Balanced Rac1 and Rhoa Activities Regulate Cell Shape and Drive Invagination Morphogenesis in Epithelia

Balanced Rac1 and RhoA activities regulate cell shape and drive invagination morphogenesis in epithelia

Bharesh K. Chauhana,b, Ming Louc, Yi Zhengd, and Richard A. Langa,b,e,1

aDivisions of Pediatric Ophthalmology, eDevelopmental Biology, and dExperimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; bDepartment of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, OH 45229; and cDepartment of Chemistry and Physics, Lamar University, Beaumont TX 77710

Edited* by David D. Sabatini, New York University School of Medicine, New York, NY, and approved September 14, 2011 (received for review June 7, 2011)

Epithelial bending is a central feature of morphogenesis in animals. neural tube formation. Epithelial sheets first establish and main- Here we show that mutual antagonism by the small Rho GTPases tain apicobasal polarity, chiefly by the Par complex (16) regulated Rac1 and RhoA determines cell shape, tissue curvature, and invag- by the Rho GTPases, to localize cytoskeletal components and ination activity in the model epithelium of the developing mouse signaling machinery necessary for invagination (17). The actual lens. The epithelial cells of the invaginating lens placode normally process of invagination requires cell shape changes that drive elongate and change from a cylindrical to an apically constricted, folding of a tissue sheet; apical constriction is believed to be Drosophila conical shape. RhoA mutant lens placode cells are both longer and important for driving invagination (17). Studies on less apically constricted than control cells, thereby reducing epithe- gastrulation have shown that the upstream factor RhoGEF1 lial curvature and invagination. By contrast, Rac1 mutant lens pla- stimulates Rho1, thereby activating Drok/ROCK and conse- quently nonmuscle myosin to similarly constrict epithelial cell code cells are shorter and more apically restricted than controls, apices (17). Mechanisms of apical constriction mediated by resulting in increased epithelial curvature and precocious lens ves- fi fi phosphorylated myosin have also been veri ed for vertebrate in- icle closure. Quanti cation of RhoA- and Rac1-dependent pathway vagination (17). For maturing polarized epithelia, actomyosin markers over the apical–basal axis of lens pit cells showed that in α RhoA complexes engage adherens junctions through -catenin and eplin mutant epithelial cells there was a Rac1 pathway gain of (18) and then circumferential actin belts replace radial actin arrays fi function and vice versa. These ndings suggest that mutual antag- by an unknown mechanism (19). In Drosophila ventral furrow onism produces balanced activities of RhoA-generated apical con- formation, these contractile actin belts ensure apical constriction striction and Rac1-dependent cell elongation that controls cell of cells across the ventral furrow to initiate tissue bending. Re- shape and thus curvature of the invaginating epithelium. The ubiq- cently, it has shown that the actomyosin belts display a pulsing uity of the Rho family GTPases suggests that these mechanisms are behavior, rather than contracting simultaneously, to drive apical likely to apply generally where epithelial morphogenesis occurs. constriction (20). Early studies of lens placode morphogenesis revealed apical ctin remodeling is principally regulated by the highly con- constriction during invagination (21). Recently, a gene-trapped Shroom3 fi Aserved small Rho GTPase family (1, 2), which in the in- mouse with a disruption in (22) veri ed that lens pit cells terconvertible GTP-bound active state, binds to specific effector undergo Shroom3-mediated apical constriction (23), and another fi proteins that determine their cellular function in signal trans- study showed that Cdc42-dependent contractile lopodia, ema- duction pathways (3). Early reports in cultured fibroblasts showed nating from the basal lens pit, function as physical tethers to co- that overexpression of RhoA, Rac1, and Cdc42, the three most- ordinate invagination of presumptive lens and retina (24). Here, studied members, induced the formation of contractile stress we used conditional mouse genetics to assess RhoA and Rac1 fibers, protrusive lamellipodia, and probing filopodia, respectively roles in the developing mouse lens. This showed that RhoA con- (2). These and later studies identified distinctions in the mecha- trols apical cell width and that Rac1 controls cell length. By quantifying RhoA and Rac1-dependent pathway markers over the nisms by which these three small Rho GTPases remodel the actin – cytoskeleton. For RhoA, activation at the rear of cells stimulates apical basal axis of lens pit cells we showed that in RhoA mutant epithelial cells, there was a Rac1 pathway gain of function and vice the downstream effector Rho-associated kinase (ROCK) (3), fi thereby up-regulating phosphomyosin activity on preexisting fila- versa. These ndings suggest that mutual antagonism produces mentous actin (F-actin) and enhancing formation of contractile balanced activities of RhoA-generated apical constriction and