Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle.

Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. Here we describe a new tissue exhibiting planar cell polarity - the denticles, hair-like projections of the Drosophila embryonic epidermis. We describe in real time the changes in the actin cytoskeleton that underlie denticle development, and compare this with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. We present an initial characterization of the localization of several actin regulators during denticle development. We find that several core planar cell polarity proteins are asymmetrically localized during the process. Finally, we define roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity.</p

Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs.

The regulation of signal transduction plays a key role in cell fate choices, and its disregulation contributes to oncogenesis. This duality is exemplified by the tumor suppressor APC. Originally identified for its role in colon tumors, APC family members were subsequently shown to negatively regulate Wnt signaling in both development and disease. The analysis of the normal roles of APC proteins is complicated by the presence of two APC family members in flies and mice. Previous work demonstrated that, in some tissues, single mutations in each gene have no effect, raising the question of whether there is functional overlap between the two APCs or whether APC-independent mechanisms of Wnt regulation exist. We addressed this by eliminating the function of both Drosophila APC genes simultaneously. We find that APC1 and APC2 play overlapping roles in regulating Wingless signaling in the embryonic epidermis and the imaginal discs. Surprisingly, APC1 function in embryos occurs at levels of expression nearly too low to detect. Further, the overlapping functions exist despite striking differences in the intracellular localization of the two APC family members.</p

Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila.

Adenomatous polyposis coli (APC) is mutated in colon cancers. During normal development, APC proteins are essential negative regulators of Wnt signaling and have cytoskeletal functions. Many functions have been proposed for APC proteins, but these have often rested on dominant-negative or partial loss-of-function approaches. Thus, despite intense interest in APC, significant questions remain about its full range of cellular functions and about how mutations in the gene affect these. We isolated six new alleles of Drosophila APC2. Two resemble the truncation alleles found in human tumors and one is a protein null. We generated ovaries and embryos null for both APC2 and APC1, and assessed the consequences of total loss of APC function, allowing us to test several previous hypotheses. Surprisingly, although complete loss of APC1 and APC2 resulted in strong activation of Wingless signaling, it did not substantially alter cell viability, cadherin-based adhesion, spindle morphology, orientation or selection of division plane, as predicted from previous studies. We also tested the hypothesis that truncated APC proteins found in tumors are dominant negative. Two mutant proteins have dominant effects on cytoskeletal regulation, affecting Wnt-independent nuclear retention in syncytial embryos. However, they do not have dominant-negative effects on Wnt signaling.</p

<i>par-2</i> mutants.

<p>A. DIC time-lapse images of wild-type <i>par-2(or373 </i>ts<i>)</i>, <i>par-2(or539 </i>ts<i>)</i>, and <i>par-2(or640 </i>ts<i>)</i> embryos. The blastomeres in the <i>par-2</i> mutants were of similar size at the two cell stage and initiated mitosis simultaneously, in contrast to the wild type. The <i>par-2(or373 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 5 hours prior to imaging. The <i>par-2(or539 </i>ts<i>)</i> and par-<i>2(or540 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 30 minutes prior to imaging. Arrows indicate mitotic spindles at the two cell stage. Times in min:sec are given relative to AB NEBD. Scale bar, 10 µm. B. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; Normal one cell embryo; 2; assymetric two cell embyro, 3; asynchronous two cell divisions. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites transferred to the restrictive temperature for 30 minutes. C. Amino acid alteration in the <i>par-2(or373 </i>ts<i>)</i> mutant. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

<i>mei-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>mei-1(or642 </i>ts<i>) and mei-1(or646 </i>ts<i>)</i> embryos. In the <i>mei-1</i> mutants the polar bodies were large and misshapen and embryos contained multiple [top <i>mei-1(or642 </i>ts<i>)</i> embryo and <i>mei-1(or646 </i>ts<i>)</i>] or zero maternal pronuclei (second <i>mei-1(or642 </i>ts<i>)</i> embryo). The two <i>mei-1(or642 </i>ts<i>)</i> embryos were obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes, the <i>mei-1(or646 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 7 hours prior to imaging. White arrowheads indicates polar bodies, black arrowheads indicate multiple maternal pronuclei, the black arrow denotes multiple nuclei per cell at the two cell stage, and the “p” refers to the paternal pronucleus in an embryo lacking a maternal pronucleus. Times in min:sec are given relative to nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Defect maps of individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal polar body size, 2; normal pronuclear number, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes. C. Amino acid alteration in the mutants. Asterisk indicates the changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein.</p

The phenotypes of the TS mutants when grown at the restrictive temperature from the L1 larval stage.

