altered as a result of a frame-shift mutation. The altered cytoplasmic tail in KitLSl17H has minor effects on female fertility and other cell lineages but results in male sterility (Brannan et al., 1992; Huang et al., 1990; Tajima et al., 1998a). The conserved cytoplasmic domain sequence of KitL in various species and the nature of the KitLSl17H mutation suggested an important role for the cytoplasmic domain of KitL.
Previously, we demonstrated that the cytoplasmic domain sequence is important for biosynthetic processing of KitL through the ER and Golgi complex to the cell surface (Tajima et al., 1998a). More recently, in vitro studies involving site directed mutagenesis of KitL cytoplasmic domain revealed that a single leucine at position 263 is necessary and sufficient to drive KitL to the basolateral membrane (Wehrle-Haller and Imhof, 2001). In addition, the terminal valine residue of the cytoplasmic tail is critical for the exit of KitL from the ER (Paulhe et al., 2004). Together, it was evident that the cytoplasmic tail contained important cues for the processing and display of KitL at the membrane. Interestingly, in cell types such as the Sertoli cells, KitL expression is highly polarized (Manova et al., 1993). Immunohistochemical studies in the testis showed that the KitL protein accumulates in the basolateral region of Sertoli cells at the time when Kit expressing spermatogonia begin to mature (Manova et al., 1993). Presumably, a defect in polarized KitL localization would have an impact on the different cellular targets of KitL that depend on a strict localization pattern of KitL. In order to investigate the role of the leucine 263 in the polarized presentation of KitL in vivo, we employed a knock-in strategy to produce KitLL263A/L263A mouse. Earlier, we also generated a mouse lacking exon 9 of the KitL cytoplasmic tail. Here we compare the phenotypes of KitLL263A/L263A and KitL Δ9/Δ9. Our results show that polarized expression of KitL is important for spermatogenesis and hematopoiesis but is dispensable for other Kit dependent cell lineages, highlighting the importance of cellular context in manifestation of the phenotype.
A 6.1 Kb SalI-EcoRI genomic DNA fragment of KitL including exons 7–9 was subcloned into a pBluescript DTA plasmid and used to build the targeting constructs. KitLL263A knock-in mouse: site directed mutagenesis was performed on a 1.6 Kb HindIII-BglII fragment including exon 9, mutating leucine 263 to alanine (L263A). A neomycin resistance cassette flanked by loxP sites was inserted 0.5 Kb downstream of exon 9, for positive selection. KitLΔ9 knock-in mouse: a BamHI-BglII fragment of 1.4 Kb including exon 9 was replaced by the BamHI-BamHI neomycin resistance cassette. CJ7 embryonic stem (ES) cells were electroporated with linearized targeting constructs of KitLL263A or KitLΔ9 using standard protocols. Neomycin resistant clones were isolated and analyzed for homologous recombination. Sequencing analysis was performed to confirm the L263A mutation. Correctly targeted ES cell clones were microinjected into C57BL/6J blastocysts and male mice displaying 85 –100% chimerism were backcrossed to C57BL/6J females for germline transmission. The floxed neomycin cassette was excised in vivo by mating heterozygous mutant males with EIIa-cre transgenic females. A PCR based detection of the loxP site was used to genotype KitLL263A. A PCR product of 350 bp was obtained for the wild-type allele and 450 bp for the mutated allele using the primers FP gtg ttg gtc cgt att agt ggg ttc att t; RP cat cag att ttg ttc tta gtt tat cct c. KitL Δ9 mice could be easily distinguished based on coat color; homozygous mutant mice were white and heterozygous mice had a belly spot. When molecular genotyping was required, a dual PCR strategy was employed using the primers for loxP amplification (described above) and the primers: FP tga aga gga taa tga gat and RP gat ggt gtg ggt gat aac, the latter giving a product of 200 bp. Two-step PCR of DNA from KitL Δ9/Δ9 mice gave 200 bp product, wild-type DNA resulted in a 350 bp product and with KitL Δ9/+ two products of 200 bp and 350 bp were obtained.
