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BOR - Papers in Press, published online ahead of print May 25, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042846
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BIOLOGY OF REPRODUCTION 73, 639–647 (2005)
DOI: 10.1095/biolreprod.105.042846
© 2005 by the Society for the Study of Reproduction, Inc.

Analysis of Hypomorphic KitlSl Mutants Suggests Different Requirements for KITL in Proliferation and Migration of Mouse Primordial Germ Cells1

Aparna Mahakali Zama 3 , F. Parker Hudson, III 4 , and Mary A. Bedell 2 

Department of Genetics, University of Georgia, Athens, Georgia 30602-7223


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Germ cell development in mice is initiated when a small number of primordial germ cells (PGCs) are set aside from somatic cells during gastrulation. In the subsequent 4 to 5 days, PGCs enter the hindgut, undergo a directed migration away from the hindgut into the developing gonads, and undergo a massive increase in cell number. It is well established that Kit ligand (KITL, also known as stem cell factor and mast cell growth factor) is required for the survival and proliferation of PGCs. However, there is little information on a direct role for KITL in PGC migration. By comparing the effects of multiple Kitl mutations, including two N-ethyl-N-nitrosourea-induced hypomorphic mutations, we were able to distinguish stages of PGC development that are preferentially affected by certain mutations. We provide evidence that the requirements for KITL in proliferation are different in PGCs before and after they start migrating, and different levels of KITL function are required to support PGC proliferation and migration. This study illustrates the usefulness of an allelic series of mutations to dissect developmental processes and suggests that these mutants may be useful for further studies of molecular mechanisms of KITL functions in gametogenesis.

developmental biology, embryo, gametogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Primordial germ cells (PGCs) of mice are first identifiable in the extraembryonic mesoderm at Embryonic Day (E) 7.25–E7.5 as a small cluster of alkaline phosphatase (AP)-positive cells, which then migrate into the posterior portion of the primitive streak during the next 24 h ([1], reviewed in [2]). PGCs then become incorporated into the endoderm of the developing hindgut [3] and they remain associated with the hindgut for about 2 days. Beginning at E9.0–E9.5, PGCs undergo an active, directed migration from the dorsal axis of the hindgut to the genital ridges [4, 5]. Upon entry into the genital ridges at around E10.5– E11.5, PGCs become nonmotile and coalesce with somatic cells that have begun to sexually differentiate. PGCs rapidly proliferate during all stages of migration and for a short time after colonization of the gonad, such that they expand from an initial population of about 45 cells at E7.5 to a final population of about 25 000 cells at E13.5 (reviewed in [6]). Although a number of autonomous and nonautonomous factors have been shown to be required for proper migration and proliferation of PGCs [3, 4, 7–10], many details of the molecular and cellular mechanisms of these processes remain to be elucidated.

Decades of research have shown that the Steel (Sl) and Dominant white spotting (W) loci of mice are essential for embryonic and postnatal aspects of gametogenesis, as well as for development of hematopoietic cells and melanocytes (reviewed in [11–13]). The Sl locus encodes KITL, which is a member of the short-chain helical cytokine family [14, 15], and the W locus encodes KIT, which is a member of the PDGFRB superfamily of receptor tyrosine kinases (reviewed in [13, 16]). In mouse embryos, Kitl is expressed in somatic tissues in a gradient pattern along the pathway of PGC migration, with the highest levels in the genital ridges [17, 18], whereas Kit is expressed on PGCs from E7.5 through their migratory phase and until shortly after they cease proliferation in the gonads [19]. Mice with Sl and W mutations have long been known to have reduced numbers of PGCs [9, 13, 20–23], and KIT/KITL interactions have been shown to suppress apoptosis and increase proliferation of cultured PGCs [18, 24–27]. However, only two previous studies have suggested a specific role for KITL and KIT in PGC migration. In Sl-d/Sl-d embryos, ectopic PGCs located away from the normal migratory route were observed [20], and in W-e/W-e embryos, PGCs with abnormal retention and clumping in the hindgut and delayed migration were observed [21].

The large allelic series of mutants at KitlSl and KitW loci [28, 29] provides a valuable genetic resource to gain more information about gametogenesis. Our laboratory recently identified eight intragenic KitlSl mutations that exert graded effects on peripheral red blood cell (RBC) counts, thereby causing different degrees of hypoplastic anemia [30–32]. In the present study we used these novel mutations, as well as several other KitlSl mutations used previously in studies of PGC development, to provide insights into the roles of KITL during PGC migration in vivo. The results of this study suggest that there is only a partial requirement for KITL in proliferation of PGCs in the hindgut and movement of PGCs from the hindgut. However, once PGCs leave the hindgut, they become absolutely dependent on KITL for their proliferation. Of interest, hypomorphic Kitl mutations that have equivalent effects on PGC proliferation differ with respect to their effects on migration of PGCs, suggesting that different levels of KITL function are required to support these two aspects of PGC development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

The molecular defects in each KitlSl allele used in these experiments and their effects on viability of homozygous mutant mice have been described previously [23, 30–34] and are summarized in Table 1. For simplicity, KitlSl mutant alleles are indicated by their allele names (e.g., Sl-17H and Sl-39R) rather than their formal names (e.g., KitlSl-17H and KitlSl-39R). At birth, mice homozygous for lethal mutations had RBC counts that were not significantly different from those of Sl-gb/Sl-gb mice, whereas mice homozygous for viable mutations had RBC counts that were significantly greater than those of Sl-gb/Sl-gb mice [30, 31].


