encode similar proteins (59% identity) and have similar developmental functions [21
resides next to lagB1
, which encodes another predicted transmembrane protein with several Ig repeats, and lagD1
resides next to lagE1
– a close homolog of lagB1
(68% identity) [25
]. In both gene pairs, the ORFs face away from each other, separated by a short intergenic sequence. To test whether these gene pairs could be involved in kin discrimination, we measured their polymorphism levels in wild isolates. We sequenced these genes in wild isolates and found that lagB1
are polymorphic () whereas lagD1
are nearly invariant (data not shown). The ratio between non-synonymous (dN) and synonymous (dS) substitutions is an indicator of evolutionary processes, whereby dN/dS > 1 suggests positive selection [26
]. We found that lagB1
contain regions with dN/dS ratios as high as 2–4 (), which is comparable with values found in mammalian MHC genes [9
]. In lagD1
the ratios were smaller than 1 (data not shown). Therefore, lagB1
are probably evolving under positive selection, possibly balancing selection considering the high number of polymorphic alleles [8
], and the homologs lagD1
must be under purifying selection. Polymorphism is an essential feature of recognition proteins because it provides the molecular basis for self-identity. We therefore conclude that lagB1
are more likely to be involved in kin-recognition than lagD1
mRNAs are developmentally regulated [21
], so we tested the regulation of the respective tandem genes, lagB1
. shows that the lagB1
expression patterns are nearly indistinguishable, with mRNA levels peaking at 8-12 hrs, corresponding to the transition from loose aggregate to tight aggregate. shows that lagD1
mRNA levels are also nearly indistinguishable with a peak at 16 hrs, corresponding to the finger stage of development. The tandem genes are therefore coordinately regulated, probably due to common regulatory elements that reside between their ORFs.
Developmental regulation and function of lag-genes
is essential for aggregation and for subsequent development [19
], so we tested whether lagB1
was also involved in development. We disrupted lagB1
and compared the mutant to the parental AX4 strain and to lagC1−
cells failed to progress beyond the loose aggregate stage after 12 and 17 hrs of development, similar to lagC1−
, whereas the wild type formed tipped aggregates and slugs at the respective times (). A few lagB1−
mounds formed small, gnarled fruiting bodies after 40 hrs (). Neither mutant produced spores in the first 24 hrs of development (data not shown), but 2% of the lagB1−
cells formed spores after 30 hrs (). Therefore, lagC1
are essential for aggregation and for subsequent development, suggesting common functions.
The proximity of lagC1 and lagB1 raised the possibility that disrupting one gene might have inadvertently disrupted the other. To test that possibility, we measured lagB1 mRNA in lagC1− cells and vice versa. lagB1 mRNA was present in the lagC1− cells, although the levels were lower and the mRNA persisted relative to the wild type (). lagC1 mRNA was present in the lagB1− cells at levels similar to the wild type (). These observations indicate that deleting one gene did not directly inactivate the other. The reduced levels and the persistence of lagB1 mRNA in lagC1− cells probably reflect the delayed development of the mutants, but it is formally possible that they contributed directly to the observed phenotypes.
The patterns of sequence polymorphism and gene expression, and the phenotypes of the null-mutants suggested that lagB1
are likely to function together in kin discrimination. To test that possibility we followed cells in chimerae between differentially labeled strains. In control chimerae, AX4-GFP and AX4-RFP cells exhibited equal mixing of green- and red-fluorescent cells at the aggregation stage (10-12 hrs) and at the finger stage (16-19 hrs), indicating that the fluorescent markers do not cause segregation (). Mixing AX4-RFP with lagB1−
–GFP yielded a different pattern: the red-fluorescent wild type and the green-fluorescent mutant co-aggregated at first, but then segregated into regions enriched in either red- or green-fluorescent cells during mound formation (). The mounds progressed into slugs that contained cells from both strains, but the lagB1−
cells were enriched in the middle of the slugs (). This enrichment in the prespore region suggested that the mutant may cheat on the wild type [14
], but a direct test did not support this hypothesis (data not shown).
Segregation of cell-cell adhesion mutants from wild type and from lagC1− cells
The results observed with lagC1− were even more dramatic. Cells from the two strains co-aggregated at first, but then segregated within the loose aggregates (). Several hours later, the AX4-RFP cells formed migrating slugs that contained a few lagC1−-GFP cells and migrated away, leaving behind mounds of mainly lagC1−-GFP cells (). These results suggest that lagC1 and lagB1 play a role in kin-discrimination, although lagB1 plays a lesser role than lagC1.