Plasma T concentrations were measured by a tritium-based radioimmunoassay (RIA) as previously described (Cochran et al., 1981). Intratesticular testosterone concentration was measured using the method of Knorr et al (Knorr et al., 1970). In brief, testes were homogenized in 5 mL of 70% methanol. Tracer steroid (1000 counts per minute [cpm] of tritiated testosterone) was added to the homogenate to correct for recovery. The tubes were centrifuged at 1800 × g, and the supernatant was aspirated and dried under nitrogen to remove the methanol. The ether extracts were then resuspended in 400 μL of RIA buffer, and 100 μL was removed for measurement of recovery (the cpm value in 100 μL × 4 ÷ 1000). The remaining 300 μL was used for RIA.
Plasma samples were obtained by retro-orbital bleeding of mice. Samples were analyzed for soluble KitL (sKitL) levels using an ELISA kit (R&D Systems).
Protein extracts from cerebellums of wild-type, KitLL263A/L263A and KitLΔ9/Δ9 were prepared as described previously (Sommer et al., 2003). An antibody raised against the N-terminal region of KitL (sc 9132, Santa Cruz Biotechnology, USA) was used for detection of KitL and actin (sc 1616, Santa Cruz Biotechnology, USA) was used as loading control.
KitL antibody was generated at Pocono Rabbit Farm and Laboratory Inc. by injecting rabbits with the C-terminal KitL peptide YMLQQKEREFQEV. The KitL antiserum and pre-immune serum were partially purified and the KitL antiserum was further subjected to affinity purification. Testes were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), cryoprotected in 30% sucrose and embedded in OCT. Cryosections of 10–12 μm were subjected to antigen retrieval with Pronase E (Sigma) and incubated with KitL antibody or pre-immune serum. Subsequently, the sections were incubated with Alexa 488 conjugated secondary antibody (Molecular Probes) followed by counterstaining of nuclei with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). For PCNA (Dako) staining, testes fixed in 4% PFA and embedded in paraffin were sectioned at 10–12 μm. Following routine deparaffinization and rehydration, the sections were subjected to staining with PCNA antibody using the automated staining processor Discovery from Ventana Medical Systems. Sections were counterstained with DAPI. For BrdU staining, adult mice or 9d pups were injected twice intraperitoneally with 50 mg of BrdU (Sigma) per Kg body weight at a 30 minute interval. After 1h the testes were dissected, fixed in 4% PFA and processed in paraffin. BrdU incorporation was detected with a monoclonal antibody against BrdU (Roche). Sections were incubated with biotinylated secondary antibody and streptavidin –HRP followed by detection with diaminobenzidine (Sigma). BrdU staining was carried out using the automated staining processor Discovery from Ventana Medical Systems. TUNEL staining was done as described previously (Manova K et al., 1998). In brief, testes fixed in 4% PFA were processed in paraffin. Deparaffinized sections were treated with proteinase K (20 μg/ml) in PBS and bleached in 0.1% H2O2 to destroy endogenous peroxidase activity. Nicks present in the DNA of apoptotic cells were end-labeled for 1h at 37°C with terminal deoxynucleotidyltransferase (Boehringer Mannheim) and biotin 16-dUTP. Sections were incubated in ABC Vectastain reagent (Vector Laboratories). Detection of staining was achieved with diaminobenzidine followed by counterstaining with Gill’s hematoxylin.
Testes were fixed in Bouin’s solution or 4% paraformaldehyde (PFA), embedded in paraffin and stained by periodic acid-Schiff reaction (PAS) according to standard protocol. Peripheral blood parameters were analyzed as described previously (Tajima et al., 1998b). Skin mast cell numbers were determined by toluidine blue staining as described previously (Tajima et al., 1998b). Mast cells per cm skin between the epidermis and the panniculus were counted in several independent sections and averaged.
Images were acquired with a Zeiss Axiocam HRC camera mounted on a Zeiss Axioplan2 microscope or Leica TCS SP2 AOBS confocal microscope. Images of serial skin and testis sections for counting mast cells or BrdU and TUNEL positive cells were obtained with a Zeiss Axiovert 200M ‘Metamorph’ microscope.
All anti-mouse monoclonal antibodies used are from BD Pharmingen. Appropriate labeled isotype controls and single/double color stained cells were always used to define the specific gates. FACScalibur or FACScan (BD Biosciences) was used for analysis.