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  		TABLE 1. Summary of KitlSI mutations and their effects on E11.5 PGCs in whole-mount preparations

Mice heterozygous for each of the KitlSl mutations are maintained on a C3H/HeNCr background in a pathogen-free facility accredited by the Association for Assessment of Laboratory Animal Care at the University of Georgia. All procedures with mice were approved by the Institutional Animal Care and Use Committee of the University of Georgia. Matings were set up between heterozygotes carrying each of the mutations, and females were checked daily for copulatory plugs. The morning the plug was found was designated E0.5. Embryos were dissected at E9.5, E10.5, and E11.5; the caudal portions were processed for PGC analyses, and the rostral portions were used for genotyping. At each age, Kitl+/Kitl+ (wild-type, wt) littermates were used as controls for KitlSl/KitlSl (Sl/Sl) embryos and at least three embryos homozygous from each allele were analyzed. To reduce interembryo variation in development at E9.5 and E10.5, somites were counted, and only embryos with 19–21 somites at E9.5 and 35–37 somites at E10.5 were used for experiments.

DNA Genotyping

DNA extractions and polymerase chain reaction (PCR)-based genotyping methods were performed as previously described [30, 31]. An allele-specific PCR method was performed for genotyping Sl-17H embryos (details are available upon request).

Whole-Mount Histochemistry

Embryo dissection, fixation, and staining were performed essentially as described previously [35, 36]. Briefly, the caudal portions of E11.5 embryos were dissected and the internal organs were removed to expose the genital ridges. Embryos were fixed in an 8:1 mixture of ethanol and acetic acid for 30 min at 4°C, then washed with cold 100% ethanol, and stored in 100% ethanol at –20°C until further use. Before staining for alkaline phosphatase (AP) activity, the tissues were rehydrated for 2 h in 70% ethanol and washed in distilled water for 30 min. The embryos were stained for 15–20 min with freshly made AP staining solution (see below). Staining in the neural tube was used as an internal control.

Immunostaining

Embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 1 h, then washed twice in PBS, and stored overnight in 5% sucrose at 4°C. Next, the samples were transferred to 15% sucrose in PBS overnight at 4°C, then placed in OCT (Tissue Tek) and frozen at –80°C. Frozen sections of 14 µm were prepared on slides and stained for AP activity or for BrdU immunodetection (see below).

Alkaline Phosphatase Staining

Whole-mount embryos or frozen sections were stained with freshly made AP staining solution consisting of 0.5 mg/ml Fast Red (Sigma, St. Louis, MO) and 0.1 mg/ml alpha-naphthyl phosphate (Sigma), 4 mM MgCl2, and 10 mM borax [37] for 15 to 30 min at room temperature. Slides with sections had coverslips placed over them, while whole-mounts were stored in 50% glycerol with 0.01% sodium azide. Digital images of stained whole-mount embryos were captured with a QImaging MicroPublisher camera, and images of stained sections were captured with a Zeiss Axiocam or an Optronics MagnaFire camera. Slight modifications for brightness and contrast of the images were made using Adobe Photoshop software.

BrdU Labeling and Immunodetection

Pregnant females were injected i.p. with BrdU at 10 mg/kg body weight (Zymed). After 2 h, the mice were killed by CO2 asphyxiation, and the embryos were dissected and processed for frozen sections as described above, except that they were fixed overnight at 4° C in 4% PFA. After drying at room temperature, sections were stained for AP activity (as above), then washed in PBS until the OCT was completely removed. Incorporated BrdU was detected according to the manufacturer's instructions (Zymed). Briefly, the sections were treated sequentially with 0.25 mg/ml trypsin solution and 4 N HCl, then washed in PBS, blocked (CAS block, Zymed), and then incubated with anti-BrdU monoclonal antibody (1:100 dilution, Zymed) for 1 h at room temperature. After washing in PBS, the sections were incubated in streptavidin-fluorescein isothiocyanate (FITC; Vector Labs, Burlingame, CA) at 20 µg/ml for 30 min at room temperature, washed in PBS, and mounted in Prolong antifade medium (Molecular Probes, Eugene, OR). Analysis was performed with a Leica TS2 Laser Scanning Confocal System. PGCs were visualized at 580–647 nm for Fast Red (AP-positive) and 520 nm for FITC (BrdU-positive).

Statistics

Unpaired, two-tailed t-tests were performed on all values obtained from three to five embryos of each age and genotype using Prism software (Graphpad). P values of < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Screening of New KitlSl Mutations for Their Effects on Postmigratory PGCs

To semiquantitatively compare the effects of 11 KitlSl mutations (see Table 1) on PGCs at the end of their migratory stage, we used a whole-mount staining method for AP activity in genital ridges of E11.5 embryos. We first examined embryos homozygous for Sl-gb, which are deleted for the entire Kitl coding region [34], and for Sl-17H, in which E11.5 PGC numbers were previously shown to be only 22% of wt [23]. In contrast to numerous AP-positive PGCs in genital ridges of wt embryos (Fig. 1A), no AP-positive PGCs were observed in genital ridges of Sl-gb/Sl-gb embryos (Fig. 1D) and intermediate numbers of AP-positive PGCs were observed in Sl-17H/Sl-17H embryos and in Sl-gb/+ embryos (not shown). In analyzing the remaining mutants, the effects on PGCs were considered to be moderate if genital ridges had about the same number of AP-positive PGCs as that found in Sl-17H/Sl-17H embryos or severe if genital ridges had only a few PGCs or were devoid of PGCs. Representative images of genital ridges with moderate (Sl-39R/Sl-39R) and severe (Sl-30R/ Sl-30R) effects on PGCs are shown in Figure 1, B and C, respectively.



