Genetics, Vol. 160, 1535-1547, April 2002, Copyright © 2002
The Function of the frizzled Pathway in the Drosophila Wing Is Dependent on inturned and fuzzy
Haeryun Lee1,a and
Paul N. Adlera
a Biology Department and Cancer Center, University of Virginia, Charlottesville, Virginia 22903
Corresponding author:
Paul N. Adler, Gilmer Hall, Rm. 245, University of Virginia, Charlottesville, VA 22903., pna{at}virginia.edu (E-mail)
Communicating editor: T. W. CLINE
 | ABSTRACT |
|---|
The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. We have investigated the genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes). Previous data showed that the inturned-like genes were epistatic to the frizzled-like genes when the entire wing was mutant. We extended those experiments and examined the behavior of frizzled clones in mutant wings. We found the domineering nonautonomy of frizzled clones was not altered when the clone cells were simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it was blocked when the entire wing was mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. We found inturned, fuzzy, and multiple wing hairs were required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes we determined the consequences of altering the activity of frizzled-like genes in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhanced the phenotypes of hypomorphic inturned and fuzzy mutants. We also examined the relationship between the frizzled-like and inturned-like genes in other regions of the fly. For some body regions and cell types (e.g., abdomen) the inturned-like genes were epistatic to the frizzled-like genes, but in other body regions (e.g., eye) that was not the case. Thus, the genetic control of tissue polarity is body region specific.
THE adult Drosophila epidermis secretes a cuticle with a rich morphology and dramatic planar polarity (also known as tissue polarity). A number of different cuticular structures share this polarity. Cells that secrete epidermal hairs cover much of the body. These noninnervated structures typically point distally on appendages and posteriorly on the trunk. Hair development has principally been studied on the wing. Sensory bristles are found in most regions of the fly. These multicell sense organs contain a prominent shaft that usually points distally on appendages and posteriorly on the trunk. Planar polarity is also manifested in the eye by the arrangement of photoreceptor cells in ommatidia.
Mutations in a number of genes disrupt the polarity of all three of these cellular structures, which has led to the suggestion that a common genetic pathway regulates polarity in all parts of the epidermis. Among the genes where mutations affect all three cell types are frizzled (fz), starry night [stan; aka flamingo (fmi)], dishevelled (dsh), and Van Gogh [Vang; aka strabismus (stbm); GUBB and GARCIA-BELLIDO 1982; ADLER et al. 1987; WONG and ADLER 1993; KLINGENSMITH et al. 1994; THEISEN et al. 1994; ZHENG et al. 1995; TAYLOR et al. 1998; WOLFF and RUBIN 1998; USUI et al. 1999]. Mutations in other genes disrupt tissue polarity in one or two of these cell types. For example, mutations in inturned (in), fuzzy (fy), and fritz (frtz) affect hairs and bristles (GUBB and GARCIA-BELLIDO 1982 
; WONG and ADLER 1993 ; PARK et al. 1996 ; COLLIER and GUBB 1997 ; COLLIER et al. 1997 ), mutations in diego (dgo) affect hairs and ommatidia (FEIGUIN et al. 2001 ), and mutations in multiple wing hairs (mwh) affect only hairs (GUBB and GARCIA-BELLIDO 1982 ). One interpretation of these observations is that genes that affect all three cell types are part of a functional core group of genes, while genes that affect only one or two cell types are part of the cell type-specific interpretation of the common pathway (ADLER 1992 ; SHULMAN et al. 1998 ). There are, however, some difficulties with this interpretation.
The cellular basis for the development of planar polarity is different in all three of these cell types. The distal polarity of wing hairs is a consequence of wing cells forming hairs at the distal-most part of the cell. Mutations that alter hair polarity do so by altering the subcellular location for hair initiation (WONG and ADLER 1993 ). The polarity of sensory bristles is a consequence of the orientation of mitotic spindles in the differentiative cell divisions in the sense organ lineage. Mutations that alter bristle polarity have been found to alter the orientation of the sensory precursor cell (PI) division (GHO and SCHWEISGUTH 1998 ; GHO et al. 1999 ). The chirality of ommatidia is dependent on which of two cells adopt the R3 vs. R4 cell identity (ZHENG et al. 1995 ; WHERLI and TOMLINSON 1998 ). This subsequently determines the orientation of ommatidial rotation. Mutations that alter ommatidial polarity disrupt the R3 vs. R4 cell fate decision.
The fz pathway has both cell autonomous and cell nonautonomous functions in the specification of wing hair polarity (VINSON and ADLER 1987 ). Clones of cells mutant for fz or Vang typically alter the polarity of wild-type hairs that surround the clones. In the case of fz the wild-type hairs appear to be attracted to the clone while for Vang the wild-type hairs appear to be repulsed by the clone (TAYLOR et al. 1998 ). This led to the suggestion that fz clones fail to produce a polarity signal while Vang clones produce too much of the signal (or vice versa; ADLER et al. 2000A ). However, little is known about the nature of the putative signal. Downstream are a group of genes that function cell autonomously and encode proteins that accumulate asymmetrically in wing cells. Fz (which is a serpentine receptor; VINSON et al. 1989 ) and Dsh (which is a PDZ domain containing protein; AXELROD et al. 1998 ; BOUTROS et al. 1998 ) accumulate at the distal side of the cell and Fmi/Stan (which is a protocadherin; CHAE et al. 1999 ; USUI et al. 1999 ) and Dgo (which contains ankyrin repeats; FEIGUIN et al. 2001 ) appear to accumulate at both the distal and proximal sides (USUI et al. 1999 ; AXELROD 2001 ; FEIGUIN et al. 2001 ; SHIMADA et al. 2001 ; STRUTT 2001 ). The function of all of these genes is a corequirement for the asymmetric localization of others. This asymmetric accumulation could provide a cortical mark to specify the location for hair initiation. However, there is some doubt that this cortical mark is a general mechanism as dgo does not produce a tissue polarity phenotype in bristles (FEIGUIN et al. 2001 ) and Fmi/Stan does not accumulate asymmetrically in the PI cell in the bristle lineage (LU et al. 1999 ).
