Keywords: gcna antibody
Description: The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes,
The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell.
Egg and sperm derive from precursors in the early embryo called primordial germ cells (PGCs). The mechanisms underlying the migration of PGCs through the embryo to the forming gonads remain unclear. In a genetic screen, we identified a role for the receptor Ror2 and its ligand Wnt5a in promoting PGC colonization of the embryonic gonads. By ex vivo culture, we show that Ror2 acts autonomously in PGCs to enhance their polarized response to the chemotactic factor SCF. Asymmetric distribution of ROR2 within PGCs in vitro and in vivo suggests that signaling via Ror2 locally amplifies cell polarity in response to other directional cues. These studies identify a novel relationship between Ror2 and cKit signaling in polarized migration.
Primordial germ cells (PGCs) are embryonic precursors of the gametes that arise before other major cell lineages in most multicellular animals . This early specification necessitates a lengthy migration through the developing embryo in order to reach the nascent ovaries or testes. In mice, epiblast-derived cells seal their germline commitment at the embryo periphery ∼e7.25, then enter the forming endoderm and travel through the elongating hindgut epithelium. PGCs make a coordinated exodus into the surrounding mesentery at e9.5 and then converge on the gonadal ridges between e10.5 and e11.5. Though exquisitely coordinated, this process is also imperfect; by e12 when migration is over, stragglers consistently remain outside the gonad in midline tissues, and are eliminated by apoptosis . The importance of balanced regulation of PGC survival and migration is evident by the consequences of dysregulation: failure to survive or reach the gonad can lead to sterility, whereas inappropriate survival can lead to germ cell tumors . . The molecular mechanisms underlying the migration of these evolutionarily essential but relatively inaccessible cells remain largely unknown in the mammalian germline. Here we conducted a forward genetic screen for germ cell defects in mouse embryos and identified an allele of Ror2 .
Ror2 is a highly conserved receptor tyrosine kinase with homologs in many metazoans from Aplysia to Drosophila to humans . Widely expressed during development, Ror2 has been implicated in chondrocyte differentiation, cochlear, craniofacial, heart, limb and gut morphogenesis in mice and humans  –. Work in a number of different organisms suggests that Ror2 signaling affects cell polarity. In the developing mouse gut epithelium, the protein exhibits apicobasal polarity in its distribution . Polarity is requisite for cells undergoing directed migration, cell division in a particular orientation, as in asymmetric divisions, and for the organization or shape of cells with respect to their neighbors, for example in convergent extension. Defects in cell shape and convergent extension have been reported in the mouse gut, organ of Corti, and Xenopus gastrula as a result of Ror2 signaling loss .  –. Ror2-mediated polarized cell division has been reported in C. elegans . A role for Ror2 signaling in directional migration has been reported in the mammalian palate  and in several cell lines, via c-Jun N-terminal Kinase and the actin-binding protein FilaminA  – .
Phenotypic resemblance between mouse embryos with targeted deletions of Ror2 and those deficient for Wnt5a first suggested that these genes share a common pathway . . . . Biochemical approaches later confirmed ligand-receptor interactions between Wnt5a and Ror2 via the cysteine-rich (frizzled-like) extracellular domain of Ror2 . Indeed, the expression patterns of Wnt5a and Ror2 virtually overlap in the primitive streak, tail mesoderm and limb buds of midgestation mouse embryos  –. Wnt5a was similarly invoked in aspects of cell polarity, including orienftation of cell division in the limb . convergent extension movements and cell shape in the Xenopus gastrula . . and polarized migration in a melanoma cell line . . Many of these different Wnt5a-Ror2 pathway mutants exhibit similarly altered distribution of polarity mediators, such as Disheveled . . . the Dlg-Lgl complex . . Van Gogh . or adhesion receptor complexes  .
The identification of the Ror2 Y324C mutant in an unbiased screen for PGC defects brings to light a previously unrecognized function of Ror2 in germ cell development. We show here that Ror2 and its putative ligand Wnt5a promote efficient migration of PGCs to the embryonic gonads. These studies demonstrate a cell intrinsic function for Ror2 in potentiating the polarized response to secreted KitL, drawing a new link between Ror2 and Kit signaling in PGC migration.