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  		FIG. 1. Whole-mount embryos at E11.5 stained for AP activity. E11.5 embryos homozygous for each Kitl allele (indicated in lower left) were exposed at the genital ridges and AP histochemistry performed. Arrows point to clumps of PGCs and the asterisk indicates a tear in the tissue. In comparison to wt embryos (A), AP-positive PGCs were present but were in reduced numbers in genital ridges of Sl-39R/Sl-39R embryos (B) and Sl-36R/Sl-36R and Sl-d/Sl-d embryos (not shown). In contrast, Sl-30R/Sl-30R embryos (C) and Sl-gb/Sl-gb (null) embryos (D) were nearly devoid of AP-positive PGCs. Embryos homozygous for each of the other five KitlSl mutations also were nearly devoid of PGCs at this age (images not shown, see Table 1 for summary). GR, genital ridge; NT, neural tube. Original magnification x5

The results of the screen for PGC defects in all E11.5 KitlSl mutants are summarized in Table 1. E11.5 embryos homozygous for the Sl-5R, Sl-22R, Sl-28R, Sl-30R, Sl-31R, and Sl-42R mutations were found to have severe effects on PGCs, whereas embryos homozygous for the Sl-36R, Sl-39R, and Sl-d mutations, as well as the Sl-17H mutation were found to have moderate effects on PGCs. These relative effects of the different mutations on PGCs parallel the effects of the same mutations on mouse viability and anemia [30, 31]. The remainder of our studies of PGC development focused on comparisons of the effects of the clearly hypomorphic mutations (Sl-36R, Sl-39R, and Sl-d) and the complete null (Sl-gb).

Graded Effects of Hypomorphic KitlSl Mutations on PGC Numbers in E11.5 Embryos

To examine the effects of the hypomorphic mutations on PGC development in more detail, we quantified postmigratory AP-positive PGCs in serial sections prepared from the caudal regions of E11.5 embryos spanning the entire genital ridges. Representative images of stained sections are shown in Figure 2, A–E and the numbers of PGCs in the genital ridges are tabulated in Figure 2F. In wt embryos at E11.5, virtually all PGCs had migrated to the genital ridge, with only a few scattered PGCs en route to the genital ridges (Fig. 2A), and the average number of PGCs per embryo was 1166 ± 91 (Fig. 2F). Sectioned genital ridges of Sl-gb homozygotes had no PGCs (Fig. 2, B and F), which is consistent with the absence of PGCs in whole-mount preparations of these null mutants (see above). In contrast, the average numbers of PGCs in genital ridges of Sl-39R and Sl-36R homozygotes (Fig. 2F) were 238 ± 33 (20% of wt) and 302 ± 46 (26% of wt), respectively, which are not significantly different from each other and are similar to values reported previously for E11.5 Sl-17H/Sl-17H embryos [23]. However, PGC numbers in Sl-d homozygotes were only 38 ± 15 (3% of wt), which is significantly lower than in Sl-39R and Sl-36R homozygotes. Thus, the numbers of postmigratory PGCs are reduced in the Sl-36R, Sl-39R, and Sl-d mutants, but these effects are all milder than in Sl-gb mutants. Furthermore, the effects on postmigratory PGCs in Sl-39R and Sl-36R mutants are equivalent to each other but are milder than those of Sl-d mutants.



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  		FIG. 2. Quantification of PGCs in genital ridges of E11.5 embryos. Serial transverse sections were prepared from the region of embryos homozygous for each allele (indicated in lower left in A–E) containing the genital ridges and stained for AP activity. Numerous AP-positive PGCs were found in wt embryos (A) while Sl-gb/Sl-gb embryos (B) had no AP-positive PGCs. Embryos homozygous for each of the hypomorphic mutations (Sl-d [C], Sl-39R [D], and Sl-36R [E]) had PGC numbers that were substantially reduced compared to wt embryos. In F, the bars represent the mean and SEM for total number of genital ridge PGCs per embryo for at least three embryos homozygous for each KitlSl allele. Compared to wt embryos, embryos homozygous for each KitlSl mutation had significantly reduced numbers of PGCs in the genital ridge. The percentage of wt values for each homozygous mutant are Sl-36R, 26%; Sl-39R, 20%; Sl-d, 3%; and Sl-gb, 0%. Asterisks indicate significant differences between wt and each homozygous mutant from unpaired t-tests (**P < 0.01; ***P < 0.001). Original magnification A–E x20