Mutations in a number of genes, including in and fy, result in both polarity abnormalities and many cells forming more than one hair (we refer to these genes as the in-like genes). Earlier studies showed that loss-of-function mutations in fy, in, and mwh are epistatic to loss-of-function mutations in fz, dsh, and Vang (WONG and ADLER 1993 ; TAYLOR et al. 1998 ). This was interpreted as in, fy, and mwh being downstream of fz and dsh. Consistent with this are observations showing that the asymmetric accumulation of Fz, Dsh, and Fmi is not altered in in, fy, or mwh mutants (USUI et al. 1999 ; AXELROD 2001 ; FEIGUIN et al. 2001 ; SHIMADA et al. 2001 ; STRUTT 2001 ). In this article we report a series of experiments where we have further probed the relationship between fz, dsh, prickle (pk), spiny-legs (sple), and stan/fmi and in, fy, and mwh in the wing. We have also examined the relationship between fz and in and fy in other body regions. Results from several different experimental paradigms supported the hypotheses that in and fy function downstream of fz, dsh, stan, pk, and sple in the wing and that they are required for the transduction of the fz signal to regulate hair morphogenesis and hence the actin and microtubule cytoskeletons. We further found that the situation is quite complex when we consider other body regions. In the thorax and abdomen the relationship between fz and in and fy appeared similar to that in the wing. However, the situation was different in the eye and the leg, where in and fy were not epistatic to fz.
| MATERIALS AND METHODS |
|---|
Fly culture and strains:
Flies were grown on standard media at 25° or 18°. Many of the mutations used in this article were isolated in this lab. These included mutations in in, fz, fy, mwh, pk, and strb. Other mutant, deficiency, or GAL4 enhancer trap stocks were obtained from the stock center at Indiana University. Unless noted, all mutations used were phenotypic nulls or in the case of dsh1 the strongest viable allele. UAS-dsh was obtained from J. Axelrod, UAS-stan/fmi was obtained from the T. Uemura lab, and UAS-pk and UAS-sple were from D. Gubb. Several of the genes used in this article have multiple names. We use Vang instead of stbm and stan instead of fmi. This usage is in line with their FlyBase listings.
Mitotic clone analyses:
Loss-of-function clones of fz, dsh, in, and mwh were generated using FLP/FRT-based mitotic recombination (XU and RUBIN 1993 ). tricornered (trc), starburst (strb), and forked (f36) were used to mark the clones in the wing, abdomen, and thorax. Clones were induced by heat shock for 1 hr at 37° at 2–4 days after egg lay (AEL). The genotypes in which we examined clones are listed below. In experiments not described in depth in RESULTS we examined the ability to induce FLP/FRT-mediated mitotic crossing over for two different chromosome arms in the same wing cell lineage. In these experiments we used ultrahairA (on 2L; ADLER et al. 2000B ) and tricornered (on 3L; GENG et al. 2000 ). These mutations produce very different cellular phenotypes so it was quite easy to identify the doubly mutant cells that had an additive phenotype. In our original experiments we used two heat shocks but we found that this was not necessary. The frequency of clones where recombination took place on both arms simultaneously (as compared to the frequency of clones where recombination took place on one arm) was much higher than random. We found in these experiments that FRT40 and FRT42 resulted in a frequency of mitotic clones much higher than that of FRT80 (XU and RUBIN 1993 ). This was consistent with our past experience with these chromosomes. On the basis of clone frequency and size we estimate that approximately 1/100 wing cells were recombinant for ultA (FRT40) and 1/1000 for trc (FTR80). Among the trc clones, 30% (18/60) were also mutant for ultA. This is 30 times more frequent than would be expected if exchanges on the two chromosomes were independent. Our interpretation of this result is that only a subpopulation of cells is competent for crossing over and the frequency per FRT site for these cells is quite high. We note that the induction of recombination on two separate chromosome arms has been described previously (SELVA and PERRIMON 2000 ).
- fz trc in clones: w hsflp; fzP21 trc1 inIH56 FRT80/FRT80
- fz mwh strb clones: w hsflp; mwh1 fzR52 strb1 FRT80/FRT80
- dsh forked; fz strb clones: dsh1 f36a FRT18/FRT18; hsflp/+; fzR52 strb1 FRT80/FRT80
- fz trc in clones in an inturned wing: w hsflp; fzP21 trc1 inIH56 FRT80/inIH56 FRT80
- fz mwh strb clones in a mwh wing: w hsflp; mwh1 fzR52 strb1 FRT80/mwh1 FRT80
- fz strb clones in a dsh wing: dsh1/Y; hsflp/+; fzR52 strb1 FRT80/FRT80
- fz strb clones in a fy wing: w hsflp; fy2; fzR52 strb1 FRT80/FRT80
Waxing experiments:
To induce a gradient of expression of polarity genes from heat-shock-regulated transgenes (hs-fz, hs-dsh, and hs-in), hot wax was applied to the distal tip of the wing of developing pupae 24–28 hr after prepupa formation (ADLER et al. 1997 ).
Mounting of the wing and other body parts:
Adult wings were mounted in euparal after dehydration in 100% ethanol. Abdomens, thoraces, and legs were mounted in Gary's Magic Mount after dehydration in 100% ethanol. Mounted samples were examined under the bright field microscope. Their images were taken with Spot digital camera (Diagnostics) and processed in the Adobe Photoshop 5.0.
Scoring of multiple hair phenotypes:
Unless otherwise noted, the area with 100 cells (20 x 5 cells) in the "C" region centered above the posterior cross vein was selected for counting the number or fraction of the cells with multiple hairs.
Injection of microtubule antagonists into pupae:
Vinblastine and colchicine were injected into pupae as described previously (GENG et al. 2000 ).
| RESULTS |
|---|
inturned and fuzzy are needed in cells receiving and responding to a frizzled dependent intercellular signal:
The cell nonautonomous function of fz is seen when clones of cells mutant for fz are generated in the wing (VINSON and ADLER 1987 ). The normal distal polarity of wild-type hairs located distal to the clone is altered so that these hairs appear to be attracted to the clone (ADLER et al. 1997 ). This seems likely to be due to the clone cells failing to produce an intercellular signal (or to producing excess signal). To determine if genes such as in were required in the clone cells for the production of the domineering nonautonomy we induced and examined fz trc in mutant clones (trc was used to mark the clone cells; GENG et al. 2000 ). Twelve of 14 clones displayed the domineering nonautonomy characteristic of fz clones (Fig 1, c–e). This led us to conclude that in was not required for fz clone cells to produce domineering nonautonomy. Previously we found that mwh fz clones also displayed typical fz domineering nonautonomy, but in those experiments there was no independent marker to identify clone cells (VINSON and ADLER 1987 ). We therefore reexamined this by inducing mwh fz strb clones (strb was used to mark the clone; PARK et al. 1996 ). All 17 of the mwh fz strb clones examined displayed typical fz domineering nonautonomy (Fig 1G). Thus, like in, mwh is not required for fz clone cells to produce domineering nonautonomy.