As an unbiased approach to identifying new genes involved in mouse germ cell development, we conducted a genome-wide recessive ethylnitrosourea (ENU) mutagenesis screen for PGC defects in e9.5 embryos . One of the mutations identified based on the presence of ectopic PGCs mapped to the region of Ror2. An A to G transition in exon 7 at nucleotide 1203 causes a tyrosine to cysteine substitution at position 324 (Y324C) of the ROR2 predicted protein ( Figure 1A ). This missense mutation falls in the kringle domain, a conserved structural motif in the ROR2 extracellular domain. Ror2 Y324C homozygous embryos exhibit defects in tail elongation ( Figure 1B, 1C ) and somite segmentation, similar to the Ror2 targeted deletion allele ( Figure 1D ) .  ; like the knockout, Ror2 Y324C mutants die perinatally. Ror2 immunoblotting on e10.5 embryo lysates revealed a double band at approximately 200 kD; both bands were present in similar amounts between WT and Ror2 Y324C mutants ( Figure 1E ). In humans, missense mutations in the hRor2 cysteine rich, kringle and tyrosine kinase domains that are associated with Robinow syndrome cause the protein to be retained in the endoplasmic reticulum . We examined the expression of ROR2 at e11.5 by intracellular staining with an antibody directed against the cytoplasmic tail of the receptor; by flow cytometry signal was present at similar levels in WT and Ror2 Y324C ( Figure 1F. right). These experiments suggest that the mutation does not affect protein stability but do not discriminate between its normal or abnormal subcellular localization.
To determine whether Ror2 is expressed in PGCs, we employed a transgenic mouse strain, Oct4ΔPE-EGFP. which expresses Enhanced Green Fluorescent Protein (GFP) under a modified Oct4 reporter that is specific to PGCs during mid-gestation .  ( Figure 1F ). By flow cytometry, ROR2 intracellular staining was present within the GFP + population at e11.5 ( Figure 1F ). Furthermore, when Oct4ΔPE-EGFP + PGCs were purified flow cytometrically, Ror2 transcript could be detected by semi-quantitative RT-PCR; more transcript appeared to be present in GFP negative cells from embryo tails (denoted ‘soma’; Figure 1G ), where high levels of Ror2 have been previously detected by in situ hybridization . The purity of sorted PGCs was confirmed by RT-PCR for Oct4. which was absent in somatic cells, and KitL. which was confined to soma ( Figure 1G ). ROR2 protein was similarly detected in histologic sections with two different antibodies; signal appeared to be concentrated at the apical surface of the hindgut and somites  and in the ventral neural tube in wild type embryos ( Figure 1H–1H′ ), as previously reported . ROR2 was also present throughout the e10.5 dorsal mesentery and enriched at the membrane of wild type PGCs ( Figure 1I, 1I′ ). These studies confirm the expression of Ror2 mRNA and protein in migratory and postmigratory PGCs, as suggested by previous microarray data . and demonstrate the stable expression of Ror2 Y324C mutant protein.
A major ligand for Ror2 is believed to be Wnt5a. Wnt5a mRNA expression in the tail and hindgut of the embryo overlaps that of Ror2. although precisely which cells secrete Wnt5a remains unclear  –. By RT-PCR we determined that Wnt5a transcript is present in sorted Oct4ΔPE-EGFP + PGCs, although it is more abundant in GFP negative somatic cells of the tail and hindgut ( Figure 1G ). In histological sections stained with a WNT5A antibody, we observed bright foci as well as intercellular signal in the intestine and gonadal ridges ( Figure 1J–1J′ ), which both lie on the PGC migratory route. Upon closer examination, WNT5A could be detected at variable levels at or near the surface of PGCs ( Figure 1J. inset). These results collectively identify a role for Ror2 in PGC development and raise the possibility that PGCs perceive paracrine or autocrine WNT5A signals via the Ror2 receptor.