Defects in Total Numbers of PGCs in KitlSl Mutants at E9.5

To investigate whether PGCs in KitlSl mutants were affected at the stage just before their migration from the hindgut, we determined the numbers and locations of AP-positive PGCs in E9.5 (19–21 somite) embryos. Consistent with previous reports [4, 5], PGCs in E9.5 wt embryos were found primarily within the hindgut, either associated closely with the hindgut epithelium (Fig. 3A) or in the dorsal axis of the hindgut (Fig. 3B), and E9.5 wt embryos contained an average of 401 ± 73 PGCs per embryo (Fig. 4A). In E9.5 embryos homozygous for Sl-gb and Sl-d, PGCs were found predominantly in the ventral axis of the hindgut (Fig. 3, C and D). In contrast, PGCs were found in the proper locations in both Sl-39R/Sl-39R and Sl-36R/Sl-36R E9.5 embryos (Fig. 3, E and F). The total numbers of PGCs in E9.5 embryos homozygous for each of the KitlSl mutations were significantly decreased compared to those of wt embryos (Fig. 4A). These values in E9.5 Sl-gb/Sl-gb and Sl-d/Sl-d embryos averaged 76 ± 2 (19% of wt) and 90 ± 7 (22% of wt) PGCs per embryo, respectively (Fig. 4A), which were not significantly different from each other. However, the average number of PGCs in E9.5 Sl-39R/Sl-39R and Sl-36R/Sl-36R embryos was 126 ± 33 (31% of wt) and 177 ± 14 (44% of wt), respectively (Fig. 4A). As with the PGC numbers in the E11.5 genital ridge (see above), the PGC numbers in the E9.5 hindguts of the Sl-39R and Sl-36R mutants were both significantly (P = 0.002) higher than those of the other two mutants, but were not significantly different from each other.



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  		FIG. 3. Localization of PGCs in E9.5 in wt and Sl/Sl embryos. Transverse sections of embryos homozygous for each of the indicated KitlSl mutations were stained for AP activity. PGCs in normal locations are indicated with arrowheads and PGCs in ectopic locations are indicated with arrows. In A, the direction of the dorsal (D) and ventral (V) axis is shown as a double-headed arrow. HG, hindgut. In wt embryos, most PGCs were found associated with the hindgut epithelium (A) or located in the dorsal axis of the hindgut with only a few in ectopic sites (B). In Sl-gb/Sl-gb (C) and Sl-d/Sl-d (D) embryos, PGCs were in the ventral axis of the hindgut or in ectopic sites, but were not found in the dorsal axis of the hindgut. In Sl-39R/Sl-39R (E) and Sl-36R/Sl-36R (F) embryos, PGCs were distributed in a pattern similar to that of wt embryos, either associated with the hindgut epithelium or in the dorsal axis. Original magnification x20



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  		FIG. 4. Numbers and locations of PGCs in E9.5 and E10.5 embryos. In A and B, the total number of PGCs per embryo (mean ± SEM) are shown for E9.5 and E10.5, respectively. In C, the fold increases between PGC numbers at E9.5 and E10.5 are shown for each genotype. In D, the percentage of PGCs (mean ± SEM) mobilized from the hindgut was calculated by counting the number of PGCs in locations other than hindgut versus the total number of PGCs. In all panels, asterisks indicate significant differences from unpaired t-tests (*P < 0.05, **P < 0.01, ***P < 0.001). In A, B, and D, pair-wise comparisons were made between wt and each mutant, while in (C), pair-wise comparisons in PGC numbers were made between each genotype at E10.5 versus E9.5. Note that all mutants at both ages had PGC numbers that were significantly reduced from those of wt embryos (A and B). Pair-wise comparisons between each hypomorphic mutant and Sl-gb/Sl-gb were also conducted (not shown). At E9.5, none of the values in mutants were significantly different from each other while at E10.5, values for Sl-36R/Sl-36R and Sl-39R/Sl-39R mutants, but not Sl-d/Sl-d, were significantly different from those of Sl-gb/ Sl-gb. Significant differences in PGC numbers at E10.5 versus E9.5 (C) were observed in wt, Sl-36R/Sl-36R, and Sl-d/Sl-d mutants, but not in Sl-39R/Sl-39R and Sl-gb/Sl-gb. In addition, the percentage of PGCs at E10.5 that had migrated from the hindgut (D) were equivalent between wt and Sl-36R/Sl-36R embryos, but were significantly reduced in the other three mutants

Defects in Migration and Total Numbers of PGCs in KitlSl Mutants at E10.5

The results described above indicate that the KitlSl mutations differ with respect to their effects on PGCs before the onset of migration. To investigate the effects of these mutations on PGCs after the onset of their migration, we determined the numbers and locations of PGCs in sections of 35–37 somite embryos at E10.5. Representative images of E10.5 sections stained for AP activity are shown in Figure 5, the total numbers of PGCs per E10.5 embryo are summarized in Figure 4B, and the fold expansions in PGC numbers between E9.5 and E10.5 are shown in Figure 4C.



