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Tissue- and development-specific induction and turnover of hsp70 transcripts from loci 87A and 87C after heat shock and during recovery in Drosophila melanogaster

S. C. Lakhotia^* and K. V. Prasanth

Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

View larger version (69K): [in a new window]   Fig. 1. RNA:RNA in situ hybridization in wild-type larval salivary gland polytene chromosome spreads using digoxigenin-labelled hsp70 cDNA without the 3'UTR (A), 87A3'UTR (B) or 87C3'UTR (C) riboprobes. The arrows indicate the 87A and 87C loci. Note the absence of any cross-hybridization of the 3'UTR probes.

View larger version (55K): [in a new window]   Fig. 2. Heat-shock-induced expression of hsp70 genes from the 87A and 87C loci in wild-type embryos of developmental stages 5–16 as revealed by RNA:RNA in situ hybridization (RISH) using 3'UTR riboprobes of hsp70 genes from the 87A (A,C,E,G,I,K,N,Q,T,W) or the 87C (B,D,F,H,J,L,O,R,U,X) locus. Stages of the embryos are indicated on the right (D, L and V in this column refer to the dorsal, lateral or ventral views, respectively, of the embryo). The arrow in A points to the pole cells. The black arrowheads in E and F and the small white arrowheads in F, G and H indicate the neuroblasts in the procephalic region and ventral nerve cord, respectively. The white arrows in I and J indicate the proneural clusters in stage 11 embryos. The white arrowheads in K, R and U refer to the glial cells. The small black arrows in N, O and P point to the tracheal pits, while the long black arrows in Q, R, S and T show the peripheral nervous system cells. Insets in T, U and V show the clusters of neuroblasts (T and V) and glial cells (U) from the respective embryos. Embryos in M, P, S and V are immunostained with the BP104 antibody to show the embryonic central and peripheral nervous systems for comparison with RISH patterns in K–U. Scale bar (applicable to all parts of the figure, except the insets in T, U and V, which are at higher magnification), 200 µm.

View larger version (69K): [in a new window]   Fig. 3. Differential expression of hsp70 genes in larval gut as revealed by RNA:RNA in situ hybridization (RISH) using the 87A3'UTR (A,C,F), the 87C3'UTR (B,D,G) or the cDNA (E,H) riboprobe after 40 min of heat shock (HS) (A,B) or after 2 h (C,D,E) or 4 h (F,G H) of recovery (rec) from a 40 min heat shock. Insets in A and B show cells from the gut region at a higher magnification: inset 1 corresponds to cells from the middle part of the midgut (the two 3'UTR probes give a similar perinuclear signal in the cytoplasm); inset 2 shows a cell from the distal part of the midgut with strong 87A3'UTR hybridization; inset 3 is a cell from a similar position to that in inset 2 showing weak hybridization with the 87C3'UTR riboprobe. Scale bars (which apply to all images in a given row, except the insets), 200 µm.

View larger version (78K): [in a new window]   Fig. 4. (A–I) Expression of hsp70 genes following heat shock (HS) (A,G) or after 30 min (B,D–F,H) or 1 h (C,I) of recovery (REC) from heat shock in late third-instar larval eye-antennal (A–C,G–I), wing (D) or leg (E) imaginal discs or in brain ganglia (F) as revealed by in situ hybridization with the 87A3'UTR (A–F) or the cDNA (pPW18) (G–I) riboprobe. In situ hybridization patterns with the 87C3'UTR riboprobe were similar to those seen with the 87A3'UTR riboprobe. Note the specific hybridization of the 3'UTR riboprobe with clusters of dividing cells during recovery from heat shock (B–F) in all the imaginal discs and the brain. Insets in D and F show clusters of cells in the wing disc and the optic lobes of brain ganglia, respectively, at higher magnification, demonstrating perinuclear cytoplasmic hybridization with the 3'UTR riboprobe. (J–L) Eye discs of late third-instar larvae immunostained with the 7Fb antibody to show the distribution of Hsp70 in heat-shocked (J) discs and in those allowed to recover from heat shock for 2 h (K) or 4 h (L). Scale bar, 200 µm.

View larger version (92K): [in a new window]   Fig. 5. Expression of hsp70 genes in larval testes as revealed by RNA:RNA in situ hybridization using the cDNA (pPW18) (A,E,P) 87A3'UTR (C,G,I,K,N) or 87C3'UTR (D,H,J,L,O) riboprobe and the in situ profile of Hsp70 as revealed by immunostaining with the 7Fb antibody (7Fb Ab) (B,F,M,Q) in unstressed control (CON) (A,B) or heat-shocked (HS) (C–F) testes or those allowed to recover for 30 min (G,H), 1 h (I,J), 2 h (K–M) or 4 h (N–Q) at room temperature (22°C) after a 40 min heat shock. Note the constitutive presence of Hsp70 in the anterior region (spermatogonial cells) in unstressed testes and also the greater induction of the locus 87A hsp70 genes in these cells following heat shock. Scale bar, 500 µm.

View larger version (105K): [in a new window]   Fig. 6. The in situ expression of hsp70 genes revealed by RNA:RNA in situ hybridization using cDNA (pPW18) riboprobe (A–D and I–L) and the in situ profile of Hsp70 seen after immunostaining with the 7Fb antibody (7Fb Ab) (E–H and M–P) in the distal (A–H) and proximal (I–P) parts of adult testes (tes) and seminal vesicles (sv) in unstressed (A,E,I,M) controls (CON) or after 40 min of heat shock (B,F,J,M) and in tissues allowed to recover for 2 h (C,G,K,O) or 4 h (D,H,L,P) at room temperature (22°C) after a 40 min heat shock. The insets in A and B show the expression of hsp70 in spermatogonial cells (spg), while those in C and D show the cyst cells (cs) at higher magnification. The arrowhead in E shows the constitutive presence of Hsp70 in the spermatogonial cells. Insets in K and L show the expression of hsp70 in cyst cells (cs) in the seminal vesicles. Scale bar (applicable to all images, except to insets), 200 µm.

View larger version (121K): [in a new window]   Fig. 7. The in situ profile of Hsp70 in unstressed testes of wild-type larvae (A–H) or pupae (I–L) of different ages (marked on each panel in hours after egg hatching) and in an adult (M) revealed by immunostaining with the 7Fb antibody. A higher-magnification image of the boxed area in J is shown in L. Expression of hsp70 genes in the spermatogonial cells of the unstressed pupal testis detected by RNA:RNA in situ hybridization using the cDNA riboprobe is shown in N. Scale bars (which apply to all images in a given row, except L and N), 200 µm.