actin (4). For activated Rac1 and Cdc42, each binds to a particular Rac1-dependent cell elongation in controlling cell shape and thus effector protein at the cell leading edge. Rac1 indirectly associates curvature of the invaginating epithelium. with WASP family verprolin-homologous protein (WAVE) (3) or Results activates the effector p21-activated kinase (PAK) that suppresses BIOLOGY Invaginating Lens Epithelial Cells Show Trailing and Leading Edge local formation of contractile actin (2), and Cdc42 binds directly to DEVELOPMENTAL neural Wiskott-Aldrich syndrome protein (N-WASP) or WASP Markers of Migration. Prompted by work on single migrating cells (3). These latter three effectors bind an actin nucleator (Arp2/3 or and by studies showing that GTPases regulate epithelial mor- fl – diaphanous-related formins) (5) and promote generation of dis- phogenesis in the sea urchin (25), the y (26 28), and mouse (29), tinct F-actin types; Y-branched F-actin networks for Rac1 and we determined whether lens placode cells had a polarized distri- bundled F-actin for Cdc42. However, recent biosensor detection bution of molecules similar to single migrating cells. We labeled studies show that RhoA, together with Rac1 and Cdc42, are active the embryonic day E10.5 lens pit for established leading edge and with spatial and temporal distinctions at the leading edge of migrating cells (6), whereas myosin has an additional role in actin complex disassembly at the trailing edge (7). Interestingly, Rac1 Author contributions: B.K.C. and R.A.L. designed research; B.K.C. performed research; Y.Z. and RhoA have also been shown in migrating cells to exhibit contributed new reagents/analytic tools; B.K.C., M.L., and R.A.L. analyzed data; and B.K.C. mutual antagonism (8–13), mediated by p190Rho–GTPase-acti- and R.A.L. wrote the paper. vating protein (GAP) (10), the kinase sticky (13), or FilGAP (8). The authors declare no conflict of interest. The mechanisms for cell migration are coming into sharper *This Direct Submission article had a prearranged editor. focus (14, 15), in contrast to those regulating developmental Freely available online through the PNAS open access option. morphogenesis. Epithelial invagination is a conserved type of 1To whom corrrspondence should be addressed. E-mail: [email protected]. morphogenesis found, for example, during ascidian gastrulation, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Drosophila mesoderm invagination, mammalian inner ear and 1073/pnas.1108993108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1108993108 PNAS | November 8, 2011 | vol. 108 | no. 45 | 18289–18294 Downloaded by guest on October 1, 2021 trailing edge markers. Arpc2 is a subunit of the Rac1 or Cdc42- dependent actin nucleator Arp2/3 (15). Cortactin is an Arpc2 interactor that enhances actin branch formation (30). Both Arpc2 and cortactin identify lamellipodia, whereas myosin IIB identifies the preceding lamellae at the leading edge of migrating cells. Labeling of the lens pit for these markers (Fig. S1 A–D) revealed that all three were found at the base of the epithelial cells. Al- though any one example does not reveal this finding, quantification and averaging of labeling intensity over a 5-μm line interval for many examples (Fig. S1 B, yellow dashed lines, and C) showed that as in single migrating cells (31), the peak intensity of myosin IIB labeling appeared displaced toward the cell center compared with Arpc2 and cortactin. This combination and spacing of markers suggest that the base of lens pit cells might be similar functionally to the protrusive edge of a single migrating cell. The prior demonstration that Cdc42-dependent filopodia project from the basal surface of lens pit epithelial cells (24) is also consistent with this hypothesis. To determine whether lens pit epithelial cells might also have RhoA-dependent complexes (1), we labeled the E10.5 lens pit for phospho-myosin regulatory light chain (MRLC), myosin IIB, and Fig. 1. Rac1 and RhoA mutations have opposite effects on epithelial cur- fl E fl fl F-actin. According to immuno uorescence visualization (Fig. S1 , vature. (A–C) The appearance of control (A), Le-cre; RhoA / (B), and Le-cre; fl fl F,andH)andquantification of that labeling (Fig. S1G)allthree Rac1 / (C) lens pits at E10.5. (D) Curves representing the average shape of markers were found at the apex of lens pit epithelial cells in over- the apical surface of E10.5 lens pits from control (gray), Le-cre; RhoAfl/fl (red), lapping peaks that were within 1 μm of the apical edge. Although and Le-cre; Rac1fl/fl (blue) and Le-cre; Rac1fl/fl; RhoAfl/fl (green). Although phospho-MRLC was also found at the base of lens pit epithelial individual examples of any genotype show some variability in lens pit shape, cells, the majority of the activity was at the apex (Fig. S1H). Col- coordinate geometry averaging shows that the RhoA mutant pit has less lectively, these labeling studies indicated that the epithelial cells of curvature, whereas that for Rac1 has more. Quantification in E10.5 lens pits the lens placode cells have a similar molecular signature as single (n = 10) of cell numbers. migrating cells.