We next characterized the phenotypes of PGCs in Ror2 Y324C mutants. In e10.5 embryos stained with SSEA1 antibody . . PGCs can be visualized migrating through the dorsal mesentery ( Figure 2A ). In Ror2 Y324C mutants, PGCs do not migrate rostrally, but remain in the mesentery surrounding the caudal hindgut ( Figure 2B ), as well as on the surface of the tail and in the allantois (Figure S3B, S3C ). At e11.5, immunostaining with GCNA (a marker of postmigratory PGCs  ) revealed a reduction in the number of PGCs within Ror2 Y324C gonad primordia compared to wild type; furthermore, the distribution of Ror2 Y324C PGCs was skewed toward the caudal end of the gonad and extragonadal PGCs were increased in midline tissues ( Figure 2E, 2F ). At e12.5, male and female Ror2 Y324C gonads appeared less densely populated with PGCs ( Figure 2I, 2J. female not shown).
We developed techniques for the quantification of PGCs in the entire embryo or embryonic gonad with confocal imaging and 3D analysis (see Methods). The mean number ± standard deviation of PGCs in mutants at e10.25 (443±73) was similar to wild type (551±157; Figure 2D ), in spite of their abnormal distribution. However, at e11.5, the number of Ror2 Y324C PGCs in gonads was diminished (1243±369) compared to wild type (2598±265, p = 0.0002; Figure 2H ). At e12.5 this difference persisted (p = 0.009), as 7058±2282 PGCs were counted in wild type gonads and 3825±1144 in Ror2 Y324C ( Figure 2L ); male and female were combined here, as their numbers were similar. The PGC estimates and corresponding doubling time found in wild type embryos (13.4–16.7 hours) are similar to those reported previously . The doubling time for Ror2 Y324C PGCs falls within this range for postmigratory PGCs, but was more protracted from e10.25–11.5 (20 hours), predicting an earlier decline in proliferation or rise in apoptosis. We compared the phenotype of Ror2 Y324C PGCs to that of a targeted Ror2 knockout allele . At e11 and e12, we observed a similar PGC decrease compared to age-matched wild type C57Bl/6 littermates (Figure S1 ). Despite genetic background differences, the PGC deficit in Ror2 −/− embryos was indistinguishable from that resulting from our point mutation. This similarity suggests that Ror2 Y324C is a strong loss of function allele.
Previous work has shown that Ror2 lies downstream of Wnt5a both biochemically and genetically . . . Therefore, we examined the PGCs of Wnt5a null mutants and found a more pronounced and earlier deficit compared to Ror2. At e10.5, Wnt5a −/− PGCs were similarly caudally distributed ( Figure 2C ) but were already depleted in number (242±121) compared to wild type ( Figure 2D ). We noted significant reductions at e11.5, when 310±148 PGCs were present in Wnt5a gonads ( Figure 2G, 2H ), and by e12.5 this number increased to 1587±985 ( Figure 2K, 2L ). Consistent with biochemical data . . the greater severity of the Wnt5a germ cell phenotype suggests that this ligand operates through other receptors besides Ror2. Together, these studies demonstrate that Wnt5a and Ror2 mutants are phenocopies in the PGC compartment, which corroborates their function there as ligand and receptor.
To investigate the cellular mechanism underlying the PGC deficit in Ror2 and Wnt5a mutants, we extended our quantitative imaging in the embryonic gonad to include markers of proliferation and death. We performed triple immunofluorescence for GCNA, as well as phospho-histone H3 (PHH3), and cleaved PARP to quantify subsets of mitotic and apoptotic PGCs, respectively (Figure S2 ). No differences were observed in cPARP expression among postmigratory PGCs in wild type, Ror2. or Wnt5a gonads ( Figure 3 ). However, analysis of e10.5 embryo sections revealed an increase in cPARP expression among still migratory PGCs in Ror2 Y324C (11.9±1.6%) and Wnt5a (11.4±4.8%) relative to wild type (4.3±1.3%; Figure 3A. staining shown in Figure S3 ). This discrete wave of apoptosis preceded any observed loss in cell number in Ror2 mutants. We next compared the frequencies of programmed cell death between PGCs within and outside the e11.5 gonad. Caspase3 staining in histologic sections revealed similar frequencies in properly localized PGCs, but increased apoptosis among extragonadal Ror2 Y324C germ cells (14.0±1%) compared to wild type (2.4±2.4%) and Wnt5a (4.9±1.2%; Figure 3B ). Examination of PGC proliferation by PHH3 staining did not reveal significant differences in the frequency of proliferating PGCs between wild-type, Wnt5a. or Ror2 Y324C embryos at e10.25 or e11.5 ( Figure 3C ); despite several hundred PGCs counted in each genotype at each stage, the variation was large. Together these results demonstrate that increased apoptosis rather than reduced proliferation contributes to the PGC deficit in Ror2 and Wnt5a mutants.