Thymi were mechanically dissociated to a single cell suspension, washed and resuspended in staining buffer. The triple negative subsets were resolved by staining 1.5×106 thymocytes with a mix of lineage specific monoclonal antibodies (anti-mouse Ter119, B220, Mac-1, Gr-1, CD4, CD8, CD3) PE conjugated, anti-mouse CD44 Cy-chrome conjugated and anti-mouse CD25 FITC conjugated. CD44 and CD25 expression was analyzed on the gated lineage negative cells.
Bone marrow (BM) was flushed from femurs with PBS, and a single cell suspension was obtained by gentle pipetting and passage through a nylon strainer (Falcon). 1.5×106 cells resuspended in staining buffer were incubated for 10 min at 4°C with 1 μg of murine Fc block and then labeled with the appropriate monoclonal antibody mix consisting of anti-mouse B220 APC/anti-CD43 Cy-chrome/anti-mouse IgM FITC or anti-B220 APC/anti-CD43 Cy-chrome/CD24 FITC/BP-1 PE. If necessary after incubation mature red cells were depleted by hypotonic lysis (BD Pharmingen). When not immediately analyzed the labeled cells were fixed by CytoFix (BD Pharmingen) according to the manufacturer’s instruction.
Peritoneal mast cells were obtained by peritoneal lavage with 5 ml of cold PBS and resuspended in staining buffer (PBS without Ca2+ and Mg2+, 3% FCS, 0.02% NaN3). Cells were stained for Kit with PE-conjugated anti-Kit antibody and for FceRI by incubation with mouse IgE anti-DNP (clone SPE-7; Sigma-Aldrich) followed by FITC-conjugated IgE monoclonal antibody.
Methylcellulose cultures were carried out by using the technique described earlier (Nakahata et al., 1982). 2.5 ml of culture mixture containing 3 × 104 nucleated peritoneal cells, 0.8% methylcellulose (Stem cell technologies), 30% fetal bovine serum, Iscove’s medium, 1% deionized bovine serum albumin (Sigma),10−4 M mercaptoethanol and recombinant mouse KitL, were plated in Corning plates (0.5 ml/well). Plates were incubated at 37°C, 5% CO2. Colonies were scored on day 14. The mast cell nature of the colony was confirmed by cytospin and toluidine blue staining.
The Student’s t test assuming unequal variances was used to determine the significance of differences between groups. Groups were judged to differ significantly at P < 0.05.
In order to investigate the in vivo role of KitL cytoplasmic domain, two mutant mice were generated, one lacking exon 9 (KitLΔ9/Δ9) which encodes the C-terminal tail sequence and another incorporating substitution of leucine 263 to alanine (KitLL263A/L263A) (fig 1A–B). The absence of exon 9 or presence of the L263A mutation was confirmed by sequencing. In case of KitLΔ9/Δ9 however, splicing resulted in addition of 23 extraneous amino acids following exon 8 sequence (fig 1C). The deletion of the C-terminal tail in the KitL Δ9/Δ9 mice affected melanogenesis (fig 1D). KitLΔ9/Δ9 mice were white with black eyes and residual pigmentation was seen on ear tips and snout while in heterozygous KitLΔ9/+ mice pigmentation was unaffected except for a white belly spot. The pigmentation phenotype of the KitLΔ9/Δ9 mice is similar to the KitLSl17H/Sl17H (Wehrle-Haller and Weston, 1999) and the KitLSld (Brannan et al., 1991; Flanagan et al., 1991). In contrast, no pigmentation phenotype was observed in KitLL263A/L263A mice. To assess whether the mutations had any effect on the KitL protein levels, Western blot analysis was carried out using extracts from the cerebellum, a tissue known for high KitL expression. The total KitL expression levels in the cerebellum as assessed by Western blot were comparable in wild-type and mutant mice (fig 1E).