View larger version (78K):
In this window
In a new window
Download PPT slide
|
Figure 1.
in, mwh, and dsh are not required for fz clone cells to alter the polarity of neighbors. (a) Wild-type wing. Note that every wing cell forms a single hair that points distally. (b) A diagram of the wing regions (A, B, C, C', D, D', and E) divided by wing veins. The domineering nonautonomy of fz clones marked by trc (c) or strb (d; clones are outlined) alters the polarity of wild-type hairs located distal to the clone. (e) A fz in double mutant clone marked by trc shows the domineering nonautonomy typical of fz clones. (f) An in mutant clone marked by trc acts cell autonomously. (g) A fz mwh double mutant clone marked by strb displays the typical fz domineering nonautonomy. (h) A mwh clone acts cell autonomously. (i) A dsh; fz double mutant clone marked by f and strb produces domineering nonautonomy. (j) A dsh clone marked by forked acts cell autonomously. (k) A higher magnification view of a dsh f clone. (l) A higher magnification of a dsh f; fz strb clone. (m) A higher magnification view of a fz strb clone. Note in these higher magnification views the more severe phenotype (smaller and clustered or split) fz strb hairs compared to the dsh f; fz strb doubly mutant cells. f appears to partially suppress the extreme hair phenotype of strb. (n) An ultA clone. (o) A pair of ultA; trc doubly mutant cells. (p) A trc clone. Note the larger size of the polyploid ultA cells compared to the wild-type cells (multiple hairs produced by ultA cells are as long as or longer than the single hairs formed by wild type). Note the remarkable phenotype of the polyploid ultA; trc cells. All wings are positioned with proximal to the left, distal to the right, anterior up, and posterior down. The clonal boundaries are marked with a line according to the presence of the wing cell marker phenotype. Clones of >10 cells were selected to determine the nonautonomy.
|
|
To determine if dsh was required for fz clone cells to produce domineering nonautonomy we wanted to generate clones that were doubly mutant for dsh f and fz strb by the simultaneous induction of FLP/FRT-based crossing over on the X chromosome and on 3L. To determine if this was feasible we carried out a test experiment using ultA (ADLER et al. 2000B ) and trc (GENG et al. 2000 ), which provide easily distinguishable phenotypes (Fig 1, n–p). We found that the simultaneous induction of cells where there were recombination events on both arms was more frequent than random (these experiments are described in more detail in MATERIALS AND METHODS). We were able to identify the dsh; fz doubly mutant clone cells by their hair morphology (f; strb hairs are short, thin, and bent, while f hairs are bent and strb hairs are very short, frequently split, or multipled and thin). As was the case for the fz in and mwh fz clones the domineering nonautonomy of fz was not blocked by the cell also being mutant for dsh (11/11 clones; Fig 1I).
To determine if in was required for cells to respond to the presence of mutant fz cells we induced fz trc in clones in a fz trc in/in wing. We could identify the clone due to the hair morphology phenotype of trc. While in mutations result in altered hair polarity, over most of the wing the in mutant pattern is consistent enough to enable us to determine if the abnormal intercellular signal expected from the fz trc clone resulted in domineering nonautonomy. The fz trc clones did not alter the polarity of the surrounding cells (11/11 clones; Fig 2A). Thus, we concluded that in was required for cells to detect or respond to fz clone cells. Similar results were obtained when we induced fz clones in wings mutant for dsh (5/5 clones; Fig 2E), fy (22/22), and mwh (4/5 clones; Fig 2C). Hence we concluded that these genes were also essential for cells to detect or respond to fz clone cells. Previous experiments had found mutations in stan and Vang also blocked or suppressed the domineering nonautonomy of fz (TAYLOR et al. 1998 ; CHAE et al. 1999 ). The results for these six genes stand in contrast to the findings for pk and ds, which did not block the domineering nonautonomy of fz clones, although mutations in pk and ds did alter the anatomical direction of the domineering nonautonomy (ADLER et al. 1998 , ADLER et al. 2000A ).
View larger version (148K):
In this window
In a new window
Download PPT slide
|
Figure 2.
in, mwh, and dsh are required in cells to receive or respond to altered fz activity of neighboring cells. (a) The presence of a clone of fzP21 trc cells in an inIH56/Df wing fails to alter the hair polarity of the neighboring cells. (b) For comparison with a, we show the same region (E region) of an inIH56/Df mutant wing without a clone. (c) The presence of a fzR52 strb clone in a mwh background does not alter the polarity of neighboring cells. (d) For comparison with c, we show the same region (D region) of a mwh wing without a clone. (e) The presence of a fzR52 strb clone in a dsh background does not alter the polarity of neighboring cells. (f) For comparison with e we show the same region (D region) of a dsh wing without a clone. All wings are positioned with proximal to the left, distal to the right, anterior up, and posterior down. Clones with >10 cells were chosen to score for the nonautonomy.
|
|
The domineering nonautonomy of fz clones suggests that cells can assess the level of fz activity of neighboring cells and respond to this. When a gradient of fz expression is induced, hair polarity is directed to point from cells of higher levels toward cells of lower levels (ADLER et al. 1997 ). To determine if the activity of in, fy, and mwh was required for cells to respond to a gradient of fz expression we induced such a gradient in a wing that was mutant for any of these genes. We found no effect on hair polarity of directing a gradient of fz expression in wings mutant for in (Fig 3, A–C), fy (Fig 3, D–F), or mwh (data not shown). We concluded that these genes were required for cells to respond to altered levels of fz expression, suggesting that they functioned downstream of fz for planar polarity in the wing. We note that previous experiments showed that mutations in dsh, stan, and Vang similarly blocked the ability of cells to respond to a gradient of fz expression (ADLER et al. 1997 ; TAYLOR et al. 1998 ; CHAE et al. 1999 ).