Restriction of the observed burst of programmed cell death to migratory PGCs, together with its absence in gonadal PGCs, suggested that the location of mutant germ cells could be a factor in their elimination. On one hand, migrating mutant PGCs could be more sensitive to the reduced levels of survival factors such as KITL and SDF1 in the dorsal mesentery as compared to the gonad . . . . where they are more protected from death. On the other hand, inefficient migration may lead to an accumulation of ectopic Ror2 PGCs, which die in an environment lacking survival factors . To distinguish between these possibilities, we rescued PGC apoptosis in Ror2 mutants by generating double mutants with a targeted knockout of the pro-death gene Bax. Previous work established an increase in ectopic PGCs in e11.5 Bax single mutants due to the lack of apoptosis of mis-migrated PGCs, although the total number of PGCs remained unchanged . Genetic ablation of Bax in Ror2 Y324C mutants increased the number of midline and ectopic PGCs, but did not restore the number of PGCs in the gonads. At e11.5, 1815±362 PGCs were counted in Ror2; Bax double mutant gonads, which did not differ from 1275±359 in stage matched Ror2 littermates (p = 0.07; Figure 3D ). Although Bax does not rescue PGCs in Ror2 gonads, a significant increase in the total number of PGCs in the entire aorta-gonad-mesonephros region of double mutants compared to Ror2 single mutants (p = 0.036; data not shown) reflects rescue of ectopic PGC death throughout the midline in Ror2; Bax ( Figure 3G. compared to Figure 3E, 3F ). This result suggests that defects in migration are primary to the defects in PGC survival in Ror2 mutants.
We next directly compared the efficiency of PGC migration in mutants. When quantified in histological sections at e11.5 ( Figure 3H, 3I ), ectopic (extragonadal) PGCs comprised over 70% of the total PGCs in Wnt5a mutants, and 30% in Ror 2 Y324C. compared to less than 5% in wild type ( Figure 3J ). Poor cell trafficking could therefore account for the loss of gonadal PGCs of both mutants at e11.5. However, it remained unclear whether morphologic differences in the caudal hindgut of both mutants cause the observed migration defects. Indeed, morphological and molecular analysis revealed a shortening and widening of the Ror2 Y324C caudal hindgut at e9.5 ( Figure 1E. Figure S4 ), which corresponds to the PGC exodus from the hindgut. Upon examining embryos before hindgut formation, we confirmed that the location and number of early PGCs were indistinguishable from wild type in Ror 2 Y324C as well as Wnt5a at e7.5–8.0 (Figure S5 ). By e9.0, we observed ectopic PGCs accumulated in the allantois, throughout the tail mesoderm, and caudal hindgut of Ror2 Y324C mutants (Figure S6A –S6C ). However, this phenotype does not distinguish between the possibilities of an intrinsic PGC migration defect versus a structural abnormality that hindered the passage of PGCs from the allantois into the hindgut pocket.  .
Given the previously demonstrated expression of Wnt5a throughout the allantois and primitive streak . we wondered whether it could act chemotactically to draw PGCs from the allantois into the hindgut. To address this possibility, we implanted beads coated with WNT5A into the caudal region of e8.0 embryos. Control BSA-coated beads delivered to the hindgut pocket did not disrupt embryo or PGC development over 24 h culture (Figure S6D, S6G ). Beads soaked in recombinant WNT5A or concentrated conditioned medium did not alter the course of PGCs, whether placed directly in or near their path (Figure S6E, S6H ). Consistent with previous reports . beads similarly impregnated with the known chemoattractants SDF1and Stem Cell Factor (SCF, or secreted KitL) affected migration of PGCs at close range, inducing occasional deviation from their normal route (Figure S6F, S6I ). Although we did not assess biological activity of Wnt5a-soaked beads, when PGCs were explanted and cultured over 24 hours, we did measure a modest increase in their number in the presence of recombinant WNT5A, suggesting that WNT5A is biologically active (Figure S6J ). Collectively these results could indicate that WNT5A may not act as a direct chemotactic cue for PGCs; rather, they suggest that Wnt5a and Ror2 could have a permissive role to allow the response of PGCs to other navigation signals.