Testis size in the KitLΔ9/Δ9 mice was reduced by almost 90% compared to wild-type (fig 2A). The reduction in testis size was less severe in the KitLL263A/L263A mice, nonetheless a 50% reduction in testis size was observed compared to wild-type. Further, histological analysis revealed normal spermatogenesis in the KitLL263A/L263A while tubules in the KitLΔ9/Δ9 mice predominantly lacked germ cells (fig 2B). The epididymis also lacked sperm in the KitLΔ9/Δ9 mice. Postnatally, the KitLΔ9/Δ9 testis contained multi-layered seminiferous epithelia with mature sperm lining the lumen and the first wave of spermatogenesis was normal. However, a gradual loss of germ cells was observed starting at 4 weeks, resulting in empty tubules by 8 weeks and the males were subsequently infertile (data not shown). In this respect, the testicular phenotype observed in KitLΔ9/Δ9 mice is very similar to the KitLSl17H/Sl17H. Unlike KitLΔ9/Δ9 mice, the effects of the L263A mutation on testicular histology were subtle. We assessed whether the reduction in testis size in the KitLL263A/L263A mice was a result of an altered hormonal profile. Previously in the KitY719F/Y719F mice we observed altered testosterone levels, and showed that Kit signaling could promote Leydig cell steroidogenesis at least in vitro (Rothschild et al., 2003). In contrast to KitY719F/Y719F mice plasma and testicular testosterone concentrations remained unchanged in the wild-type and KitLL263A/L263A males (fig 2C–D). Female KitLL263A/L263A mice were fertile and produced litters of normal size (data not shown).
KitL localization in the testis is presumed to change with age. In addition to an apical distribution, increased basal localization of KitL is observed between postnatal d19 to d24 (Manova et al., 1993). Since basal KitL expression can be readily assessed at this age, we analyzed KitL expression in KitLL263A/L263A mice between d19 and d21 (fig 3A–C). In the wild-type testis, KitL staining was uniformly distributed along the basal membrane of all the tubules. However, in the KitLL263A/L263A mutants, basal KitL staining was significantly reduced but was not entirely absent. Wild-type testis sections treated with pre-immune serum lacked KitL staining. In the adult testis, KitL expression is more complex and is believed to vary with the stage of the tubule (Manova et al., 1993). A clear adluminal localization of KitL could be seen in both the wild-type and mutant Sertoli cells (fig 3D–E). In the wild-type testis KitL was localized in both the adluminal and basal faces of the Sertoli cell membrane (fig 3D, inset), however, in the mutant, KitL decorated the long apical processes of the Sertoli cells but staining was reduced or absent along the basal membrane (fig 3E, inset).
We first tested whether apoptosis was the cause for the reduced testis size in the KitLL263A/L263A mice. Testes of two age groups were analyzed: adult and pups of postnatal 6–9d when spermatogonia are mitotically active and are yet to initiate meiosis. Interestingly, there was no difference in the number of TUNEL positive cells in the 6–9d wild-type and mutant groups, and even in the adult, quantitative evaluation revealed no statistically significant difference between wild-type and mutant mice (fig 4A–F). Next, we investigated whether proliferation of spermatogonia was affected in the mutant (fig 4G–L). BrdU incorporation, as assessed by the number of BrdU positive cells per testis section was also similar in wild-type and mutants in the 6–9d group. However, in the adult, BrdU incorporation was significantly reduced in the mutant compared to wild-type. We then used PCNA, a known marker of proliferation to determine the status of proliferating spermatogonia in the adult wild-type and mutant testis (fig 4M–N). In contrast to the uniform pattern of PCNA expression in the wild-type, distinct patches of PCNA-absent regions were observed in the KitLL263A/L263A testis. Together, the BrdU incorporation and PCNA expression point to a proliferation defect in the KitLL263A/L263A testis.
In the KitLL263A/L263A, the proliferation of spermatogonia was reduced, however at no age were the tubules completely empty. This is in contrast to the KitLΔ9/Δ9 mutant where tubules become progressively empty with age (data not shown). We analyzed if reducing KitL levels would intensify the testis phenotype in the adult KitLL263A mice. Hence, we crossed KitL+/Sl mouse with KitLL263A/L263A and analyzed the testes of the resulting offspring, i.e., KitL+/Sl and KitLL263A/Sl (fig 5A–K). While the testis and epididymis of the adult KitLL263A/L263A and KitL+/Sl mice had germ cells and mature sperm, the reduction of KitL levels by half in the KitLL263A/Sl mouse resulted in a more severe testis phenotype compared to KitL+/Sl or KitLL263A/L263A. The testis phenotype of KitLL263A/Sl was comparable to the testis of KitLΔ9/Δ9 mice with absence of developing germ cells in the testis and mature sperm in the epididymis. In contrast, the testes of 4 week old wild-type, KitL+/Sl and KitLL263A/Sl mice looked morphologically similar.
