Germline-specific labeling of the somatic chromosomes by protein phosphatase 2A and histone H3S28 phosphorylation in Acricotopus lucidus

Abstract

Additional chromosomes limited to the germline (=Ks) were established as a special form of germline-soma differentiation in the Orthocladiinae, a subfamily of the Chironomidae (Bauer and Beermann in Z Naturforsch 7b: 557-563, 1952). The Ks together with the somatic chromosomes (=Ss) pass through a complex chromosome cycle with elimination at mitosis and a monopolar migration of all Ks. The dissimilar behavior of Ks and Ss in these exceptional mitoses initiated the search for differential chromosome marks in the orthocladiid Acricotopus lucidus. The search, using immunofluorescence, revealed that in metaphases of male gonial mitoses, and both meiotic divisions, the Ss are fully labeled by protein phosphatase 2A (PP2A) and histone H3S28ph, while in metaphases of somatic cells both marks were detected only at the centromeres of the Ss. In another orthocladiid, Psectrocladius obvius, the same labeling pattern of the Ss as in A. lucidus was established for H3S28ph, but not for PP2A, which was localised solely at the centromeres. In Chironomus nuditaris, a species possessing no Ks, PP2A and H3S28ph signals were always restricted to the centromeres. High levels of H3K4me3, a marker of transcriptionally competent chromatin, were detected on the Ss in metaphases I of C. nuditaris, while in both orthocladiids, the Ss in metaphases I were devoid of H3K4me3 signals. This strongly supports an earlier idea of a silencing of the Ss in male meiosis of A. lucidus suggesting the possibility of extending this concept to the Orthocladiinae. The germline-soma differentiation in A. lucidus is not only made apparent by the occurrence of Ks but also by a germline-specific labeling of the Ss by PP2A and H3S28ph.

Keywords: Germline-specific chromosome marks; Germline–soma differentiation; Orthocladiinae; Somatic chromosomes.

Fig. 1

PP2A completely covers the six Ss in germline cells of A. lucidus visualised by immunofluorescence using antibodies against the subunit PP2A-A. a In a spermatogonial metaphase and b an anaphase. c In a differential spermatogonial mitosis with monopolar (>) moving Ks and remaining Ss. d In an early prophase I nucleus of a primary spermatocyte (sp I). The decondensed Ss in the interphase nucleus of the aberrant spermatocyte (asp) are devoid of PP2A signals. e Primary and aberrant spermatocytes are connected by a permanent cytoplasmic canal (arrowhead). Mitochondria (red) are transported via the canal from the aberrant to the primary spermatocyte. Microtubules (green). Combined fluorescence-phase contrast image. In a the paired Ss are marked with dots. Chromosomes are stained with DAPI and pseudocoloured red. a, b, c, e Same magnification. Scale bars = 10 μm

Fig. 2

a–d PP2A labeling of the Ss in meiosis of A. lucidus detected by antibodies against the subunits PP2A-A and PP2A-C. a–c Complete labeling of the three S-bivalents (dots in a) in metaphases I of primary spermatocytes (sp I). b No signals were found at the condensed Ss of the aberrant spermatocyte (asp) using the polyclonal PP2A-A antibody. c The monoclonal PP2A-C antibody detected PP2A at the centromeres of the Ss in both aberrant spermatocytes. The centromeric PP2A fluorescence signals of the Ss in the aberrant spermatocytes were selectively enhanced. The enhanced area is marked by dots. For details, see text. d Complete PP2A labeling of the Ss in metaphases II of secondary spermatocytes (sp II). e In the nuclei of young spermatids (sp) defined areas exhibit a clear PP2A labeling. Two of the four spermatids are shown. f In mitotic metaphases of brain ganglia cells the PP2A signals are limited to the centromeres of the Ss. The homologous Ss are tightly paired. Scale bars = 10 μm

Fig. 3

Immunodetection of PP2A in a, d, g metaphases I of primary spermatocytes (S-bivalents, dots) and b, e, h mitotic metaphases of larval brain cells of A. lucidus, P. obvius and C. nuditaris, and c, f aberrant spermatocytes of A. lucidus and P. obvius using an antibody against PP2A-C. a Complete labeling of the entire Ss by PP2A in metaphase I of A. lucidus. For further details, see text. Scale bar = 10 μm

Fig. 4

Immunodetection of H3S28ph in a, d, g metaphases I of primary spermatocytes, (S-bivalents, dots) and b, e, h mitotic metaphases of larval brain cells of A. lucidus, P. obvius and C. nuditaris, and c, f aberrant spermatocytes of A. lucidus and P. obvius. a, d The Ss of A. lucidus and P. obvius are completely labeled by the H3S28ph antibody. g Insert. Prophase I spread of C. nuditaris clearly showing the four S-bivalents. For further details, see text. Scale bar = 10 μm

Fig. 5

Immunodetection of H3K4me3 in a, d, g metaphases I of primary spermatocytes (S-bivalents, dots) and b, e, h mitotic metaphases of larval brain cells of A. lucidus, P. obvius and C. nuditaris, and c, f aberrant spermatocytes of A. lucidus and P. obvius a, d The Ss of A. lucidus and P. obvius are devoid of H3K4me3 signals. a Insert. Section of a spermatogonial metaphase with Ss (dots) showing intense H3K4me3 labeling. For further details, see text. 9, germline-limited chromosome K9. Scale bar = 10 μm

Fig. 6

Immunodetection of PP2A and H3S28ph in female germline and somatic cells of A. lucidus. In differential oogonial mitoses all Ss (dots) are completely labeled by a PP2A and b H3S28ph antibodies. In mitotic metaphases of c brain ganglia and d follicle cells PP2A signals are only present at the centromeres of the Ss. Scale bar = 10 μm

Fig. 7

Immunodetection of H3S28ph in regular and aberrant spermatocytes in meiosis of P. obvius. a Image with three syncytial complexes (1–3) of primary (sp I) and aberrant spermatocytes (asp). In mid anaphase I (1) of the reduction division in the primary spermatocyte the Ss are still completely labeled by H3S28ph in contrast to the lagging Ks, while in late anaphase I (2, 3) the H3S28ph signals have largely disappeared from the Ss. The connected aberrant spermatocytes passing through mitosis with equal segregation of the six Ss. In early and mid anaphase (1, 2) the Ss are completely labeled by H3S28ph, while in late anaphase (3) the mark begins to disappear. b In meiosis II the H3S28ph-labeled Ss and the unlabeled Ks of the two secondary spermatocytes (sp II) segregate equally, while the two aberrant spermatocytes with the condensed and H3S28ph-labeled one-chromatid Ss remain undivided. Scale bar = 10 μm