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An anterior function for the Drosophila posterior determinant Pumilio

Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, 2500 Speedway, Austin, TX 78712, USA

View larger version (39K): [in a new window]   Fig. 1. The Drosophila bcd 3'UTR contains conserved NRE sequences. Diagram of bcd and hb 3'UTRs with size bar (top). NREs with conserved A and B boxes (hatched areas) and spacers (intervening gray areas). The bcd 3'UTR contains a bipartite NRE sequence and a downstream B box (1 1/2 NREs). Sequence alignment of the bcd and hb NRE regions (below) with conserved nucleotides (capitalized) and identical residues (*). Sequence names are the species with a bcd or hb prefix. Sequences in the EMBL/GenBank database: bcd, X14458, M32121, M32124, M32123, X55735, X78058, M32122, M32126, M3125; hb, Y00274, AJ00535, AJ00536, AJ00534, X15359. Numbers are the first nucleotide aligned in their database entry.

View larger version (53K): [in a new window]   Fig. 2. Endogenous bcd mRNA deadenylation is delayed developmentally in pum– mutants. (A) The bcd 3'UTR and PAT assay internal control (bcd, to scale) with 1 1/2 NREs (arrow) and bcd-specific oligo priming site (PATbcd). (B) Internally controlled bcd PAT assay profiles from wild type (lanes 1-9) and pum– (lanes 1'-9') samples including ovarian (1,1') and embryonic (2-9,2'-9') RNAs (20°C). C1, cDNA synthesis control (only internal control RNA); C2, PCR control (no template). Equal volumes of the final reactions were resolved on acrylamide-urea gel and autoradiographed. ‘m’, 32P-labeled marker. (C) osk PAT assay profile in wild type and pum– mutants. The same cDNAs as B were amplified with an osk-specific primer (Sallés et al., 1994). osk deadenylation does not change noticeably in pum– mutants. We observed a slight increase in osk stability (8-9 versus 8'-9'). C1, bcd cDNA control; C2, osk PCR control (no template).

View larger version (67K): [in a new window]   Fig. 3. bcd mRNA is stabilized in pum– mutants. RNAs used in Fig. 2 analyzed by northern blot. (A) Probed for bcd and rpA1. bcd is stabilized in pum– mutants compared with wild type (lane numbering as in Fig. 2). Similar results were obtained for pum13/pum13, pum1/pum13 and pumMsc/pumFC8 embryos. (B) Reprobing for hb shows the embryo populations are developing synchronously and hbmat is also stabilized in pum– mutants. (C) Western blot of wild type (lanes 1-9) and pum– (1'-9') embryo proteins probed for Bicoid (time course identical to Fig. 2, and A,B). Lane C, in vitro translated unlabelled Bicoid protein. The lower panel (same gel) shows a secondary antibody crossreacting band that acts as a loading control.

View larger version (46K): [in a new window]   Fig. 4. Transgenic mRNAs with mutated NREs escape Pumilio regulation. (A) The bcd NRE (A and B boxes shaded) and NRE transgenic mutant constructs (NRE1, NRE2; dinucleotide mutations noted). (B) Northern blot of timed samples from wild-type transgenic (wt*, lanes 1-8), NRE1 (lanes 1'-8') and NRE2 (lanes 1''-8'') embryos probed for bcd and rpA1. Both NRE1 and NRE2 transgenics contain bcd at later developmental times. m, size markers. (C) Western blot of wild-type transgenic (wt*, lanes 5-10), NRE1 (5'-10') and NRE2 (5''-10'') mutant embryo extracts probed for Bicoid. The developmental period overlaps with and extends beyond Fig. 4B. The lowest band (secondary antibody crossreaction) acts as a loading control. Transgenics contain a full complement of both endogenous and transgenic bcd mRNAs and Bicoid protein. The wild-type* and NRE1 embryos are homozygotes; the NRE2 mutant line is heterozygote for the bcd transgene.