KitL/Kit play a critical role in mast cell development and function. Therefore we analyzed skin and peritoneal mast cells in KitL mutant mice. In the skin sections of KitL263A/L263A mast cell numbers per cm skin were increased compared to wild-type as assessed by toluidine blue staining (fig 6A–B). In contrast, mast cells were severely reduced in skin sections of KitLΔ9/Δ9 (fig 6C–E). The peritoneum is the most sensitive mast cell compartment in connection with Kit function. In many Kit/KitL mutants peritoneal mast cell number is altered in the absence of significant mast cell deficits in other regions. Mast cells were identified as Kithigh/FceRI+ cells by flow cytometry and confirmed by alcian blue/safranin staining following peritoneal lavage. KitLΔ9/Δ9 mice showed a reduced number of peritoneal mast cells as often observed in Kit/KitL loss of function mutants. Similar to the skin in KitL263A/L263A mice peritoneal mast cells were increased in number (fig 6F). KitL is a critical growth factor for mast cell proliferation and survival. It therefore follows that a change in the level of KitL might affect mast cell numbers. Hence we assessed soluble KitL (sKitL) levels in the plasma samples of the two mutants and wild-type littermates. While there was a two-fold reduction of sKitL in KitLΔ9/Δ9 mice there was no significant difference in sKitL level between KitL263A/L263A and wild-type (fig 6G). Furthermore, clonogenic mast cell progenitors in the peritoneum of wild-type and KitL263A/L263A mice were assessed by mast cell colony assay. The number of clonogenic mast cell progenitors were increased in the peritoneum of KitL263A/L263A mice reflecting the increased numbers of mast cells in the peritoneum (fig 6H).
There is an essential and non-redundant role for Kit in adult mouse lymphopoiesis. Although impaired thymus cellularity in KitLSl17H/Sl17H mice was reported previously (Kapur R. 1999), early T cell differentiation and especially B lymphopoiesis have never been investigated in detail. We analyzed, by flow cytometry, T and B cell differentiation processes in the thymi and BM of KitL263A/L263A and KitLΔ9/Δ9 mutant mice. Similar to KitLSl17H/Sl17H mice, thymus cellularity, and thus, the CD4+/CD8+ double positive cells, representing more than 90% of total thymocytes, was significantly decreased in mutant KitLΔ9/Δ9 but not in KitL263A/L263A mice (fig 7A–C). The early T-lymphoid differentiation steps were investigated by analyzing the triple negative (CD3-/CD8-/CD4- TN) subsets as described previously. The TN 1–4 fractions were resolved by analysis of the cell surface markers, CD44 and CD25 into: TN1 (CD44+CD25−), TN2 (CD44+CD25+), TN3 (CD44−CD25+), and TN4 (CD44−CD25−) (Godfrey et al., 1993). Within the triple negative subsets in KitLΔ9/Δ9 mice CD25+ cells were strongly reduced with a corresponding increase of the CD44+/CD25− (TN1) cells. The increase of the TN1 subset was relative due to depletion of the downstream TN subsets (TN2, TN3 and TN4).
Early B cell development in the bone marrow was analyzed as described previously (Agosti et al., 2004; Hardy et al., 1991). Both the KitLΔ9/Δ9 and KitLL263A/L263A mice did not show any significant alteration in any of the early B-cell subsets (data not shown).
The processing of KitL into membrane and soluble forms and their impact on Kit dependent lineages has been well studied using KitLSld and KitLSl17H mutants (Brannan et al., 1992; Tajima et al., 1998a; Tajima et al., 1991). It is evident from these mutants that the cytoplasmic tail is critical for KitL presentation on the cell surface. In agreement with this, in vitro experiments showed that cytoplasmic tail deletion mutants of KitL caused improper trafficking of the protein from ER to Golgi (Tajima et al., 1998a). More recently mutagenesis of residues in the KitL cytoplasmic tail identified two distinct amino acids leucine 263 and valine 273 critical for polarized KitL localization and ER export respectively (Paulhe et al., 2004; Wehrle-Haller and Imhof, 2001). The substitution of leucine 263 to alanine in MDCK cells did not affect KitL apical localization but resulted in lack of basolateral expression. Thus, the KitLL263A mouse has made possible for the first time to study the ramifications of mis-polarized KitL expression in different lineages without the mutation presumably having any impact on KitL expression.
