Figure 1.
Expression patterns of gcm and repo genes during glial cell lineage differentiation.
(A) Gcm is expressed in neuroglioblasts, glioblasts, and transiently in immature glia cells [8], [24]. Exceptionally, expression of gcm is observed in a very few number of interneurons in the ventral nerve cord from L1 to adult stage [9]. (B) A scheme of the expression pattern of repo. In addition, Repo is detected in longitudinal glioblasts and glial cells [25]. (C) Existence of different types of glial cell lineage and neuronal cell lineage cells at developmental stages.
Figure 2.
Effects of glial expressions of hHtt103Q on survival, locomotive and spontaneous activities of adult flies.
(A) The survival curves of adult flies show that the flies expressing hHtt103Q with gcm-Gal4 driver died by Day 6 and the flies expressing hHtt103Q with repo-Gal4 driver died by Day 14. (B) Negative geotactic responses of hHtt103Q or hHtt20Q expressing flies. Because gcm; hHtt103Q flies died within 1 week as shown in A, we could not test them at 7 days. Asterisk: p<0.05 in Student's t-test. The mean+SE and the number of flies used for each experiment are shown. (C) and (D) indicate spontaneous activity at Day 2 of control and repo; hHtt103Q flies, respectively. Open and filled boxes under the graph indicate Light and Dark cycle (L∶D = 12 hr∶12 hr). We could not test gcm; hHtt103Q flies due to the low birth rate.
Table 1.
The number of adult flies which expressing hHtt103Q induced by gcm-Gal4 is reduced.
Figure 3.
hHtt103Q expression in glial cell lineage induces pathological change in the central nervous system.
(A) In repo; hHtt103Q flies, mutant Htt stained by anti-hHtt N-18 antibody was clearly colocalized with a glial cell marker, repo stained by anti-repo antibody (upper panels, arrow heads). High magnification shows a negative relationship between the expression level of hHtt103Q and that of repo (middle panels, dotted squares numbered). Glial cells marked with squares in middle panels are magnified in lower panels. Four examples of high-Htt103Q cells (1∼4) and 4 examples of high-repo cells (5∼8) are shown. (B) Morphological changes of brains in the gcm; hHtt103Q and repo; hHtt103Q flies. The coronal sections of the head were stained with toluidine blue, anti-repo antibody (glial cell marker), and anti-elav antibody (pan-neuron marker). Arrows in repo; hHtt103Q flies indicate vacuolar changes.
Figure 4.
Glial cell lineage expression of hAtx1-82Q induces abnormal development.
(A) The graph shows survival from larvae to adult fly of the hAtx1-82Q expressing (repo; hATX1-82Q and gcm; hATX1-82Q) or non-expressing siblings. The repo; hATX1-82Q and gcm; hATX1-82Q larvae (F1) are generated by crossing UAS-hATX1-82Q virgin females and repo-Gal4/TM3, GFP Sb or gcm-Gal4/CyO, GFP males. repo+ and gcm+ are F1 of WT virgin females and repo-Gal4/TM3, GFP Sb or gcm-Gal4/CyO, GFP males. The ratio of GFP(−)/GFP(+) larvae was calculated. The gray line in the graph is the ideal ratio for no toxicity. (B) Head size in gcm; hATX1-82Q at mature pupa stage was evaluated. Vertical and horizontal arrows indicate the length of pupae and the width of their heads, respectively. Wild type (control), repo; hATX1-82Q F1 and gcm; hATX1-82Q F1-pupae (GFP-negative) were examined. The lower panels show the heads after removing the capsule. (C) Histograms show the head width /pupa length ratio (%) of pupae. The frequency of ratio was plotted in each histogram, and the numbers of examined control, repo; hATX1-82Q and gcm; hATX1-82Q were 15, 16 and 54, respectively.
Figure 5.
Morphological analysis of the small head phenotype in pupae expressing hAtx1-82Q in glial lineage cells.
(A) The head structure of normal pupa stained by toluidine blue. The squares indicate brain and retina regions for immunohistochemistry shown in figure 6, 7. (B) Representative morphologies of the head of the gcm; hATX1-82Q (mild and severe cases) and repo; hATX1-82Q pupae are shown (toluidine blue staining).
Figure 6.
hAtx1-82Q expression in glial cell lineage induces pathological change in the central nervous system.
(A) Double-staining with anti-human Atx1 (H21) and anti-repo antibodies shows co-localization of mutant hAtx1 and repo proteins in glial cells of repo; hATX1-82Q pupae (arrowhead). A small part cells expressed only repo or hAtx1 protein (arrow). The former would non-expressors and the latter would be ghost cells with hAtx1 inclusions. (B) Immunohistochemistry with anti-repo and anti-elav antibodies showing glial cells and neurons, respectively. The right higher magnifications show photoreceptor cells in the retina, which were remarkably reduced in repo; hATX1-82Q pupae.
Figure 7.
hAtx1-82Q expression in glial cell lineage induces a change in a pattern of axon.
Axons from photoreceptor neurons were stained with an antibody specific for photoreceptor neurons (MAb24B10). The number of axons and their alignment were remarkably distorted especially in repo; hATX1 82Q pupae. Lamina and medulla were remarkably deformed (white and black arrows, respectively).