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  		FIG. 5. Localization of PGCs in E10.5 in wt and Sl/Sl embryos. Transverse sections of embryos homozygous for each of the indicated KitlSl alleles were stained for AP activity. PGCs with normal morphology are shown with arrowheads and PGCs that are disintegrating and appear apoptic are shown with arrows. A portion of each panel was enlarged and shown as an inset to reveal details of the PGC morphology. HG, hindgut; DM, dorsal mesentery; GR, genital ridge. In wt embryos at E10.5, all PGCs had left the hindgut and were located either within the dorsal mesentery or were about to enter the genital ridge (A), or had arrived at the genital ridge and had begun to coalesce with the somatic cells of the presumptive gonad (B). In Sl-gb/Sl-gb (C) and Sl-d/Sl-d (D) embryos, most PGCs were found in the hindgut and only a few were on the normal migratory route. In Sl-39R/Sl-39R embryos (E), most PGCs had been retained in the HG but a few migrated through the DM. In Sl-36R/Sl-36R embryos (F), the pattern of PGC migration was similar to that of wt embryos. Note the large number of apoptotic and disintegrating cells (arrows) in Sl-gb/Sl-gb (C), Sl-d/Sl-d (D), and Sl-39R/Sl-39R (E) embryos. More details of these abnormal cells are shown in Figure 6f'. Original magnification x20

In E10.5 wt embryos, there was an average of 1237 ± 169 PGCs per embryo (Fig. 4B), with most PGCs found in the dorsal portions of the mesentery (Fig. 5A) and within or near the genital ridges (Fig. 5B). This value at E10.5 is a 3-fold increase over the value observed in E9.5 wt embryos (Fig. 4C). In E10.5 embryos homozygous for each KitlSl mutation, the total numbers of PGCs were all significantly less than in wt at this age (Fig. 4B), and ranged from 78 ± 3 (4% of wt) in Sl-gb/Sl-gb embryos to 291 ± 39 (24% of wt) in Sl-36R/Sl-36R embryos. However, there are interesting differences between the effects of different mutations on the locations, total numbers, and expansion of PGCs between E9.5 and E10.5. In Sl-gb/Sl-gb and Sl-d/ Sl-d embryos, only a few PGCs were found outside of the hindgut and most of those retained in the hindgut had an abnormal morphology (Fig. 5, C and D). The PGC numbers in E10.5 Sl-gb/Sl-gb embryos averaged only 78 ± 3 PGCs (Fig. 4B), which was not a significant change from the 76 ± 2 PGCs observed at E9.5 (Fig. 4C). Surprisingly, the total PGC numbers in E10.5 Sl-d/Sl-d embryos were only 45 ± 7, which is about a 50% reduction from the 90 ± 7 PGCs observed in E9.5 Sl-d/Sl-d embryos (Fig. 4C). In E10.5 Sl-39R/Sl-39R and Sl-36R/Sl-36R embryos, some PGCs were found on the migratory path toward the genital ridges (Fig. 5, E and F), but many PGCs with an abnormal morphology were retained in the hindgut of the former mutants (Fig. 5E). In E10.5 Sl-39R/Sl-39R embryos, the average number of PGCs per embryo was 156 ± 26 (Fig. 4B), which is only slightly more (1.2-fold) than the number observed at E9.5 (Fig. 4C). However, in E10.5 Sl-36R/Sl-36R embryos, there was an average of 291 ± 39 PGCs per embryo. This value was significantly greater than any of the other mutants at E10.5 (Fig. 4B), and represents a significant (P = 0.034) increase of 1.6-fold over the E9.5 Sl-36R/Sl-36R value of 177 ± 14 (Fig. 4C). Thus, Sl-36R mutants were the only Kitl mutants to show a significant increase in PGC numbers between E9.5 and E10.5.

To quantify the extent of PGC migration, we determined the fraction of PGCs that had migrated from the hindgut compared with the total number of PGCs per embryo (Fig. 4D). In E10.5 wt embryos, 93% of the PGCs were found to have migrated from the hindgut (Fig. 4D). In E10.5 Sl-36R/Sl-36R embryos, 83% of the PGCs had migrated out of the hindgut, and this value was not significantly different from that of wt embryos (Fig. 4D). In contrast, the percentages of migrating PGCs observed in embryos homozygous for the Sl-39R, Sl-d, and Sl-gb mutations were significantly reduced compared to wt, with only 50%, 45%, and 31% of PGCs, respectively, found outside of the hindgut at E10.5 (Fig. 4D). These values in the Sl-39R, Sl-d, and Sl-gb mutants were not significantly different from each other.

In summary, there are four main observations from the analyses of E9.5 and E10.5 embryos. First, PGCs were present in embryos homozygous for each of the KitlSl mutations (including Sl-gb, the null mutation) at both E9.5 and E10.5, albeit at significantly reduced levels compared to wt embryos. Second, Sl-36R and Sl-39R embryos had significantly more PGCs at both ages than Sl-gb embryos. Third, PGC numbers at E10.5 versus E9.5 were not significantly different in Sl-39R and Sl-gb embryos, but were significantly decreased in Sl-d embryos, and significantly increased in Sl-36R embryos. Fourth, the percentage of PGCs that had migrated from the hindgut in Sl-36R embryos was nearly the same as in wt embryos, but in Sl-39R, Sl-d, and Sl-gb embryos was reduced to 30%–50% of wt embryos.