In the testis, junctional proteins of the Sertoli cells participate to form the blood-testis barrier which effectively divides the seminiferous epithelium into basal and adluminal compartments. The basal compartment of the seminiferous tubules is highly specialized and is occupied by the spermatogonia and early spermatocytes and also forms a niche for the developing spermatogonia (Ogawa et al., 2005). The formation of the tight junction complex then divides the Sertoli cell membrane into basal and adluminal membrane regions with a possible role in conferring polarity to the Sertoli cells (Cereijido et al., 1998). However, unlike other polarized epithelia, it is difficult to distinguish the lateral and apical faces of the Sertoli cell and the shape of this unique cell is largely dependent on the innervating germ cells. Tight junctions between the Sertoli cells start to be formed by P10, resulting in many fully formed junctional specializations by P14 and mature junctional complexes by P24 (Byers S, 1993). We analyzed KitL expression in Sertoli cells in the adult and at P19-21 when the tight junction formation is not yet completed. In the young and adult testis adluminal and basal staining of KitL in Sertoli cells was intense in the wild-type in contrast to the KitLL263A/L263A testis where basal KitL expression at the same ages was disrupted or weak although adluminal expression of KitL was unaffected. In contrast in MDCK cells, the L263A mutation severely reduced KitL expression along the basolateral membrane (Wehrle-Haller and Imhof, 2001). It is possible that efficient sorting of KitL in vivo by L263 is dependent upon KitL levels.
In the testis KitL is required for spermatogonial function and presumably also for Leydig cells both of which express Kit. Leydig cells are the major source of testosterone, which is a critical hormone for spermatogenesis. Earlier studies by us had shown that KitL could enhance testosterone production of Leydig cells in vitro and in addition the hormonal profile was altered in KitY719F/Y719F mice (Kissel et al., 2000). However, the testosterone level in the plasma and within the testis was unaffected by the L263A mutation. It is unlikely that sKitL from Sertoli cells is a major source of ligand for the Leydig cells, which on the other hand may derive sKitL from the interstitium that contains blood vessels. However sKitL levels in the plasma of the wild-type and KitLL263A/L263A mice were unchanged. Given the unaltered hormonal profile in the KitLL263A/L263A, it was likely that the reduction in testis size was due to reduced expression of KitL on the Sertoli cell basal membrane and the consequent effect on spermatogonial proliferation and differentiation. Primordial germ cell (PGC) number and their migration are also dependent on Kit signaling. Although PGC number was reduced in KitLΔ9/Δ9 as observed in KitLSl17H/Sl17H, PGC numbers were unaffected in the KitLL263A/L263A (data not shown).
Kit is highly expressed in type A spermatogonia followed by type B spermatogonia and at lower levels in spermatocytes (Manova et al., 1990; Vincent et al., 1998). In the postnatal testis starting at P5 type A spermatogonia proliferate and differentiate in a Kit dependent fashion and characterization of KitY719F/Y719F mice revealed that PI3-kinase signaling is critical for proliferation and survival of spermatogonia (Kissel et al., 2000). Furthermore, Kit may be important for differentiation of type B spermatogonia to pre-leptotene spermatocytes (Vincent et al., 1998). The proliferation and differentiation of spermatogonia up to meiosis occurs within the basal compartment of the seminiferous tubules. We therefore questioned whether the reduction of KitL in the basal membrane of Sertoli cells had an impact on spermatogonial proliferation and survival. Whereas proliferation of spermatogonia at age P6-9 was unchanged in the mutant, in the adult BrdU positive cells and PCNA positive cells were reduced in the KitLL263A/L263A mice. Kit-KitL is also essential for spermatogonial survival and in agreement with this lack of p53 function has been shown to rescue the Kit loss of function phenotype in KitWv/Wv mice (Jordan et al., 1999). However, in the KitLL263A/L263A testis apoptosis did not significantly differ from the wild-type. In accordance with the reduction in spermatogonial proliferation, testis size was reduced in KitLL263A/L263A. On the other hand, in the KitLΔ9/Δ9 as in the KitLSl17H/Sl17H mice progressive alteration in testicular morphology was observed (data not shown), with complete absence of differentiating germ cells by 8 weeks post-natal (Brannan et al., 1992). In KitLΔ9/Δ9 and KitLSl17H/Sl17H this extreme testicular