1<p>We determined the L1 upshift phenotype by plating hypochlorite-synchronized L1 larvae and incubating them at 26°C until they reached adulthood. Abbreviations: Egl: egg laying-defective, Emb: embryonic lethal, Pvl: protruding vulva, Sb: small broods, Ste: sterile.</p

<i>zyg-1</i> mutants.

<p>A. DIC time-lapse images of wild-type, <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or297 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos. In the <i>zyg-1</i> mutants the two cell stage blastomeres assembled monopolar spindles, cytokinesis failed, and there were multiple nuclei present at the four cell equivilent stage. The <i>zyg-1(or278 </i>ts<i>)</i>, <i>zyg-1(or409 </i>ts<i>)</i>, and <i>zyg-1(or1018 </i>ts<i>)</i> embryos were obtained from hermaphrodites shifted to the restrictive temperature for 5–6 hours. The <i>zyg-1(or297 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 30 minutes prior to imaging. Black arrows indicate normal bipolar spindles in the wild-type embryo and white arrowheads indicate multiple nuclei present at the four cell equivalent stage. Times in min:sec are given relative to AB nuclear envelope breakdown (NEBD). Scale bar, 10 µm. B. Amino acid alterations in the mutants. Asterisks indicate the changed residues. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect maps for the <i>zyg-1</i> mutants.Individual embryos observed during time-lapse recordings: embryos are listed on the left and phenotypes are listed on the top: 1; normal two cell embryo, 2; bipolar spindles at two cell stage, 3; one nucleus per cell at four cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at the restrictive temperature for 30 minutes.</p

Determination if the TS mutations are potentially fast-acting.

1<p>We determined if an allele was potentially fast-acting in the following manner: We mounted embryos produced at 15°C on microscope slides and immediately made time-lapse videomicrographs at a room maintained at 24°C. If defects similar to those observed after long temperature shifts were found in at least 20% of the embryos and if there was little embryonic lethality at 15°C, we conclude that the allele may be fast-acting. We have labeled these cases as “Yes”. However, if there was significant embryonic lethality at 15°C, we cannot conclude that the presence of cellular defects after short upshifts is due to the upshift or to defects that occur even at 15°C. We have labeled these cases as “Unclear”.</p>2<p>For <i>mei-1, par-2,</i> and <i>zyg-1</i>, we incubated mutant worms at 26°C for 30 minutes prior to imaging (instead of the usual∼1 min. upshift) because the gene products appeared to be required prior to when we started our imaging (pronuclear migration).</p>3<p>High lethality at the permissive temperature precludes making a determination.</p>4<p>The low penetrance of severe defects precludes making a determination.</p

A <i>plk-1</i> mutant.

<p>A. DIC time-lapse images of wild-type and <i>plk-1(or683 </i>ts<i>)</i> embryos. In the <i>plk-1</i> mutant the nuclear centrosomal complex (NCC) failed to rotate, a transverse P₀ spindle assembled, and the daughter blastomeres were binucleate. The <i>plk-1(or683 </i>ts<i>)</i> embryo was obtained from a hermaphrodite shifted to the restrictive temperature for 6 hours prior to imaging. Black dots represent centrosomes/spindle poles and asterisks denote multiple nuclei per cell at the two cell stage. Times in min:sec are given relative to NEBD. Scale bar, 10 µm. B. Amino acid alteration in the mutant. Asterisk indicates changed residue. Homologous proteins are aligned below the <i>C. elegans</i> protein. C. Defect map for individual embryos observed during time-lapse recordings, embryos are listed on the left and phenotypes are listed on the top: 1; nuclear centrosomal complex rotation, 2; spindle alignment, 3; one nucleus per cell at two cell stage. In the long upshifts, hermaphrodites were transferred to the restrictive temperature for 5–8 hours. In the short upshifts, embryos were harvested from hermaphrodites grown at 15°C and immediately mounted on agar pads for imaging, which took ∼1 min.</p

Summary of the TS mutant loci and comparison of previously available alleles.

1<p>Information obtained from: <a href="http://www.wormbase.org" target="_blank">http://www.wormbase.org</a>.</p