Rates of Proliferation of PGCs in Different Regions of the Migratory Pathway

Although it is well established that KITL regulates survival and proliferation of cultured PGCs (reviewed in [9, 10, 38]), there is little information regarding its requirements in PGCs at different stages of their development in vivo. To determine whether KitlSl mutations differ with respect to their effects on proliferation of premigratory versus migratory PGCs, we used BrdU incorporation to identify PGCs in S phase in hindgut, mesentery, and genital ridges of embryos at E10.5 and E11.5. Sections of wt, Sl-gb/Sl-gb, Sl-d/Sl-d, Sl-39R/Sl-39R, and Sl-36R/Sl-36R embryos were double-stained to reveal AP-positive PGCs and BrdU-positive cells and sections examined by confocal microscopy. In Figure 6, representative images of BrdU-positive (arrowhead) and BrdU-negative (arrow) PGCs in different locations of wt and Sl-39R/Sl-39R embryo sections are shown, including PGCs in the genital ridge and dorsal mesentery (Fig. 6, A, a, and a') and hindgut (Fig. 6, B and b) of E10.5 wt embryos, PGCs in the dorsal mesentery (Fig. 6, D and d) and hindgut (Fig. 6, F and f) of E10.5 Sl-39R/ Sl-39R embryos, and PGCs in the genital ridges of E11.5 wt (Fig. 6C) and E11.5 Sl-39R/Sl-39R (Fig. 6E) embryos.



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  		FIG. 6. BrdU incorporation in PGCs in different locations of embryos at E10.5 and E11.5. Pregnant females were injected with BrdU and sections of their embryos stained with Fast Red for AP activity (red) and with an FITC-labeled antibody to BrdU (green). Confocal images are shown with both channels merged. Images of wt embryos are shown in the top two rows and images of Sl-39R/Sl-39R embryos are shown in the bottom two rows. Arrowheads point to BrdU-positive PGCs and arrows point to BrdU-negative PGCs. Barbed arrows point to disintegrating, BrdU-negative PGCs that are probably apoptotic (f' only). GR, genital ridges; DM, dorsal mesentery; HG, hindgut; HGL, hindgut lumen. A, B: show images of an E10.5 wt embryo, and the boxes indicate enlarged regions shown in a, a', and b. D, F: show images of an E10.5 Sl-39R/Sl-39R embryo, and the boxes indicate enlarged regions shown in d, f, and f'. C, E: show images of genital ridges from E11.5 wt and Sl-39R/Sl-39R embryos, respectively. At all locations and ages, numerous BrdU-positive PGCs were observed but their frequency was generally higher in wt embryos than in Sl-39R/ Sl-39R. Note the many disintegrating, BrdU-negative PGCs (barbed arrows) in the Sl-39R/Sl-39R hindgut at E10.5 (f'), similar to those shown in Figure 5E. Original magnification A, B, D, and F x40; C and E x63

The proliferation index (i.e., the number of BrdU-positive PGCs versus the total number of PGCs counted), was calculated for PGCs in three regions of E10.5 and E11.5 embryos, and the results are summarized in Figure 7. PGCs in the E10.5 hindgut (Fig. 7A) are premigratory, PGCs in the E10.5 mesentery and genital ridge (Fig. 7B) are in the process of migrating or have just completed their migration, and PGCs in the E11.5 genital ridge (Fig. 7C) are postmigratory. While the proliferation index for premigratory PGCs in the E10.5 hindgut of wt embryos was 39% ± 10%, the corresponding values were slightly, but not significantly, reduced in all homozygous mutants (Fig. 7A). Of particular interest, 14% of the PGCs in the E10.5 hindgut of Sl-gb/Sl-gb embryos were BrdU-positive (Fig. 7A), indicating that premigratory PGCs in the E10.5 hindgut were able to proliferate even in the complete absence of KITL. In striking contrast, no migratory PGCs in the E10.5 mesentery and genital ridges of Sl-gb/Sl-gb embryos were found to be BrdU-positive (Fig. 7B), suggesting that there is an absolute requirement of KITL for PGC proliferation once they leave the hindgut. However, the proliferation indices of migratory and postmigratory PGCs in each of the hypomorphic KitlSl mutants at E10.5 (Fig. 7B) and E11.5 (Fig. 7C) were all significantly reduced compared to those of E10.5 wt embryos at the same stage, and ranged from 54% to 66% of the corresponding wt values. Of interest, the proliferation index of PGCs in Sl-39R/Sl-39R embryos was 46%, which is significantly greater (P = 0.04) than the value of 32% observed in Sl-36R/Sl-36R embryos. Of all the assays conducted in the present study, the proliferation index of E11.5 PGCs is the only instance in which the defect in Sl-39R/Sl-39R embryos was milder than that of Sl-36R/Sl-36R embryos.



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  		FIG. 7. Proliferation indices of PGCs along their migratory pathway in E10.5 and E11.5 embryos. At each age, serial sections of embryos homozygous for each KitlSl allele (indicated below the bars) were processed for AP activity and BrdU immunodetection (see Fig. 6 for examples) and the percentage of BrdU-positive PGCs versus the total number of PGCs were calculated. A range of 35–95 total PGCs in mutant embryos and 100–200 total PGCs in wt embryos were scored in this way, and the means and SEM for at least three embryos of each genotype and age are shown. For data in each panel, asterisks indicate significant differences between wt embryos and embryos homozygous for each of the KitlSl mutations in unpaired t-tests (*P < 0.05, **P < 0.01, and ***P < 0.001). Note that in the E10.5 hindgut (A), none of the mutants had BrdU-positive indices that were significantly different from those of wt, whereas in PGCs that had left the hindgut (B), all of the mutants had BrdU-positive indices that were significantly lower than in wt. These values at E10.5 in the three hypomorphic mutations were all substantially higher than in the null mutant (Sl-gb/Sl-gb), but none were significantly different from each other (not shown). Similarly to E10.5, the BrdU-positive indices of PGCs in the E11.5 genital ridges (C) of all mutants were significantly lower than in wt embryos (note that no PGCs were found in genital ridges of Sl-gb/Sl-gb embryos at this age). Of interest, the BrdU-positive indices of PGCs in the Sl-39R/Sl-39R genital ridges at E11.5 were significantly higher (not shown) than the corresponding values in the other two hypomorphic mutants

