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The receptor-like tyrosine phosphatase Lar is required for epithelial planar polarity and for axis determination with Drosophila ovarian follicles

Howard Hughes Medical Institute, Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210, USA

View larger version (32K): [in a new window]   Fig. 1. Structure and development of Drosophila ovarian follicles. (A) A drawing of a Drosophila ovariole, showing the anterior germarium (ger) followed by a string of eight successively older ovarian follicles connected by interfollicular stalks. The position of the polar cells is shown (red). Each egg chamber stage is indicated above (see Spradling, 1993). As a result of egg elongation during stages 8-14, mature stage 14 egg chambers are 20-fold longer than stage 2 egg chambers in the AP axis, but only seven times wider in the DV axis. Somatic cells are shown in green, whereas nurse cells and the oocyte are tan; the germline stem cells and forming cysts are illustrated in red and orange, respectively. (B) A magnified view of the germarium showing regions 1, 2a, 2b and 3. Cell types are indicated by the same colors as in A, except that the intercyst cells at the end of region 2b (arrow) are in dark blue, while the non-dividing somatic cells that surround region 1 and 2a are shown in light blue. (C) A magnified view of the follicular epithelium from a stage 8 follicle. The apical and basal orientation of the follicle cells can be seen with respect to the basement membrane (red) and the nurse cells.

View larger version (84K): [in a new window]   Fig. 2. bola mutations disrupt egg elongation and basal actin alignment. Scanning electron micrographs of wild type (A) and homozygous bola2 mutant (C) stage 14 egg chambers illustrate that bola follicles remain round, instead of elongating like wild type. A graph of the ratio (R) of the AP axis to the DV axis in wild type (unbroken line) and bola (broken line) egg chambers is plotted versus the time in hours (H) after stage 7 (inset). Individual bola follicle cells fail to elongate as indicated by the shape of individual follicle cell imprints in the eggshell (compare B (wild type) with D (bola)). Alexa-phalloidin staining in the germarium (E) shows abundant basal actin fibers aligned perpendicular to the AP axis, but newly budded stage 2 follicles contain swirling, unpolarized actin (arrow). At stage 4 (F), the orientation of basal actin in follicle cells still lacks a coordinated pattern; however, by stage 7 (H), it is polarized perpendicular to the AP axis of the egg chamber (J). By contrast, in bola egg chambers, the basal actin at stage 7 (I) and later (not shown) usually does not become polarized (K). However, the nurse cell actin filaments in stage 10 bola follicles appear normal (G). a, anterior; ooc, oocyte; p, posterior; S4, stage 4; S7, stage 7; wt, wild type.

View larger version (35K): [in a new window]   Fig. 3. bola mutations are viable alleles of Lar. The 129 kb Lar transcription unit is shown, along with the positions of the bola1 and bola2 P element insertions. bola1 lies 21.0 kb upstream from the previously described 5' end of Lar. bola2 is located 5.4 kb downstream from the 3' end of exon 1 in the first intron. (B) bola mutations abolish ovarian expression of Lar. A northern blot of ovarian RNA is shown that has been probed with Lar and cup cDNAs. The expression of the 8.4 kb Lar mRNA is specifically lost in the Larbola1 ovaries. Similar results were obtained for Larbola2 (not shown). (C,D) Lar is expressed in somatic and germline cells of the ovary. Whole-mount in situ hybridization was carried out of ovaries using a Lar cDNA probe. Both somatic cells and germ cells are labeled throughout much of oogenesis. In the germarium (C), strong expression starts in region 2b follicle cells. By stage 1 and in later egg chambers, Lar is expressed uniformly in both germ cells and follicle cells (C,D).

View larger version (100K): [in a new window]   Fig. 4. Lar is required for follicle formation, polar cell development and oocyte patterning. (A) Actin fibers are polarized relative to the polar cells. A stage 7 egg chamber is shown that was stained with anti-FasIII (red) to mark polar cells (see also inset) and phalloidin to mark actin (green). Actin fibers circle the polar cell pair like parallels of latitude. (B) The actin orientation around the polar cells develops gradually during stage 5-6. A stage 5 egg chamber is shown in which the basal actin fibers circle the polar region (arrowhead), but remain disoriented in the middle of the chamber (lower right). (C) In a stage 7 bola egg chamber, actin remains unoriented with respect to polar cells. (E,F) bola follicles contain extra and ectopic polar cells. A stage 7 bola follicle containing three posterior polar cells (F, magnified in inset), and a stage 8 follicle containing three polar cell pairs are shown (E). (D,G) Ectopic polar cells generated by Hedgehog misexpression can influence actin polarization. A pair of ectopic polar cells (red staining) has oriented actin in the stage 7 follicle shown in D, as shown in the magnified regions (d',d"). Not all ectopic polar cell pairs (red staining) alter actin orientation, as shown in a different follicle (G). (I,K). Two germaria from homozygous Larbola2 females stained for FasIII (red) and actin (green). In region 2b (brackets), germline cysts fail to acquire a lens shape or to span the width of the ovariole. Budding is frequently abnormal: one cell from the anterior cyst has been pinched off (K, arrow) and, rarely, long stalks form (I, arrow). (H,J) Lar is required for Oskar localization. Stage 9-10 wild-type (H) or Larbola2 (J) follicles were stained with Oskar. Normally, Oskar protein accumulates at the posterior of the oocyte (J), but in more than 50% of Larbola2 follicles, posterior Oskar localization was grossly abnormal (I)

View larger version (74K): [in a new window]   Fig. 5. Lar function is required autonomously in posterior follicle cells to localize Oskar. Egg chambers bearing follicle cell clones of Lar5.5 were stained with antibodies for Oskar (red) and ß-galactosidase (green). Normal posterior Oskar localization in stage 9 chambers was always observed if the 70 posterior-most follicle cells were wild type (A). By contrast, when posterior cells were mutant for Lar, Oskar was frequently present in a central aggregate (B,C) or absent altogether (D). When a clonal boundary split the posterior region (E), Oskar localization in the underlying oocyte was nearly cell autonomous (F; same chamber as E). The results are summarized in H. The types of clones and patterns of Oskar localization are indicated by drawings along the axes. NA, not applicable; n=83.

View larger version (110K): [in a new window]   Fig. 6. Lar function is not required autonomously in stage 7-8 follicle cells for planar polarity. Ovarioles bearing follicle cell clones of Lar5.5 were stained with Alexa-phalloidin (red) to reveal actin, and with anti-ß-galactosidase (green) to mark Lar-positive cells. Actin polarization was unaffected by the Lar genotype of the germline (A,B). When egg chambers contained large clones of Lar5.5 follicle cells, actin polarity was either normal (C,D) or globally disrupted (E,F, see Table 1). Consequently, actin organization did not locally follow clonal boundaries. Defects in cyst shape (H, arrow) and follicle budding (G,H, arrowhead) were often seen when follicle cells in regions 2b and 3 were mutant. (I) A follicle with Lar5.5 mutant polar cells (FasIII+lacZ- cell pair, inset). Despite this, the actin was normally polarized. (Actin, red; lacZ, green; FasIII, blue.) (J) Clones of LanA9-32 generate a low frequency of round eggs (inset). In all egg chambers, LanA protein was only observed on the basal side of the follicle cells (arrowhead), the location of the basement membrane. LanA, red; actin, green; vasa, blue.