Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages

Abstract

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

Figure 1. Neuroblasts and INPs divide asymmetrically in culture.

(A) Schematic of the experimental set-up. (B-G) In vivo staining of wild-type 3rd instar larval brains. Type I (B, E), type II NBs (C, F) and INPs (D, G) stained for Mira (red), PH3 (blue) and aPKC (B-D, green). Par 6-GFP (E-G, green) in par 6 mutant background. (H, I) Single frames from cultured NB time-lapse movies. NBs expressing UAS-Baz^S151A.S1085A::GFP, UAS-mCherry::Pon-LD. White asterisk labels NB, blue asterisk marks INP. (H) Single frames from movie of type I NB undergoing multiple rounds of division in culture. (I) Single frames from movie of type II NBs undergoing multiple rounds of division in culture. Times in hr:min. (A-H) White arrowheads label apical, yellow arrowheads basal polarity domains. Scale bars, 10 µm.

Figure 2. Neuroblasts generate in vivo-like lineages in culture.

(A, B) Drosophila larval brain lobe expressing type I (A) or type II (B) specific nuclear GFP (red) stained for Mira (green). Scale bars, 20 µm. (C, D) 20-hr NB primary cell culture stained for Dpn (red) and Mira (white). Type II NBs are identified by nuclear GFP (green, arrow), whereas type I NBs are GFP negative (arrowheads). Cell culture stainings are represented in two layers. (E, F) Image time course from representative movies capturing a dividing type I (E) or type II (F) NB. Arrows mark GMC (E) or INP (F) shortly before division. Arrowheads label daughter cells shortly after the GMC (E) or INP (F) divided. Note that the GMC leads to two daughter cells equal in size, whereas the INP divides asymmetrically generating two daughter cells that differ in size. (G) The type II NB (asterisk) generates INPs that express R9D11-CD8::GFP shortly before they divide (1st INP white arrowhead, 2nd INP yellow arrowhead). Times are in hr:min. Scale bars, 10 µm.

Figure 3. Automated 4D image analysis.

(A) Type I lineages expressing nuclear GFP. Nuclei diameters were measured before cell division (red line). Cell diameters were obtained by measuring cell diameter when nuclear GFP labeled the entire cell right after nuclear breakdown (green line). Scale bar, 10 µm. (B) Size ratio of type I NB/GMC estimated with nuclei diameter vs. size ratio estimate with cell diameter (n=5). Error bars represent standard deviation. (C, D) Examples of output images of type I (C) and type II (D) NB lineage analysis by Definiens. One frame equals three minutes. Red marks the NB, purple marks the GMC (C) or INP (D); the remaining colors mark neurons (C) or GMCs (D). Note that only the first INP was followed throughout entire INP cell cycle, whereas the following born INPs were followed only for a few frames. (E) Representative stills from type II NB lineage live imaging movie modified by IMARIS. Green ball represents the type II NB, blue balls represent INPs, purple balls represent GMCs. Red balls are in mitosis.

Figure 4. Quantitative analysis of type I and type II neural lineages.

(A) Frequency of divisions of type I and type II NBs, INPs and GMCs (n=29, n=31, n=6, n=6 respectively). Time measured in hours between consecutive nuclear breakdowns. (B) Absolute cell numbers type I and type II lineages counted after 10-hr recording period. (C) Nuclei volumes from all cell types of type I and type II lineages. NB type I n=30, NB type II n=31, INP n=34, GMC type I n=5, GMC type II n=6, neuron n=5. (D) Growth rates from birth to division of type I and type NBs, INPs and GMCs. NB type I n=30, NB type II n=34, INP n=6, GMC type I n=4) (A-D) Bars represent standard deviation. Statistical analysis done using T-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (E) Graphic representation of type I and type II lineages. Diameter ratios between the lineages and the different cell types is according to experimental measurements. GMC, ganglion mother cell; imINP, immature intermediate neural progenitor; mINP, mature INP; NB, NB. Red color represents nuclei, green color represents Earmuff expression.