Reduced PGC colonization of the gonads in Ror2 and Wnt5a mutants could result from disruptions in hindgut architecture or from intrinsic defects in PGC migration. The expression of Ror2 in both PGCs and their surrounding tissues does not provide any insight. In fibroblasts, previous work showed that WNT5A induces motility, cell shape change, and chemotaxis via Ror2 . . We did not observe PGC chemotaxis toward a WNT5A source in cultured embryos. Other work shows that WNT5A polarizes melanoma cells when a chemotactic gradient is present . We sought a direct test of migratory capacity of isolated Ror2 PGCs. However, when sorted from e9.5–10.5 embryos using the Oct4ΔPE-EGFP reporter, we did not observe any migration of wild type PGCs toward SDF1 or SCF in a transwell assay, as previously reported . However, Farini et al. also showed that SCF elicited cytoskeletal changes and membrane protrusions in isolated PGCs over a short period . We replicated this result using flow cytometrically purified Oct4ΔPE-EGFP + cells from e9.5 embryo posteriors and maintained on Matrigel in serum-free media. Without the support of feeder cells, which provide growth factors, survival was poor and PGCs appeared round and devoid of filopodia ( Figure 4A ). As reported . the addition of SCF induced morphological changes in PGCs, including the acquisition of membrane protrusions and ellipsoid shape ( Figure 4B ). We noted that the shape assumed by Ror2 PGCs cultured in these conditions differed from wild type, and therefore endeavored to quantify this morphology. Using phalloidin to define the F-actin cytoskeleton, we measured the longest cellular axis and the orthogonal short axis of the cell body; we then computed an Elongation Index (ALong −AShort )/(ALong +AShort ), which approaches zero for round cells, such as the example in Figure 4C. Elongated cells often extended filopodia or lamellopodia, which were not included in the measurement, but which usually aligned with the long axis ( Figure 4D–4D′ ). Following 7 hours of culture without SCF, a mean Elongation Index (EI) of 0.044 was observed in wild type PGCs, which increased to 0.088 in the presence of SCF (p = 0.0005; Figure 4E ). PGC elongation continued to increase in culture up to 20 hours in SCF, to a mean EI of 0.169, ( Figure 4F ). By contrast, Ror2 PGCs mutants cultured in parallel exhibited a mean EI of 0.114 in SCF, which is significantly lower than mixed wild type and heterozygous PGCs (p = 0.005). When SCF was excluded from the media, but a strip of Matrigel was introduced along one side of the culture well to produce a gradient, the elongation response of WT PGCs was similar to that in static SCF, with a mean EI of 0.20; the graded source of SCF did not increase the EI of Ror2 PGCs: mean EI of 0.11, p = 0.0004 ( Figure 4G ). Short axis dimensions did not differ between wild-type and mutant PGCs (data not shown), but as we did not assess the z-axis length, these results do not exclude the possibility that Ror2 PGCs occupy less volume instead of remaining more spherical than wild type following SCF treatment.
We also examined the capacity of PGCs to align with a chemotactic gradient. Using the long cellular axis explained above, we measured the angle between this axis and a line orthogonal to the source of SCF (schematized in Figure 4H ). When SCF was uniformly present in the media (here termed static), the orientation of WT PGCs was randomly distributed between 0 and 90° from an arbitrary line, as would be expected. However, following 20 hours in an SCF gradient, wild-type PGC orientations were biased toward lower angles; that is, they showed greater alignment parallel to the gradient ( Figure 4I. p = 0.0018). Ror2 PGCs did not preferentially orient toward the SCF source, but were randomly distributed in their orientations ( Figure 4I. p = 0.0004). Taken together, these in vitro studies reveal a compromised ability of Ror2 PGCs to respond to SCF, either by elongating or orienting toward a chemotactic gradient. Because these assays were carried out in the absence of feeder cells using Oct4-ΔPE-EGFP + cells sorted to >95% purity, the observed defects must be cell-intrinsic.