Retroviral transfer of antisense integrin alpha6 or alpha8 sequences results in laminar redistribution or clonal cell death in developing brain - PubMed (original) (raw)

Retroviral transfer of antisense integrin alpha6 or alpha8 sequences results in laminar redistribution or clonal cell death in developing brain

Z Zhang et al. J Neurosci. 1998.

Abstract

To assess the roles of two integrin alpha subunits (alpha6 and alpha8) in the developing chicken optic tectum, progenitors were infected with retroviral vectors that contained the marker gene lacZ plus antisense sequences from either the alpha6 or alpha8 integrin subunit cDNAs. On embryonic day 3 (E3), the vector was injected into tectal ventricles of chicken embryos. On E6, E7.5, E9, or later, chicken embryos were killed, and optic tecta were dissected and processed for histochemical detection of lacZ-positive cells. The antisense-bearing cell clones (descendants of a single infected progenitor) were analyzed for proliferation and migration patterns and were compared with lacZ-only vector-infected control clones. At E6, both alpha6 and alpha8 integrin antisense-containing cell clones were similar to controls. At E7.5, integrin alpha8 antisense-containing clones exhibited a cell number reduction in upper laminae (intermediate zone and tectal plate), and at E9, they exhibited a reduction in the ventricular zone as well. Integrin alpha6 antisense-containing cell clones exhibited no difference in total cell number at E9 but had a net laminar redistribution of more cells in the ventricular zone and less cells in the tectal plate. Our data show that different integrins play different roles during brain development: alpha6 integrin is essential for migration of tectal cells into specific laminae, and alpha8 integrin is essential for the survival of optic tectum cells. Also alpha8 integrin-substrate interactions may suppress early programmed cell death in premigratory and migratory neuroblasts.

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Figures

Fig. 1.

Fig. 1.

Expression of integrin subunits and substrates in the developing optic tectum. Cryosections of developing optic tectum were immunostained with antibodies against integrin α8 (A), integrin α6 (B), tenascin-C (C), and fibronectin (D). A, B, E7 tectum; C, D, E9 tectum. For all parts, the ventricular surface is down. Arrows in_D_ denote blood vessels (BV).M, Meninges; VZ, ventricular zone;IZ, intermediate zone; TP, tectal plate. See Results for details.

Fig. 2.

Fig. 2.

Retroviral vectors used in this study. pLZ14, pLZα6AS, and pLZα8AS encode gag-lacZ fusion proteins. pLZα6AS and pLZα8AS are similar to pLZ14 with the addition of an antisense sequence (filled arrow) against either the α6 or the α8 integrin subunit. Boxes indicate viral long terminal repeats. Key restriction sites used in construction of the antisense vectors are marked. See Materials and Methods and Results for details.

Fig. 3.

Fig. 3.

Analysis of α8 integrin immunostaining of optic tectum cells by flow cytometry. Tecta were injected with either LZ14 or LZα8AS on E3, and dissociated tectal cells were analyzed for α8 integrin immunostaining on E7. The top panel shows α8 integrin levels in cells infected by LZ14. The bottom panel shows α8 integrin levels in cells infected by LZα8AS (p < 0.0001).

Fig. 4.

Fig. 4.

Analysis of α6 integrin immunostaining of QT6 cells by flow cytometry. QT6 cells were infected with either LZ10 or LZα6AS and analyzed for α6 integrin after dissociation and immunofluorescent staining. The filled graph represents levels of α6 integrin in control LZ10-infected cells. The_unfilled graph_ represents levels of α6 integrin in LZα6AS-infected cells (p < 0.0001).

Fig. 5.

Fig. 5.

Appearance of LZα6AS-infected cell clones_in vivo_. Tecta were injected with LZ14 or LZα6AS on E3 and processed for X-gal histochemistry on E6 (top) and E9 (bottom). Shown are LZ14 control clones (left) or LZα6AS clones (right) in thick sections.

Fig. 6.

Fig. 6.

Relative frequency histogram of affected zones in LZα6AS-infected tecta at E9. The top panels show the relative frequency distribution of LZα6AS- and LZ14-infected cells within the ventricular zone. The bottom panels show the relative frequency distribution of LZα6AS- and LZ14-infected cells within the tectal plate.

Fig. 7.

Fig. 7.

Appearance of LZα8AS-infected cell clones_in vivo_. Tecta were injected with LZ14 or LZα8AS on E3 and processed for X-gal histochemistry on E6 (top), E7.5 (middle), or E9 (bottom). Shown are LZ14 control clones (left) or LZα8AS clones (right) in thick sections.

Fig. 8.

Fig. 8.

Relative frequency histogram of tectal clones infected with LZα8AS and LZ14 at E7.5. The _top panels_show the relative frequency distribution of LZα8AS- and LZ14-infected cells within the tectal plate. The bottom panels show the relative frequency distribution of LZα8AS- and LZ14-infected cells within the intermediate zone.

Fig. 9.

Fig. 9.

Relative frequency histogram of LZα8AS- and LZ14-infected clones at E7.5 and E9. The top two histograms display the relative frequency distributions of total cell number within clones at E7.5. The bottom two histograms display the relative frequency distributions of total cell number within clones at E9.

Fig. 10.

Fig. 10.

Disappearance of LZα8AS infected clones over time. A group of embryos were injected with either LZα8AS or LZ14 at E3, and some were killed at E6, some at E9, and some at E12. The mean cell clones per tectum at E6 are expressed as 100%, and the mean cell clones per tectum at both E9 and E12 are expressed as the mean percentage (relative to that at E6) ± SEM (error bars).

Fig. 11.

Fig. 11.

Flow cytometry analysis of LZα8AS-infected cells after FISEL+. The bottom panel shows the FISEL+ labeling pattern of normal uninfected cells from tecta that were injected with LZα8AS at E3 and processed at E8. At least two populations appear to be present, although overlapping. The top panel shows the FISEL+ labeling pattern of LZ14-infected cells (p > 0.10 between means of top and bottom panels). The middle panel shows the FISEL+ labeling pattern of LZα8AS-infected cells in the same population as the_bottom panel_. These cells showed increased FISEL+ labeling compared with the LZ14-infected controls (p < 0.0001) as well as compared with uninfected cells (p < 0.0005). Many fewer cells are shown in the top two panels than in the bottom panel because FISEL+ labeling was measured only in the small fraction of total dissociated cells that were infected and lacZ+.

Fig. 12.

Fig. 12.

Summary data. The effects of antisense α8 and α6 sequences on the clonal development of tectal cells are shown in this quantitative summary diagram. Each circle,solid or stipled, represents a cell, and the number of cells shown are the mean numbers of cells at each age. Data are taken from Tables 1-5.

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