phenotype may be due to the reduced surface expression of KitL caused by lack of cues for proper trafficking and basolateral sorting. We therefore questioned whether reduction of KitL itself might exacerbate the phenotype in the KitLL263A/L263A mice and produced compound KitLL263A/Sl mice. Interestingly, in the adult no differentiating germ cells could be seen in any of the tubules of the KitLL263A/Sl testis comparable to the KitLΔ9/Δ9 testis. In MDCK cells in vitro the level of KitL expression had been shown to affect basolateral sorting efficiency (Wehrle-Haller and Imhof, 2001). The increased severity of the testis phenotype in KitLL263A/Sl compared to KitL+/Sl or KitLL263A/L263A indicates that by reduction of KitL levels the basolateral sorting deficit becomes more apparent and accordingly, in the KitLL263A/Sl testis KitL localization in the basal membrane of Sertoli cells was not discernable (fig S1). Interestingly, in immature KitLL263A/Sl mice the testis morphology is similar to KitL+/Sl. The first wave of spermatogenesis appears to occur normally in KitLL263A/Sl and even in KitLSl17H/Sl17H (Brannan et al., 1992) and KitLΔ9/Δ9 (data not shown). However, subsequently spermatogenesis is disrupted in these mutants. In this regard, recent work has shown that the first round and the successive waves of spermatogenesis arise from two distinct differentiation programs (Yoshida et al., 2006). Interactions between Sertoli cells seem to be important in the manifestation of the effects of the sorting mutation. In the immature testis, before the formation of tight junctions in the Sertoli cells, although basal KitL may be reduced spermatogonial proliferation in KitLL263A/L263A and littermate controls is comparable. The onset of the phenotype presumably occurs after the formation of Sertoli cell tight junctions, when the compartmentalization of spermatogonia becomes very rigid and the formation of tight junctional complexes restricts the accessibility of any adluminal KitL reaching the spermatogonia in the basal compartment.
Whereas pigmentation in the KitLL263A/L263A mice was normal, KitLΔ9/Δ9 mice were depigmented and heterozygous KitLΔ9/+ mice had a small belly spot as observed in the KitLSl17H mice (Wehrle-Haller and Weston, 1999). Interestingly, pigmentation in the KitLL263A/Sl mutant is not different from KitL+/Sl, both of which have diluted coat color (fig S2). We previously showed that the extraneous cytoplasmic tail sequences in KitLSl17H diminish KitL dimerization and the same may be true for KitLΔ9 (Tajima et al., 1998a). Thus the altered KitL cytoplasmic tail sequences in the KitLSl17H and KitLΔ9 mice may contribute to the heterozygous pigmentation phenotype presumably in a dominant negative fashion. Although we have not analyzed melanocyte development in detail in the KitLL263A/L263A mice, the lack of any synergistic effect on pigmentation in KitLL263A/Sl due to reduction of KitL indicates that polarized localization of KitL may not be critical for melanoblast or melanocyte development, proliferation and survival.
In hematopoiesis Kit has roles in the hematopoietic stem cell hierarchy as well as in erythropoiesis, lymphopoiesis and in mast cell differentiation and function. Kit is expressed on hematopoietic stem and progenitor cells and Kit expression is lost during differentiation. Hematopoietic stem and progenitor cells depend on KitL in vitro for growth and survival, often in synergy with other growth factors and cytokines. Whereas Kit and KitL null mice die perinatally of anemia, Kit and KitL hypomorphic mutations develop macrocytic anemia. Although polarized expression of KitL has not been documented in cells of the hematopoietic microenvironment, it was reasonable to hypothesize an effect of altered sorting. Previous characterization of KitLSl17H/Sl17H mutant mice showed reduced homing of hematopoietic progenitors to the spleen and long-term mutant bone marrow cultures did not support hematopoietic growth (Tajima et al., 1998a). Furthermore, hemizygous KitLSl/Sl17H mice were shown to have reduced BM cellularity, peripheral RBC counts and hematocrit levels. The KitLΔ9/Δ9 mice similar to KitLSl17H/Sl17H mice have normal bone marrow cellularity, slightly reduced RBC numbers, marginally increased mean corpuscular volume and long-term bone marrow cultures showed strongly impaired hematopoietic growth (data not shown). Since KitLL263A/L263A mice did not exhibit any hematopoietic deficiencies it appears likely that polarized KitL expression may not be critical in the hematopoietic microenvironment at least under steady state conditions. Further investigations may clarify a role for basolateral KitL sorting in stress hematopoiesis.