inturned and fuzzy are needed for cells to respond to prickle, spiny-legs, and starry night:
To determine the relative positions of in, fy, and mwh with respect to several other fz-like tissue polarity genes we examined their ability to block the gain-of-function phenotypes that result from the directed expression of stan/fmi, pk, and sple. We used ptc-GAL4 to overexpress pk. This results in a band of expression in the C region of the wing, with a decreasing gradient of expression away from the center of the ptc expression domain (ADLER et al. 1997 ; USUI et al. 1999 ). This results in hairs pointing toward the midline in the C region of the wing (GUBB et al. 1999 ). This phenotype was blocked by mutations in in (Fig 3, G–I), fy, and mwh. The resulting wings (e.g., ptc-GAL4/UAS-pk; in/in) showed no effects from the directed expression of pk. We used two different GAL4 drivers (ms1096-GAL4 and act-GAL4) to direct the expression of UAS-sple. Both of these drivers result in relatively even expression across the wing (although in ms1096 the level in the dorsal cell layer is higher than that in the ventral cell layer). This results in a reversal of hair polarity over much of the wing (Fig 3J) that resembles that seen in pkD (ADLER et al. 2000A ). Once again we found that mutations in in, fy (Fig 3, J–L), and mwh were able to block this gain-of-function phenotype. We used the omb-GAL4 driver to drive expression of UAS-stan. In the wing disc omb-GAL4 drives expression in a band located centrally along the anterior/posterior axis of the wing. However, in the pupal wing the expression pattern is more complicated during the time for tissue polarity development. During this time in the distal region of the wing we see a series of alternating bands of expression and no expression. Driving expression of stan using omb-GAL4 leads to a series of bands of polarity reversals consistent with the previous results of USUI et al. 1999 who found that polarity was oriented from cells of low toward cells of high stan expression. Once again we found that mutations in in (Fig 3, M–O), fy, and mwh were able to block the consequences of overexpressing stan. These data argue that in, fy, and mwh functioned downstream of fz, pk, sple, and stan in tissue polarity.
Driving expression of dsh using the ptc-GAL4 driver results in the formation of multiple hair cells in the proximal part of the ptc expression domain (Fig 4A and Fig D). In addition, ectopic bristles are also often found, presumably due to the role of dsh in canonical wg signaling (AXELROD et al. 1996 ). When we examined UAS-dsh +/+ ptc-GAL4; in/in flies we found an additive multiple hair cell phenotype (i.e., stronger than either single phenotype) in the proximal part of the ptc domain (Fig 4C) and the presence of ectopic bristles. Equivalent results were found in UAS-dsh fy; fy ptc-GAL4 wings (Fig 4F). The formation of the ectopic bristles was not surprising, since this is likely an effect of dsh on canonical wg signaling and in and fy are not thought to play a role in wg signaling. The additive multiple hair cell phenotype was unexpected and argues either that in and fy are not downstream of dsh in the fz pathway or that when overexpressed, Dsh can bypass the requirement for in and fy function.
View larger version (51K):
In this window
In a new window
Download PPT slide
|
Figure 4.
The multiple hair phenotype of overexpressed dsh is enhanced in wings mutant for fuzzy and inturned. Driving expression of UAS-dsh using ptc-GAL4 leads to lethality when flies are raised at 25° but adults frequently eclose when raised at 18°. We find occasional multiple hair cells in the C region of the wing on such flies. This phenotype shows a little variation in different genetic backgrounds (A and D). In addition, the overexpression of dsh sometimes leads to patches of smaller cells. Mutations in fy (fy2; B) and in (inIH56/Df; E) cause some cells in this region to form multiple hairs. The multiple hair cell phenotype of overexpressed dsh is additive or synergistic with the multiple hair cell phenotypes of fy (C) and in (F).
|
|
The overexpression of fz just prior to hair initiation results in a multiple hair cell phenotype that resembles that of in and fy (this is sometimes referred to as the late fz gain of function; KRASNOW and ADLER 1994 ). When we overexpressed fz just prior to hair initiation in pupae that were mutant for a null allele of either in or fy we found an increased number of multiple hair cells (Table 1). Thus, for the late fz gain of function, as for the overexpression of dsh, either in and fy are not required for fz signal transduction or when fz is overexpressed late the requirement for in and fy can be bypassed.
The relationship between in, fy, and the fz-like genes:
To test whether the frizzled-like genes could be regulating the inturned-like genes negatively or positively, we examined the effects of both decreased and increased fz-like gene activity on hypomorphic alleles of in and fy. In these experiments we took advantage of the much higher number of multiple hair cells found in in and fy mutants compared to fz-like mutants. In all cases examined we found that mutations (null or hypomorphic) in fz, pk, Vang, stan, and dsh acted as strong enhancers of a weak in or fy phenotype as assayed by the frequency of multiple hair cells (Fig 5; Table 2). These data are consistent with the fz-like genes acting as positive regulators of in and fy.
View larger version (99K):
In this window
In a new window
Download PPT slide
|
Figure 5.
Loss-of-function mutations in fz-like genes enhance the phenotypes of weak alleles of in and fy. All micrographs are from the C region of the wing just anterior to the posterior cross vein. This is the region we used for scoring the fraction of multiple hair cells for Table 2. (A) fyJN12/fy1. (B) fyJN12/fy1; fzR54/fzK21. (C) fzR54/fzK21. (D) inII53; (E) pk1; inII53. (F) pk1. Note the enhancement of the in and fy multiple hair cell phenotypes by fz and pk.
|
|
View this table:
In this window
In a new window
|
Table 2.