RhoA and Rac1 Lens Mutants Generate Opposing Invagination Phe- straining interepithelial distance and pulling the lens pit down into notypes. The earlier observations suggest that the mechanisms the optic cup (24). To determine whether the Rac1 or RhoA of invagination might be related to mechanisms of single cell conditional mutants had defects in the generation of these filopo- migration. To test this hypothesis, we generated placode-specific dia, we determined filopodial indices and measured interepithelial Rac1 and RhoA somatic mutants in the mouse using conditional distances (Fig. S3). As shown previously, the Cdc42 mutant has alleles (Fig. S2 A and C) and the Le-cre driver. Without effective almost no filopodia (Fig. S3B) and more than double the inter- antibodies to RhoA, we used PCR genotyping of DNA isolated epithelial distance (Fig. S3 F and G, aqua bar) compared with from lens pits to show that the loxP sites were recombined in the A F G fl/fl control (Fig. S3 , ,and , gray bar). The Rac1 mutant has no RhoA ; Le-cre embryos (Fig. S2B). Using antibodies to Rac1, we fi fl/fl change in lopodial index and no change in interepithelial distance also showed that the Rac1 ; Le-cre mutants had lost Rac1 im- (Fig. S3 D, F,andG, blue bar). By contrast, the RhoA mutant has munoreactivity in the lens pit (Fig. S2 D–I). These studies also a reduction in the filopodial index that is about 66% of the control show that Rac1 immunoreactivity in the lens pit cells has a mem- value (Fig. S3 C and F, red bar). This may not be surprising given brane-association pattern (Fig. S2 F–K). that these processes contain functional contractile myosin com- Somatic deletion of Rac1 or RhoA resulted in subtle but re- plexes (24). However, this 34% reduction in filopodial index is not producible phenotypes in the shape of the lens pit at E10.5 (Fig. accompanied by a change in the interepithelial distance (Fig. S3G, 1). In the RhoA mutant, the lens pit showed a more open shape red bar). This probably means that the RhoA mutant still has suf- (Fig. 1B) compared with control (Fig. 1A). By contrast, the Rac1 ficient filopodia to constrain the interepithelial distance. Consistent mutant showed a lens pit that was more closed (Fig. 1C). Using with this argument, a focal adhesion kinase (FAK) conditional coordinate geometry, we combined many examples of curved lines mutant has even fewer filopodia (an index of 0.43, 50% of the control that represented the apical surface of the lens pit to quantify the value) but still has no change in the interepithelial distance (Fig. S3 F average shape in control and mutant examples. This confirmed and G, pale cream). On the basis of this analysis, we cannot con- that on average, lens pits of RhoA mutants showed less curvature, clude that the number or length of filopodia in the RhoA mutant has whereas those of the Rac1 mutants showed more (Fig. 1D). These any impact on invagination morphogenesis of the lens pit. changes in curvature showed that at E10.5 the shape of the lens pit We generated RhoA, Rac1 double mutants with Le-cre and was in delicate balance and could be influenced either positively or assessed cell pit shape, which revealed a lens pit shape very similar negatively in these mutants. to the RhoA single mutant (Fig. 1D). Although it was tempting to RhoA family GTPases are known to control cell cycle progression conclude that this revealed an epistatic relationship between in some contexts (32). To determine whether the Rac1 and RhoA RhoA and Rac1, further investigation showed that the double conditional mutants showed any evidence of this, we quantified cell mutant lens pit displayed dramatically reduced levels of F-actin number in comparison with wild type and, as an additional control, A D fl/fl (Fig. S4 ), abnormal patterns of cell packing (Fig. 2 ) and re- used the previously characterized Le-cre; Cdc42 conditional mu- duced levels of the adherens junction marker β-catenin (Fig. S4 E tant (24) (Fig. S3E). This showed that a normal lens pit contains and I) similar to those observed in lens-specific β-catenin somatic about 60 cells in section and that none of the mutants showed a sig- mutants (34). This all suggested that there was a fundamental loss nificant change. We also did not observe any indication of the mul- of the actin cytoskeleton in the double mutants, and that the tinucleate cells that might result from RhoA regulation of cytokinesis change in cell pit shape was unlikely to reflect the normal genetic (33). These distinct consequences of RhoA mutation are likely to relationship between the two GTPases. result in part from differential expression of RhoA effectors. fl/fl We previously showed that the Le-cre; Cdc42 conditional RhoA and Rac1 Lens Mutants Display Contrasting Cell Shapes. The mutant fails to generate filopodia that connect presumptive lens changes in cell pit shapes described above suggested that the Rac1 and retina (24). These processes aid in morphogenesis by con- and RhoA mutants would show changes in cell shape. To assess