Although the objective of this analysis was to quantify the extent of PGC proliferative defects in Sl/Sl embryos, KITL is also known to suppress apoptosis of PGCs [26]. Indeed, many PGCs were observed in hindguts of Sl-gb/Sl-gb, Sl-d/Sl-d, and Sl-39R/Sl-39R embryos (Fig. 5, C–E) that had abnormally intense staining for AP activity and appeared to be disintegrating. However, such abnormal PGCs were very infrequent in hindguts of wt (Fig. 5, A and B) and Sl-36R/Sl-36R embryos (Fig. 5F and not shown). From our confocal analyses, we also confirmed the presence of a large number of disintegrating and apoptotic PGCs in the hindgut of the Sl-39R/Sl-39R embryos (see arrows in Fig. 5E and barbed arrows in Fig. 6f'). PGCs with a similar appearance were shown previously to be nonmotile and apoptotic [5, 7, 37–40], suggesting that the abnormal PGCs in the Sl/Sl mutants are probably apoptotic.

A summary of the observations made of all of the effects of homozygous KitlSl mutations on different aspects of PGC development between E9.5 and E11.5 is shown in Table 2. In general, Sl-36R, Sl-39R, and Sl-d had milder effects than Sl-gb, with Sl-36R usually being the mildest and Sl-d being the most severe of the hypomorphic mutations. However, there are intriguing differences in relative strengths of the mutations at various PGC developmental stages. At E10.5, none of the four mutations (including Sl-gb, the null mutation) had significant effects on the number or proliferation index of premigratory PGCs in the hindgut. However, at the same age, the percentages of PGCs that had migrated out of the hindgut were severely affected by three of the mutations, while migration occurred to nearly the same extent in Sl-36R mutants and in wt embryos. While Sl-36R and Sl-39R had equivalent effects on BrdU-positive migratory PGCs at E10.5, Sl-39R had a milder effect than Sl-36R on BrdU-positive postmigratory PGCs at E11.5. These differences in degree of impact of the KitlSl mutations at various stages of PGC development suggest that there are either quantitative or qualitative changes in the activities of the KITL mutant proteins, or in the responses of PGCs to the KITL mutant proteins as PGC development progresses.


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  		TABLE 2. Relative activities of homozygous KitlSl mutations at different stages of PGC developmenta


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the most useful aspects of the KitlSl allelic series is that different hypomorphic alleles exhibit graded effects, indicating that subtle differences in activities of the different KITL mutant proteins are reflected in quantifiable biological responses by KITL-dependent cell types. In previous studies of peripheral RBCs [30] and in the present studies of PGC development (see Table 2), graded effects were observed for hypomorphic KitlSl mutations, with either or both of the Sl-36R and Sl-39R mutations consistently having milder effects than Sl-d. Each of the hypomorphic KitlSl alleles is predicted to encode KITL mutant proteins with different molecular defects (see Table 1), including a missense substitution that also affects N-linked glycosylation site (Sl-39R), premature termination due to a nonsense mutation (Sl-36R), and premature termination with an out-of-frame C-terminus due to an intragenic deletion (Sl-d). Thus far we cannot discern any correlation between relative effects on function and location or type of Kitl mutation. Preliminary studies with cultured cells revealed abnormal glycosylation patterns of Sl-39R-encoded KITL (M.A.B., unpublished observations), but whether this affects KITL function remains to be determined. Detailed studies of the various KITL mutant proteins will be needed to explain the differences in allele strength, but they could reflect alterations in localization or function of mutant KITL proteins at different developmental stages, altered interactions of other proteins with KITL mutants, or altered responses of germ cells at different developmental stages to the mutant KITL proteins.

Early studies demonstrated that PGC numbers in KitlSl [20] and KitW [41] mutants were similar to those of wt in E8 and E9 embryos; therefore, allocation and early migration of PGCs are believed to be independent of KITL and KIT (see reviews in [9, 10]). In the present study, we found that the numbers of PGCs in the E9.5 hindgut of embryos homozygous for null or hypomorphic KitlSl mutations were significantly reduced compared to those of wt embryos (Fig. 4A). This observation suggests that PGCs become at least partially KITL-dependent shortly after they arrive in the hindgut. However, Sl-gb/Sl-gb embryos have some PGCs at E9.5, albeit only about 20% of wt embryos, indicating that PGCs at this stage are not completely dependent on KITL. In addition, there are BrdU-positive PGCs in the Sl-gb/Sl-gb hindgut (Fig. 7A), indicating that PGC proliferation in the hindgut is not completely dependent on KITL. Although we did not quantify the numbers of apoptotic cells in this study, many PGCs with an abnormal morphology like that of apoptotic cells were observed in E10.5 Sl-gb/Sl-gb and Sl-d/Sl-d embryos (Fig. 5, C and D). Between E9.5 and E10.5, the PGC numbers are constant in Sl-gb/Sl-gb embryos, in contrast to the 3-fold expansion observed in wt embryos (Fig. 4C). Therefore, expansion of PGC numbers between E9.5 and E10.5 is completely dependent on KITL functions, but whether this occurs through preferential effects on apoptosis or proliferation remains to be determined.