Enhancement of weak inturned and fuzzy phenotypes by mutations in frizzled and other upstream genes
|
|
If there were a simple positive relationship between the fz-like genes and in and fy then we would expect that the overexpression of fz-like genes should suppress hypomorphic alleles of in and fy. To test this we overexpressed fz from a hs-fz transgene and found that this enhanced the multiple hair cell phenotypes of weak inII53 and fyJN12 alleles. These results argued that fz antagonized the activity of in and fy (Table 3). We similarly tested the genetic relationship between pk, sple, and fmi and weak alleles of in and fy. Overexpression of sple and stan from ms1096-GAL4 (or act-GAL4); UAS-sple and omb-GAL4; UAS-stan, respectively, in hypomorphic mutant backgrounds of in and fy resulted in an increase in the multiple hair cell phenotypes in the region of the wing where expression was driven by the GAL4 enhancer trap (Fig 3J, Fig Q, Fig T, Fig M, Fig R, and Fig U). A similar enhancement was not seen for the overexpression of pk from ptc-GAL4 UAS-pk (Fig 3, G, P, and S). These results suggested that sple and stan antagonized the activity of the in and fy. Notably the gain-of-function polarity phenotypes of overexpressed pk, sple, and stan were blocked even with weak alleles of in and fy, confirming that these genes are required for the function of the fz pathway. Taken with the data described above it is clear that the fz-like genes do not act as simple positive or negative regulators of in and fy. The interaction is reminiscent of the observations that similar phenotypes result from either overexpression or a lack of function of fz-like genes (KRASNOW and ADLER 1994 ; AXELROD et al. 1998 ; BOUTROS et al. 1998 ; GUBB et al. 1999 ; USUI et al. 1999 ).
View this table:
In this window
In a new window
|
Table 3.
Enhancement of the multiple hair cell phenotype of hypomorphic alleles of in and fy by overexpression of fz
|
|
Overexpression of dsh from UAS-dsh; ptc-GAL4 at 18° in weak in and fy backgrounds produced severe multiple hair cell phenotypes that were more severe than null in and fy alleles (data not shown). These results are reminiscent of what we saw with the null in and fy alleles.
As a further test of the dose relationship between the fz-like and in-like genes we examined the consequence of overexpressing both a fz-like and an in-like gene simultaneously. We first confirmed that the overexpression of in or fy did not produce a wing phenotype (PARK et al. 1996 ; COLLIER and GUBB 1997 ). When we coordinately overexpressed fz and in using the waxing protocol to generate a distal-to-proximal expression gradient we found an enhanced reversed polarity phenotype. Both the frequency of finding regions of polarity reversal (41/73 for hs-fz, 0/11 for hs-in, and 22/24 for hs-fz; hs-in) and the size of the region appeared to be enhanced. In an additional set of experiments we found that the simultaneous overexpression of fz and dsh using the waxing procedure also resulted in an enhanced reversed polarity phenotype (0/17 for hs-dsh, 61/69 for hs-fz; hs-dsh).
in, fy, and mwh:
Double mutants of in, fy, and frtz with mwh display a mwh phenotype (WONG and ADLER 1993 ; COLLIER et al. 1997 ). In generating such flies we noticed that in and fy flies that carry one mutant mwh gene had an enhanced multiple hair cell phenotype. We examined this in some detail and found it to be a consistent result for seven different in alleles at both 18° and 29°. We further found that an increase in mwh gene dose (the presence of a duplication) weakly suppressed the in multiple hair cell phenotype (Table 4). Since the dominant enhancement by mwh is seen for null alleles of in and fy, the enhanced phenotypes cannot be from mwh serving as an upstream activator of in or fy. The data are consistent with in and fy acting to increase the activity of mwh. This is also consistent with previous observations that argued that mwh was epistatic to in and fy (WONG and ADLER 1993 ).
View this table:
In this window
In a new window
|
Table 4.
Enhancement of the multiple hair cell phenotypes of in and fy by the reduction of mwh dose
|
|
The microtubule cytoskeleton and in and fy function:
The disruption of the microtubule cytoskeleton by treatment with vinblastine or colchicine results in many wing cells forming more than one hair (TURNER and ADLER 1998 ). At the level of the individual cell these multiple hair cells resemble those found in in and fy mutants as the hairs are formed at the cell periphery and appear to be the result of independent initiation events. The altered polarity seen in in and fy mutant cells is not induced by vinblastine or colchicine. Immunostaining has not revealed any defects in microtubule organization in in or fy mutants (P. N. ADLER, unpublished results). Thus, it seems unlikely that the in and fy phenotypes are due to disrupting the microtubule cytoskeleton and in this way indirectly affecting hair morphogenesis. However, it seemed possible that the disruption of the microtubule cytoskeleton could be inducing multiple hairs by interfering with in and/or fy function. We reasoned that if that was the case then treatment of in or fy null mutant wing cells with vinblastine or colchicine would not produce a stronger phenotype than the in or fy null cells by themselves. We injected vinblastine into in or fy mutant pupae and found an additive response. That is, injected in or fy mutant cells produced a stronger multiple hair cell phenotype than did untreated mutant cells or cells from injected wild-type pupae (Fig 6). This result shows that the microtubule disruption phenotype cannot be due to a simple interference with in/fy function and argues that the microtubule cytoskeleton has a function that is independent of the fz pathway. The additive response in this experiment stands in sharp contrast with the lack of additivity in double mutants of in, fy, frtz, and mwh.
View larger version (135K):
In this window
In a new window
Download PPT slide
|
Figure 6.
Vinblastine treatment enhances the multiple hair cell phenotype of a fy null mutant. Null mutations in fuzzy result in wing cells forming multiple hairs with abnormal polarity (top). Wild-type pupae injected with vinblastine (VB) produce multiple hair cells in the wing (bottom) without polarity defects. When the fuzzy null mutants are treated with vinblastine, they display an enhanced multiple hair cell phenotype (middle).
|
|
fz, in, and fy on the abdomen:
Mutations in most fz pathway genes produce planar polarity phenotypes in many body regions (GUBB and GARCIA-BELLIDO 1982 ; ADLER 1992 ). On the abdomen fz mutants produce altered bristle polarity, with most bristles now pointing at least partially toward the midline and not posteriorly as in wild type (GUBB and GARCIA-BELLIDO 1982 ). Abdominal hair polarity is also altered in fz mutants. The polarity phenotype seen in the abdomen of in and fy mutants is in general more severe than that of fz (GUBB and GARCIA-BELLIDO 1982 ). The tendency of bristles to point toward the midline is enhanced and, in addition to the hair polarity phenotype, most if not all hair-forming cells form more hairs than in wild type (wild-type abdominal cells often form 4–5 hairs). As is the case in the wing, in and fy are epistatic to fz as double mutants resemble in and fy and not fz. Wild-type abdominal cells located posteriorly to clones of fz strb cells (strb is a hair and bristle morphology marker) have their polarity altered and appear as if attracted to the clone (Fig 7). Similarly, wild-type cells located anteriorly to clones of pwn Vang cells have their polarity altered and appear as if repulsed by the clone (data not shown). Thus, the situation on the abdomen appears quite similar to the wing with fz and Vang showing complementary domineering nonautonomy in both body regions. To determine if in was required for the domineering nonautonomy of fz clones we examined fz trc in clones in the abdomen. As we had seen in the wing, the domineering nonautonomy of fz in the abdomen was not blocked by cells also being mutant for in (Fig 7J and Fig K). We were unable to test if in was required in the responding abdomen cells as the hair phenotype of in mutants was too severe to allow us to assess if there was a nonautonomous effect of a fz clone.