To test the kin-discrimination role of lagD1
, we mixed lagD1−
-GFP with AX4-RFP cells. The strains mixed well and remained mixed throughout development (). These results do not support a role for lagD1
in kin-discrimination, even though lagD1
shares many other properties with lagC1
], highlighting the difference between the polymorphic lagC1
gene and the nearly invariant lagD1
LagC1 is a cell-adhesion protein [22
], so we tested whether other adhesion genes participate in kin-discrimination. cadA
] and csaA
] encode two thoroughly studied cell-cell adhesion proteins [22
]. We tested the respective null-mutants in chimerae with AX4 cells and found no evidence of segregation (), arguing against a general role for cell-adhesion genes in kin-discrimination. These results are not in conflict with work that described csaA
as a greenbeard gene [29
]. In those experiments, csaA−
segregated from the wild type during development on soil but not on agar. Moreover, the sequence conservation of csaA
in wild strains (unpublished data) is inconsistent with a direct role in kin-discrimination.
The segregation of lagC1− from AX4 could have resulted from differential adhesion. To test the adhesion properties of the strains, we developed them in pure populations for 5 and 12 hrs. We disaggregated the cells, mixed them, and allowed them to reaggregate in liquid suspension. We found mixed aggregates in both cases (), indicating mutual adherence and suggesting that differential adhesion cannot account for the segregation observed in . Moreover, the 12-hrs cells became segregated within the mixed aggregates (), consistent with the proposed role of lagC1 in kin-discrimination.
have similar developmental roles because mutations in either one cause a developmental arrest at the loose aggregate stage ([19
]; ). We further tested their roles in kin-discrimination by testing chimerae between lagC1−
and other mutants (). The control experiment showed that the differential labels did not cause segregation (). To test whether lagB1
participate in one kin-discrimination pathway, we mixed differentially labeled cells of the respective mutants and observed no segregation at any stage (). Conversely, lagC1−
first co-aggregated (), but then segregated into clusters consisting mainly of one or the other strain (). The latter observation suggests that segregation is not a result of differential developmental progression because the strains segregate even though both progress to the same developmental stage.
These results suggest that lagB1
, but not lagD1
, function in one kin-discrimination pathway. This conclusion is also supported by the finding that lagB1
are physically mapped near each other, so they are likely to be inherited together in a syntenic block as seen in other kin-discrimination genes [2
], and by the observation of developmental co-regulation (), which provides the temporal opportunity for common function.
We also tested the role of other cell-adhesion genes by mixing lagC1− with cadA− or with csaA−. In both cases the cells co-aggregate initially, but then segregated into structures that consisted mainly of one strain or the other (). These results suggest that the kin-discrimination roles of lagB1 and lagC1 are specific to these genes rather than a general property of cell-cell adhesion genes.
Mixing AX4 cells with genetically dissimilar cells results in partial segregation, implying a kin-discrimination mechanism [18
]. The properties of lagB1
suggest they might participate in that mechanism. To examine the correlation between the lagB1
sequence polymorphism and segregation we computed the dissimilarities between the AX4 LagB1 and LagC1 sequences and the respective sequences in 11 wild isolates. We then computed the correlation between these dissimilarities and the published strain segregation data [18
]. We observed a positive correlation between sequence dissimilarity and segregation, although it was weaker than the correlation with genetic distances inferred from microsatellite length (Supplement Table S2
). We then searched and found protein domains within LagB1 and LagC1 that exhibited better correlations between sequence dissimilarity and strain segregation (Supplement Table S2
). In LagB1, the correlation was best between aa 239-259 and in LagC1 it was between aa 180-197 (Supplement Fig. S1
). Both regions reside near the first Ig-fold of the extracellular domain, suggesting that specific extracellular domains may have a function in segregation.
Because we tested many correlations, one or more could have been strong just by chance. However, if these correlations reflect a causative relationship, they should be predictive of strain segregation, which would be less likely if they were serendipitous. Ostrowski et al
. mixed three wild strains, QS32, QS33 and QS38, all of which segregated from AX4. QS32 and QS33 are genetically close to each other and distant from QS38. In that experiment, QS32 did not segregate from itself or from QS33, but segregated from the genetically dissimilar strain QS38 [18
]. shows that our sequence data predict low segregation of QS32 from QS33 and high segregation of QS38 from QS32. Therefore, the predictions of the sequence-based segregation model correlate well with the observations, supporting the hypothesis that LagB1 and LagC1 play a direct role in kin-discrimination.
Prediction of strain segregation from lagB1 and lagC1 sequence dissimilarity
Our data suggest that lagB1
participate in a common self- or kin-recognition mechanism in D. discoideum
. In other organisms, genetic crosses between organisms with polymorphic alleles have provided support for the role of such genes in self/nonself-recognition [3
]. Since D. discoideum
is not amenable to these types of studies, tests of the causative relationships between lag
-genes and kin-recognition would have to be accomplished by other means. Nevertheless, our findings suggest that the molecular mechanisms that regulate kin-recognition evolved before the evolutionary departure of the amoebazoa from the evolutionary line leading to animals, illustrating the critical role of these mechanisms in multicellularity and in sociality.