View larger version (87K): [in a new window]   Fig. 5. Prolonged Bicoid expression dominantly interferes with head development. Head cuticles of wild-type*, NRE1 and NRE2 transgenic embryos in a bcd+ background with maternal genotypes (phase contrast, side views, dorsal towards the top, anterior leftwards). (part I) Wild-type* tridentate mouth hooks (mh) with dorsal (large arrow) and ventral (small arrow) projections. The labrum (lr) and epistomal sclerite (eps) are labral segment markers. bcd NRE1 (part II) and NRE2 (part III) mutant transgenics fail to develop the dorsal mh projection. (Part IV) Wild-type* head with notable marked structures (Jürgens et al., 1986): mh, MxSO, AntSO, lr, dorsal arms (da), dorsal bridge (db) and posterior pharyngeal wall (ppw). Some mutant transgenic embryos with the mh defect fail to complete head involution, resulting in deformations and altered spatial relationships among cuticle structures. NRE1 (part V) and NRE2 (part VI) transgenics with a reduced head skeleton, deformed protruding lr and mh defect. Black arrows: structures residing in a different focal plane. Parts I-III visualize mh abnormality and parts IV-VI reveal head involution defects. Scale bar: 20 µm in parts I-III; 40 µm in parts IV-VI.

View larger version (82K): [in a new window]   Fig. 6. Analogous head defects in bcd NRE and pum– mutants. (A) Scanning electron microscopy of the embryonic cuticle head (frontal view) and body reference (side view) with maternal genotypes. (Part I) A wild-type head with notable structures (mh, MxSO, AntSO). (Part II) bcd– null embryos have no head structures. The globular anterior structures resemble posterior ones. (Part III) A wild-type bcd transgene (wt*) rescues the bcd– null anterior defects. NRE1 (part IV) and NRE2 (part V) mutant bcd transgenes rescue bcd– anterior development, while additionally inducing defects in some embryos, consistent with the dominant effects in Fig. 5. Protruding structures (arrow). (Part VI) A pum– (pum13/pum13) embryo has an analogous medial protrusion and an exposed sclerite resembling those in part V (large and small arrows, respectively). Body side views (right) show abdominal defects documented for pum– mutation (Barker et al., 1992; Lehmann and Nüsslein-Volhard, 1987; Wang and Lehmann, 1991; Wharton and Struhl, 1991). (B) pum1/pum13 (part I) and pum13/pum13 (part II) heads (phase contrast) do not develop the mh dorsal projection (large arrow; ventral projection, small arrow). pum13/pum13 exhibits additional head skeleton defects consistent with its relative allelic strength and its suggested dominant-negative molecular behavior (Barker et al., 1992; Wharton et al., 1998). The protruding lr (black arrow) from defective head involution is in a different focal plane. Black arrows indicate structures residing in a different focal plane. Scale bars: 120 µm in A; 20 µm in B.

View larger version (51K): [in a new window]   Fig. 7. nos– mutants have a milder effect (A) Internally controlled bcd PAT assay from wild type (lanes 1-9) and nos– (lanes 1'-9') RNAs from ovaries (lane 1,1') or embryos (lanes 2-9,2'-9', 25°C). C1, bcd cDNA control; C2, osk PCR control (no template). While nos– mutants exhibit some delayed bcd deadenylation (compare lane 8' with lane 8), the bcd polyA tail profile returns to a wild-type one in the final time point (lane 9' versus lane 9). By contrast, pum– embryos show a stronger deadenylation defect in the equivalent time course (compare the ratio lane 9:lane 9' here with lane 9:lane 9' in Fig. 2B). Note, each mutant set can only be compared with its parallel wild-type collection. (B) Western blot of wild-type (lanes 2-9) and nos– (lanes 2'-9') extracts probed for Bicoid (time course as in A). A nonspecific crossreactive band of slightly lower mobility than Bicoid appears in nos– extracts. (C) Scanning electron micrograph of nos– heads (frontal view). Four percent of nos– cuticles exhibit a protrusion analogous to pum– (severe), 29% showed a smaller variably sized protrusion (moderate) and 67% had no such abnormality (unaffected). n=52. Scale bar: 20 µm.