View larger version (124K):
In this window
In a new window
Download PPT slide
|
Figure 7.
fz and in in the abdomen, thorax, and leg. A–D show tarsal segments of wild type (A), fz (B), in1 (C), and fz in (D). The arrow points to a cuticular bleb seen in fz mutants. E–H show part of the femur of wild-type (E), fz (F), in (G), and fz in (H) flies. The arrows point to duplicated bracts and the arrowheads to bracts that are located incorrectly with respect to the proximal distal axis of the leg and the associated bristle. I shows part of a thorax from a living in fly. The arrows point to split bristles. J and K show fz strb and fz trc in clones, respectively, in the abdomen (outlined). Note in both cases the domineering nonautonomy of fz can be seen in the abnormal polarity of hairs located distal to the clone.
|
|
fz, in, and fy on the thorax:
fz mutations cause altered bristle and hair polarity on the thorax. A similar phenotype is seen in in and fy mutants, although the bristles with altered polarity most commonly point toward the midline in these mutants and the fy phenotype seems somewhat weaker. Over much of the notum in in and fy mutants hairs show altered polarity but in contrast to the wing and abdomen multiple hair cells are rare. As was seen on the abdomen both fz and Vang clones show complementary domineering nonautonomy in the thorax (data not shown). We generated fz trc in cells in the thorax and found they still showed the posteriorly directed domineering nonautonomy of fz clones (data not shown). Thus, as was seen in the wing and abdomen, in function is not required for the domineering nonautonomy of fz clones. Once again we were unable to determine if a fz clone produced a nonautonomous effect in an in mutant background.
In our examination of in, fy, and fz thoraces we identified two phenotypes where fz clearly differed from in and fy. On the wild-type scutellum hairs point posteriorly. This pattern is disrupted in fz mutants resulting in a complicated pattern. In in and fy mutants the hairs on the scutellum point anteriorly (i.e., polarity is reversed compared to wild type; data not shown). In fy; fz and fz in double mutants the in/fy polarity pattern is seen. A second in/fy phenotype not seen in fz mutants is the routine presence of several split thoracic microchaetae (Fig 7I). The split shaft is similar to that seen in tricornered and furry mutants (GENG et al. 2000 ; CONG et al. 2001 ) and does not appear to be due to a shaft duplication (unless the duplicated shafts fuse during morphogenesis). Such bristles are not routinely found in fz mutants. In fy; fz and fz in double mutants we routinely found such split bristles. Thus, in and fy are epistatic to fz for these two thoracic phenotypes.
fz, in, and fy on the leg:
In the leg, fz mutants (and Vang and dsh mutants) show several phenotypes (GUBB and GARCIA-BELLIDO 1982 ; HELD et al. 1986 ). Hair polarity is disrupted with many hairs no longer pointing distally. Occasional bristle cells also show abnormal polarity. This is most notable when the bract (a thick hair-like structure), which in a wild-type leg is always located proximal to the bristle, is found in an alternate location (e.g., distal to the bristle). While this is not common, at least one (and usually several) mispositioned bracts can be found on essentially all fz legs (Fig 7F). Mutations in fz also have a dramatic phenotype in leg joints (particularly in tarsal segments; Fig 7B). The segments are typically incomplete (missing parts) and the joints (and/or parts of joints and segments) are found duplicated with a plane of mirror image symmetry perpendicular to the proximal distal axis (HELD et al. 1986 ). We also frequently observed bulging blebs of cuticle. In contrast, in an in or fy mutant a somewhat different set of phenotypes are found. Hair polarity is abnormal, but compared to fz many more multiple hair cells are found on in and fy legs (Fig 7G). Occasional bristles with abnormal polarity are seen, but we typically do not see bristles with mispositioned bracts. The bracts are, however, often multipled in in, mwh, and fy (this phenotype is weaker in fy than the other mutants and is typically strongest in the tarsal segments; Fig 7G). Joint duplications are rare, and indeed few if any shape defects are seen in fy and mwh legs. On in legs we often see internal cuticular spheres, which may represent a mild manifestation of the joint phenotype. Rarely, we see duplicated joints. In fy; fz and fz in double mutants we find the joint defects (Fig 7D) and mispositioned bracts characteristic of fz and the multipled bracts characteristic of in and fy (Fig 7H). Thus, in the leg the phenotypes are additive.
fz and in in the eye:
Mutations in fz produce a rough eye phenotype that results from alterations in the regular arrangements of ommatidia. Ommatidia come in two chiral types defined by the arrangement of photoreceptor cells and, in any normal eye, each type is restricted to either the dorsal or the ventral half of the eye. Several defects are seen in fz mutants. These include incorrect chiral types, incorrect rotation, and symmetrical ommatidia (ZHENG et al. 1995 ). in, fy, and mwh do not have rough eyes, although a few aberrant ommatidia are found in essentially all in eyes. In in flies we have seen examples of incorrect chiral type, incorrect rotation, symmetrical ommatidia, and ommatidia with an incorrect number of photoreceptor cells (data not shown). In fz in and fy; fz double mutants the eyes are rough and display the high number of abnormal ommatidia seen in fz. Thus, for the eye phenotype fz is epistatic to in.