Although during embryonic development Kit has a redundant role in T and in B cell differentiation, in the adult mouse Kit is critical in both pro-T and pro-B cell subsets in an age-dependent manner (Agosti et al., 2004; Asamoto and Mandel, 1981; Rodewald et al., 1995; Takeda et al., 1997; Waskow et al., 2002). Whereas, in KitLΔ9/Δ9 mice early adult T-cell development was impaired, B cell development in the bone marrow of these mice was not affected. Interestingly in KitL263A/L263A mice both T and B cell development were not affected by the mutation. Therefore, the bone marrow micro-environment probably does not include KitL expressing polarized epithelial cells. In contrast, in the thymic microenvironment KitL expression may require sorting, although this is only apparent with reduced levels of KitL cell surface expression.
Mast cells arise from hematopoietic progenitors in the bone marrow, but maturation and differentiation of mast cells occurs mainly in the tissues where they reside. In contrast to other hematopoietic cell progeny they express Kit and they depend on KitL for their survival, growth and function (Oliveira and Lukacs, 2003). A mast cell deficit is a common feature of mice with Kit and KitL loss of function mutations including KitLSl17H/Sl17H mice (Tajima et al., 1998a). In agreement with this in KitLΔ9/Δ9 mice the numbers of mast cells in the skin and the peritoneum were reduced which is in accordance with the reduced plasma sKitL levels in these mice. Surprisingly in KitLL263A/L263 mice despite normal plasma sKitL levels mast cell numbers in both the dorsal skin and the peritoneum were increased. The increase in mast cell number was not due to inflammation as pro-inflammatory cytokine levels, including IL-6, IL10, TNF-α and MCP-1, were low and comparable between KitLL263A/L263 and wild-type mice (data not shown). Apical presentation of KitL itself or increased proteolytic processing and shedding, facilitated by altered sorting, might produce a localized effect generating increased KitL levels thus producing the observed phenotype. In agreement with this sKitL is known to be required for the recruitment of mast cells to the skin, and animals lacking a major proteolytic cleavage site in KitL lack dermal mast cells (Kunisada et al., 1998; Tajima et al., 1998c).
Supplementary Fig 1. KitL localization in the KitLSl/L263A testis
KitL localization (green) in the 4-week old KitLSl/L263A testis is unaffected along the adluminal region of Sertoli cells (arrowhead). Basal KitL staining of Sertoli cells is severely reduced (arrows). Section is counterstained with DAPI (blue). Boxed section is enlarged as inset showing lack of basal staining in Sertoli cells. Scale bar, 20μm.
Supplementary Fig 2. Coat color is unaffected in the hemizygous KitLSl/L263A mice
In comparison to the coat color of KitL+/+, the coat color of KitL+/Sl is distinctly reduced and often a belly spot is observed. Interestingly, the coat color in KitLSl/L263A was not further reduced (A–B).
This paper is dedicated to the memory of our colleague and friend Matthew Hardy. We thank William Wright for helpful suggestions. We would like to thank Sandra Gonzalez, Mesruh Turkekul, Afsar Barlas, Yevgeniy Romin and Tao Tong of the Molecular Cytology Facility for help with histological analyses. We thank Chantal Sottas and Maureen Sullivan for technical assistance. The authors also gratefully acknowledge Elisa de Stanchina, Huiyong Zhao and Juan Qui of Antitumor Assessment Facility for their assistance with drug administration. This work was supported by grants from the National Institutes of Health, RO1 HD 38908 and RO1 HL/DK55748 (to P.B.).
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