Characterization of axolotl lampbrush chromosomes by fluorescence in situ hybridization and immunostaining

Abstract

The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified some time ago by their relative lengths and predicted centromeres, but they have never been associated completely with the mitotic karyotype, linkage maps or genome assembly. We identified 9 of the axolotl LBCs using RNAseq to identify actively transcribed genes and 13 BAC (bacterial artificial clone) probes containing pieces of active genes. Using read coverage analysis to find candidate centromere sequences, we developed a centromere probe that localizes to all 14 centromeres. Measurements of relative LBC arm lengths and polymerase III localization patterns enabled us to identify all LBCs. This study presents a relatively simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences (from the axolotl genome assembly) to the physical LBCs by immunostaining and fluorescence in situ hybridization. Our data will facilitate a more detailed transcription analysis of individual LBC loops.

Keywords: Ambystoma mexicanum; Axolotl; BAC FISH; Centromere; Lampbrush chromosomes.

Fig. 1.

An axolotl lampbrush chromosome stained with DAPI and an antibody against RNA polymerase II. A) Pol II immunostaining highlights the loops of the LBC. B) DAPI stains the condensed chromomeres along the LBC axis. C) Overlay of DAPI (cyan) and Pol II (red). Bars = 50 μm.

Fig. 2.

BAC fluorescence in situ hybridization (FISH) on axolotl lampbrush chromosomes. A) BAC clone BbMex_4E17 (red) localizes to transcripts on a pair of loops near the telomere on the p arm of LBC 2. B) BAC clone AMMCBa_426N21 (red) localizes to transcripts on a pair of long loops on the q arm of a medium-sized chromosome. Chromosomes are counterstained with DAPI (cyan). Bars = 10 μm.

Fig. 3.

BAC fluorescence in situ hybridization (FISH) on axolotl lampbrush and mitotic chromosomes. A) BAC AMMCBa_355L20 (red) localizes to the p arm of mitotic chromosome 3 and B) to a pair of loops near the telomere on the p arm of a long LBC. C) BAC clone AMMCBa_45F11 (red) localizes between the centromere and telomere on the q arm on mitotic chromosome 13 and D) to a pair of loops on the q arm of LBC 13. Chromosomes are counterstained with DAPI (cyan). Arrows point to signal from hybridization of the probe. Bars = 10 μm.

Fig. 4.

LBC #5 hybridized with a clone against the centromere and also immunostained with an antibody against pol III. A) Pol III sites detected by antibody staining. The pattern is shown diagrammatically in Fig. 5. B) The same preparation after in situ hybridization for the centromeres (arrows). The in situ procedure reduces but does not completely eliminate pol III staining. C) DAPI stain showing DAPI-positive chromomeres. The centromeres are also DAPI-positive. Arrows point to the centromeres. Bar = 100 μm.

Fig. 5.

Ambystoma mexicanum LBC Maps. Maps of the 14 LBCs of A. mexicanum showing positions of the centromeres, the most prominent pol III loci, the four histone locus bodies, and the single nucleolus. The fraction preceding each chromosome is the position of the centromere, measured from the left end. The relative lengths and centromere positions are in approximate agreement with those originally determined by Callan (1966) with one minor exception: reversal of chromosomes 5 and 6.