fz and wing eversion:
Some fz flies have a deformed wing that is short, fat, and often kinked proximally (Fig 8, D–F). This is seen in all strong and null fz alleles we have examined. We have found that this is due to a defect in wing disc eversion. In a normal pupa the wing everts so that it extends posteriorly and ventrally (Fig 8B). In a fz mutant many pupae contain wings that extend anteriorly and sometimes dorsally (Fig 8A). A substantial fraction of such pupae fail to emerge from the pupal case due to the wing getting caught on the pupal case or the flies emerge with one torn wing. We suggest this is due to difficulty of the miseverted wing sliding out of the pupal case. The frequency of this phenotype is sensitive to genetic background and in individual backgrounds we have seen from 3 to 43% of wings from strong fz mutants affected. The wing eversion phenotype is rescued by the basal expression (25°) of a hs-fz transgene, as is the case for the wing hair polarity phenotype (KRASNOW and ADLER 1994 ). This phenotype is also seen occasionally in other tissue polarity mutants such as pk. The phenotype is rare in cell autonomous fz alleles (<1%) suggesting it is due to a defect in the nonautonomous function of fz. Flies with the miseverted wings are rarely seen (<1%) in flies mutant for in, fy, or mwh. In fy; fz and fz in double mutants we saw the low frequency of miseverted wings characteristic of in and fy. Hence for this phenotype in and fy appeared to be epistatic to fz.
View larger version (76K):
In this window
In a new window
Download PPT slide
|
Figure 8.
The miseversion phenotype of fz. A shows a fz pupa with a miseverted wing. B shows a wild-type pupa for comparison. Note how the wing in A is pointing anteriorly and partially covers up the eye. The miseversion results in adult wings of abnormal shape. Examples of these are shown in D–F. Compare these to the wild-type wing in C.
|
|
| DISCUSSION |
|---|
in and fy and their role in wing development:
The hypothesis that in, fy, and mwh functioned downstream of fz and dsh originally came from the observation that mutations in in, fy, and mwh were epistatic to mutations in fz and dsh (WONG and ADLER 1993 ). Consistent with this hypothesis it has recently been determined that the distal accumulation of Fz, Dsh, Fmi, and Dgo is not altered in in, fy, or mwh mutants (USUI et al. 1999 ; AXELROD 2001 ; FEIGUIN et al. 2001 ; SHIMADA et al. 2001 ; STRUTT 2001 ). In the experiments we reported here we found that in, fy, and mwh were required for cells to either detect or respond to the lack of fz activity in a clone of neighboring mutant cells. We also found that the function of in and fy was required for cells to respond to a directed gradient of fz, stan, pk, or sple expression. Thus, using both of these experimental approaches we found that in and fy were required for the function of the fz pathway. The simplest interpretation of this is that in and fy are essential downstream components of the fz pathway in the wing. An alternative explanation is that in and fy function in a parallel pathway and that the function of the fz pathway is dependent on this putative in pathway. There were a couple of surprising exceptional results. The overexpression of dsh or the late overexpression of fz induces the formation of multiple hair cells (KRASNOW and ADLER 1994 ) and we found these phenotypes were additive with an in or fy mutation. This could indicate that there are multiple pathways downstream of fz and dsh in the wing and that one of them does not require in or fy function. We think it more likely that this is an example of overexpression bypassing a normal requirement for downstream proteins. We suggest that In and Fy normally function as adapters to allow distally accumulated Fz and Dsh to stimulate the cytoskeleton and induce hair initiation in the correct part of the cell. When Fz or Dsh is overexpressed just prior to hair initiation the high cellular concentration might allow these proteins to bypass the need for In and Fy and to interact with and hyperactivate the hair initiation machinery.
In an in or fy mutant not only is the site of hair initiation uncoupled from the distal accumulation of Fz, Dsh, Stan, and Dgo, but many wing cells now form multiple hairs. This appears to be a failure in a refinement process that in a wild-type cell leads to hairs initiating near the distal-most point in the cell and not all along the distal edge of the cell where Fz, Dsh, Stan, and Dgo accumulate. It is interesting that the region over which hairs form in an in or fy mutant is roughly equivalent in size to the region where hairs form in a Rho kinase mutant (WINTER et al. 2001 ) or after disruption of the microtubule cytoskeleton (TURNER and ADLER 1998 ). This suggests that In/Fy may function in the activation of Drok that leads to the refinement of the area for hair initiation (WINTER et al. 2001 ).
The observation that in and fy were epistatic to fz and the frequent multiple hair cells in an in or fy wing led to the hypothesis that in and fy encoded negative regulators of hair initiation and that the activity of fz inactivated these negative regulators at the distal-most part of the cell leading to hairs initiating there (WONG and ADLER 1993 ). The ability of the late overexpression of fz to produce an in-like phenotype (KRASNOW and ADLER 1994 ) was consistent with fz being a negative regulator of in and fy. In this article we looked more directly at the dose relationship between fz-like genes and in-like genes and our results show that our earlier model was incorrect and too simplistic. We found that both increases and decreases in fz-like gene activity enhanced the phenotypes of weak in and fy alleles. It is notable that both loss- and gain-of-function mutations in the fz-like genes can result in similar wing phenotypes (KRASNOW and ADLER 1994 ; AXELROD et al. 1998 ; BOUTROS et al. 1998 ; GUBB et al. 1999 ; USUI et al. 1999 ). Thus, one interpretation of these experiments is that the gain-of-function mutations in the fz-like genes are actually producing a loss of function. This would allow one to argue that the fz-like genes act as simple positive effectors of in and fy. This is possible; however, there are observations that a gain-of-function mutation in fz is not equivalent to a loss of function. For example, a hypomorphic fz wing phenotype is enhanced by decreased dsh function while a fz gain-of-function phenotype is suppressed by decreased dsh dose (KRASNOW et al. 1995 ). We think the results described in this article are consistent with the hypothesis that In and Fy have two functions in hair morphogenesis. One is to transduce the fz signal to the cytoskeleton so that a hair forms in the distal-most part of the cell. This function would be dependent on the localized accumulation of Fz, Dsh, Fmi, and Dgo along the distal side of the cell (AXELROD 2001 ; FEIGUIN et al. 2001 ; STRUTT 2001 ) and hence would be disrupted by either gain- or loss-of-function mutations in the fz-like genes. However, there is no reason to expect the disruption of this function to result in an enhancement of the multiple hair cell phenotypes of in and fy. The second function of in and fy would be to ensure a single hair is made. One possible mechanism for this could be to activate the hypothesized refinement process. In a wild-type cell this function would not require the function of the fz-like genes and could explain the strong multihair phenotype of in and fy mutants that is not seen in fz. How could such a model for In/Fy function explain the enhancement of the multihair phenotype of weak in and fy alleles by alterations in fz activity? One possibility is that a cooperative interaction between these functions might be revealed in the sensitized genetic background of a hypomorphic mutation.
