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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01684

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Changing chromatin dynamics and nuclear organization during differentiation in Drosophila larval tissue

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA

View larger version (30K): [in a new window]   Fig. 1. Heterochromatic association in eye imaginal discs. (A) Separation of differentiated and undifferentiated cells. Image of an eye-antennal disc dissected from late third instar larvae. GFP is expressed in cells undergoing differentiation (dc) that are present posterior (pos) to the morphogenetic furrow (mf). No GFP expression is seen in the undifferentiated cells (udc) present anterior (ant) to the furrow. Dissection at the mf (demarcated by the red line) allows the separation of cells on the basis of their differentiation state. (B) To quantitate the association between bwD and 2Rh, FISH was done on nuclei obtained from differentiating and undifferentiated cells that were either bw+ or heterozygous for bwD. The distance between the two probes was measured and divided by the radius of the nucleus stained with DAPI. The distribution of the data is represented in box plots. Box plots are calibrated representations of histograms wherein each horizontal line represents the 10th, 25th, 50th (median), 75th and 90th percentiles. The numbers inside the box plots represent the total number of nuclei studied. P values to determine statistical significance of the difference in the data were determined using the Mann-Whitney test for statistical significance and are depicted above the brackets. (C) Histograms showing the distribution of the distances between 59E and 2Rh in eye imaginal disc nuclei from bwD/bw+ larvae. In differentiated cells distribution is skewed towards the lower end.

View larger version (25K): [in a new window]   Fig. 2. Heterochromatic association in CNS nuclei from developmentally arrested larvae. (A) The effect of feeding larvae erg2 yeast. Larvae were reared on wild-type yeast (top) or erg2 yeast (bottom). Irrespective of the yeast fed, the larvae enter the mid-third instar larval stage and appear to undergo normal development until 115 hours AED. At 135 hours AED the larvae fed erg2 yeast remain stalled in the third instar stage and eventually die after a few more days. By this time, larvae fed wild-type yeast have pupated (they usually pupate at 118-125 hours AED) and are ready to eclose 216 hours AED. (B) Box plots (as in Fig. 1) show data from FISH on CNS from bw+ and bwD larvae. Larvae fed erg2 yeast were dissected at 110-115 hours AED (day 1) or 130-135 hours AED (day 2). Larvae fed wild-type (WT) yeast were dissected at 110-115 hours AED (day 1). P values (Mann-Whitney) are shown above brackets for the respective sets. (C) Histograms showing the distribution of the distances between 59E and 2Rh in bwD larvae reared on WT yeast on day 1 and erg2 yeast on day 2.

View larger version (33K): [in a new window]   Fig. 3. The dynamics of chromosomal movement in differentiated and undifferentiated cells in primary cultures of eye imaginal discs. (A) Location of lacO repeats (stars) at 1F on the X and 59E on the 2nd chromosomes. The bottom circle is a schematic of these markers in interphase nuclei; d is the measure recorded at each time point. (B) Representative images from larvae containing the two above described lacO repeats and expressing mRFP-LacI fusion protein under an ubiquitin promoter. The top panel shows undifferentiated nuclei present anterior to the morphogenetic furrow and the bottom panel shows differentiated cells from ommatidial clusters present posterior to the furrow. The background fluorescence of mRFP demarcates the nucleus. The two bright dots are the two loci tagged with lacO repeats bound with mRFP-LacI fusion protein expressed under an ubiquitin promoter. (C) Plot of the average of mean square change in distance <d2> vs. the time interval (t). The distance between the center of masses of the two tagged loci was computed over time. For undifferentiated cells data was collected from 19 nuclei from 7 eye discs dissected from third instar larvae. In the case of differentiated cells, the data was obtained from 21 nuclei from 9 eye-discs. Error bars show ± 1 standard error of the mean. Comparison of the height of the graphs reveals that the two loci are confined to a smaller nuclear space in differentiated cells, suggesting that chromosomal movement is more constrained during differentiation.

View larger version (29K): [in a new window]   Fig. 4. Comparison of the dynamics of chromosomal movement among larvae reared on wild-type yeast and developmentally arrested larvae reared on erg2 yeast. No significant change in chromatin motion was observed in larvae reared on erg2 yeast when compared with those reared on wild-type yeast. For larvae reared on wild-type yeast the data is from 12 nuclei in the CNS dissected from 3 larvae. For larvae reared on erg2 on day 1 the number of nuclei was 10, dissected from 3 larvae and on day 2 the number of nuclei was 13 from 4 larvae. For fixed nuclei the numbers were 8 and 3, respectively. Error bars show ± 1 standard error of the mean.