Although the trend for the proliferation indices of PGCs in the E10.5 hindgut was to be reduced in all KitlSl mutants, these values were not significantly different from those of wt embryos (Fig. 7A). A strikingly different observation was made for PGCs that had migrated away from the hindgut in the E10.5 embryos, in which proliferation indices of PGCs in the mesentery and genital ridges of all Sl/Sl embryos were significantly less than in wt embryos (Fig. 7B). Most importantly, in E10.5 Sl-gb/Sl-gb embryos, BrdU-positive PGCs were found only in the hindgut and not in the mesentery (Fig. 7B). While the total numbers of PGCs and the percentages of migrating PGCs were about the same in E10.5 Sl-gb/Sl-gb and Sl-d/Sl-d embryos (Fig. 4, B and D), migrating BrdU-positive PGCs were observed in Sl-d/Sl-d embryos but not in Sl-gb/Sl-gb embryos (Fig. 7B). Therefore, the lack of BrdU-positive migrating PGCs in E10.5 Sl-gb/Sl-gb embryos was probably not due to an indirect effect of reduced PGC numbers and was likely the direct result of the absence of KITL. These results suggest there is an absolute requirement for KITL in PGC proliferation once these cells start migrating away from the hindgut. It has been demonstrated that synergy between KITL and other cytokines such as leukemia inhibitory factor, fibroblast growth factor, and interleukin-4 is essential for abundant growth of PGCs in vitro [18, 24, 42]. Therefore, we hypothesize that other cytokines are able to partially compensate for the absent or reduced KITL functions in the hindgut of Sl/Sl embryos. However, once PGCs begin migrating away from the hindgut, the cells become critically dependent on KITL and it does not appear that there are any in vivo compensatory mechanisms that occur in the absence of KITL.

A key question about KITL is whether it plays a direct role in PGC migration. Defects in PGC migration in Sl/Sl embryos were apparent in the E9.5 hindgut, where PGCs in Sl-gb/Sl-gb and Sl-d/Sl-d embryos did not undergo ventral-to-dorsal migration (Fig. 3, C and D). At E10.5, defects in PGC migration in all mutant embryos except for Sl-36R/ Sl-36R were even more pronounced, with more than 50% of PGCs retained in the hindgut (Fig. 4D). The observations made in the present study on PGCs in Sl/Sl mutants are strikingly similar to those reported previously in W-e/W-e mutants. In the latter mutants, there was no increase in PGC numbers between E8.5 and E10.5, PGCs in the E9.5 hindgut remained in the ventral axis and did not move dorsally, and nearly half of PGCs in E10.5 embryos failed to leave the hindgut [21]. These observations on localization of PGCs in Kitl and Kit mutant embryos suggest that KITL-KIT signaling does play an active role in migration of these cells. While previous in vitro studies indicate that KITL does not act as a chemotropic factor for PGCs [25], it has been shown to be a chemokinetic factor for melanocyte migration [43]. Thus, KITL may act to mobilize PGCs for their migration out of the hindgut. However, it is not currently known whether KITL exerts its effect on PGC migration through a direct or indirect mechanism. Of interest, the percentage of PGCs that migrated from the hindgut was equivalent between Sl-36R/Sl-36R and wt embryos. This significant difference in movement of PGCs from the hindgut of Sl-36R/Sl-36R embryos and lack of this movement in Sl-39R/Sl-39R and Sl-d/Sl-d embryos (Fig. 4D) occurs at a time when the percentage of BrdU-positive PGCs in the three mutants is virtually identical (Fig. 7, A and B). Thus, KITL function in Sl-36R/Sl-36R embryos is enough to support migration but is not enough to support normal proliferation of PGCs. To our knowledge, this is the first report that migration and proliferation of PGCs may require different aspects of KITL function.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Mia Buehr for helpful advice on the whole-mount staining, and to Monal Patel, Dai Li, Harlan Starr, Cristina Budde, and Eric Keller for their technical assistance. We also thank Dr. Nancy Manley for sharing histological equipment, and Dr. Mark Farmer for assistance with the confocal microscope.


   FOOTNOTES
 
1 Supported by grants to M.A.B. from the National Science Foundation (IBN-9728428) and National Institutes of Health (GM65393). Back

2 Correspondence: Mary A. Bedell, Department of Genetics, University of Georgia, Athens, GA 30602-7223. FAX: 706 583 0691; bedell{at}uga.edu Back

3 Current address: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160 Back

4 Current address: The Children's Hospital of Philadelphia, Abramson Research Center, Philadelphia, PA 19104 Back

Received: 15 April 2005.

First decision: 9 May 2005.

Accepted: 10 May 2005.


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 RESULTS
 DISCUSSION
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