The mechanism that leads to the domineering nonautonomy of fz and Vang clones is poorly understood. An attractive hypothesis is that such clones lead to either a lack of an intercellular signal or an excess of the signal (TAYLOR et al. 1998 ; ADLER et al. 2000A ). In the experiments reported here we found that in, mwh, and dsh are not required in the cells that compose a fz clone for the clone to produce domineering nonautonomy. This was not a surprising result as all three of these genes act cell autonomously (KLINGENSMITH et al. 1994 ; THEISEN et al. 1994 ; PARK et al. 1996 ). The result does emphasize that there is a fz function in tissue polarity that does not require the function of dsh, in, or mwh. It is possible that the cell nonautonomous function of fz in the wing involves a Ca2+-mediated fz pathway identified in vertebrates that dsh is not a part of (WINKBAUER et al. 2001 ).
Tissue and cell type-specific variation and what it says about a core pathway:
The similar complementary domineering nonautonomy of fz and Vang clones in the wing (VINSON and ADLER 1987 ; TAYLOR et al. 1998 ), abdomen, notum, and eye (ZHENG et al. 1995 ; WOLFF and RUBIN 1998 ) argues for a common mechanism for fz-dependent intercellular signaling. There are also similarities in the cell autonomous function of the fz pathway in different body regions, but there are also differences. In the wing the polarized accumulation of Fz, Dsh, Stan, and Dgo is the earliest known cell autonomous step (USUI et al. 1999 ; AXELROD 2001 ; FEIGUIN et al. 2001 ; SHIMADA et al. 2001 ). There are reasons to doubt that this is the case in other cell types. dgo, which is an essential component of the fz pathway in the wing, does not produce a bristle polarity phenotype (FEIGUIN et al. 2001 ) and Stan does not accumulate asymmetrically in the bristle cell lineage (LU et al. 1999 ).
The relationship between fz and in and fy appears quite similar in the wing, abdomen, and thorax. In all three body regions, mutations in in and fy are epistatic to mutations in fz. There is also a great deal of similarity between the phenotypic consequences of most fz pathway mutations in these regions. For in and fy the phenotypes appear equivalent in the wing and abdomen as they result in both altered hair and bristle polarity and in many cells forming more hairs than normal. In the thorax in and fy mutations also cause altered bristle and hair polarity; however, over much of the notum most epidermal cells form only a single hair. This might be due to differences in the cell biology of notum cells compared to wing and abdomen cells. The Fz protein preferentially accumulates at the posterior/anterior edges of notum cells (P. N. ADLER, unpublished results), much as it does along the distal/proximal boundaries of wing cells (STRUTT 2001 ) and it is likely that in the notum it also serves to mark the side of the cell where a hair will form. However, the notum cells have a smaller apical surface than do wing cells so the need to further refine the region for hair initiation to ensure that a single hair is formed is likely reduced (ADLER et al. 2000B ). Thus, in and fy might still function to refine the region for hair initiation in notum, but this function may be unimportant in these cells. If one considers only the wing, notum, and abdomen, the idea of a common planar polarity pathway that includes fz and the in-like genes is attractive. However, there are problems with this hypothesis. In the eye, in and fy have either a weak phenotype or no phenotype, and double mutants show the fz phenotype. In the eye it is possible that in and fy either are redundant or have little or no function. In the leg the situation is more complicated. Mutations in both in and fz have dramatic phenotypic effects but these are not equivalent. The duplicated joint phenotype that is so dramatic in fz mutants is present weakly in in and appears to be missing in fy and mwh. In considering the bract cells we find two different phenotypes. In fz mutants we find mispositioned bracts, while in in, fy, and mwh we find multipled bracts. We suspect the multipled bracts are analogous to the multiple hair cells seen in other body regions. Thus, the bract cell is one where fz and in both have important functions, but they are apparently in independent processes.
The wing eversion phenotype and its possible relationship to convergent extension phenotypes in vertebrate embryos:
In vertebrate embryos a fz-based planar polarity pathway regulates convergent extension (HEISENBERG et al. 2000 ; TADA and SMITH 2000 ; WALLINGFORD et al. 2000 ; WALLINGFORD and HARLAND 2001 ). Thus far there has been no evidence for a fz-based planar pathway having an analogous role in Drosophila. The eversion defect seen in fz mutant wings may be such an example. This is another phenotype where in and fy appear to be epistatic to fz. The observations are somewhat puzzling as the defect in fz appears to be rescued by a mutation in in or fy. Thus, it is not clear that a simple inactivation of the fz pathway is responsible for the eversion phenotype. Perhaps for this phenotype there are multiple inputs into the fz pathway upstream of in and fy and the eversion defect is due to an imbalance in the input to in and fy. Blocking the pathway at in or fy would eliminate the function of the pathway completely and suppress miseversion.
The cellular mechanisms involved in the eversion of the wing are only poorly understood, but it is possible that convergent extension plays a role (CONDIC et al. 1991 ). It is also possible that the joint defects seen in fz mutants have a similar basis. In vivo observations on everting wings and joint morphogenesis would make an important contribution toward determining if convergent extension plays a role in these morphogenetic events. The development of in vivo imaging approaches for Drosophila pupae makes such experiments possible.
| FOOTNOTES |
|---|
1 Present address: Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. 
| ACKNOWLEDGMENTS |
|---|
We thank Paul Skogland and Kevin Lease for their comments on the manuscript. We thank Randi Krasnow, Chris Turner, and Simon Collier for help during the early phases of the research reported here. This work was supported by a grant from the National Institutes of Health (GM-37136).
Manuscript received November 9, 2001; Accepted for publication January 30, 2002.
| LITERATURE CITED |
|---|
TOP
ABSTRACT
MATERIALS AND METHODS