18290 | www.pnas.org/cgi/doi/10.1073/pnas.1108993108 Chauhan et al. Downloaded by guest on October 1, 2021 Fig. 2. Rac1 and RhoA mutations have opposite effects on cell shape. (A–D) Cell profiles from the lens pits of control and somatic mutants were generated from β-catenin–labeled cryosections. The profiles were exported, measured for width and height, and combined to produce the average profiles in E.(E) Apical and basal cell dimensions are indicated (above and below the profile) relative to the control basal dimension. Similarly, the absolute dimensions in micrometers are indicated for the average control cell. Significance values for apical dimension: control (2.08 ± 0.14) to RhoA (2.74 ± 0.18), P < 0.001; control to Rac1 (1.72 ± 0.12), P = 0.043; RhoA to Rac1, P < 0.001. Significance values for cell length: control (26.1 ± 0.46) to RhoA (32.6 ± 0.37), P < 0.001; control to fl fl Rac1 (23.8 ± 0.44), P = 0.008; RhoA to Rac1, P < 0.001. Basal dimensions are not significantly different. This analysis shows that Le-cre; RhoA / cells (red) were longer and less apically constricted and that Le-cre; Rac1fl/fl cells (blue) were shorter and more apically constricted. Control cells occupied an angle of 6.0°, whereas Le-cre; RhoAfl/fl and Le-cre; Rac1fl/fl cells occupied angles of 4.0° and 7.4°, respectively.

this proposal, we recorded cell shape profiles from the entire lens cortactin, an enhancer of actin branch formation (30). Marker pit of control and mutant embryos (n = 5 lens pits, about 350 cell labeling was quantified over apical–basal line intervals that were profiles) at E10.5 (Fig. 2 A–C), and then generated average cell distributed radially around the curve of the subject lens pit. For shapes (Fig. 2E) using a coordinate system. In both RhoA and each genotype the data were obtained from 17 line intervals on Rac1 mutants, the basal cell dimension was not significantly each of five E10.5 lens pits (Fig. S5). The data from the 85 line changed (Fig. 2E). However, in RhoA mutants, the ratio of the intervals were combined by normalization to nuclear labeling apical to control basal dimension was 0.57 compared with 0.44 in with Hoechst 33258. To allow comparison of intensity profiles the control (Fig. 2E). This was a subtle, but statistically significant for epithelia of the different mutants, we also performed length change (P < 0.001). Accompanying reduced apical constriction, normalization according to established procedures (38). the RhoA mutants showed an unexpected cell elongation with Quantification of phalloidin labeling revealed that F-actin is a height index of 1.25 (Fig. 2E, red, P < 0.001). These two shape found in two intensities located at the apex (the major intensity) changes mean that the average RhoA mutant cell occupies an and at the base of control cells (Fig. 3 A and B, gray line). The angle of 4.0° compared with 6.0° in the control. This cell shape distribution of F-actin in the lens pits of single mutants was change is consistent with the documented change in the shape of changed very little (Fig. 3B, red and blue profiles) with the ex- the lens pit (Fig. 1 B and D), as cells that occupy a reduced angle ception that the Rac1 mutant showed a slight increase in the level will generate an epithelial structure with reduced curvature. of F-actin in the basal half (Fig. 3B, blue). These profiles show that The averaging of cell profiles from the lens pits of Rac1 mutant there was no fundamentally aberrant change in the ability of RhoA embryos (Fig. 2C) revealed that they were shorter than wild type or Rac1 mutant cells to assemble or distribute filamentous actin. with a height index of 0.91 (Fig. 2E,blue,P = 0.008). Un- RhoA mutant mice showed substantially reduced myosin II at expectedly, they were also more apically constricted with an index both the apex and the base (Fig. 3 C and D, red). At the cell apex, of 0.36 (P = 0.043). With these shape changes, the angle occupied this change was consistent with a reduced level of phospho-MRLC by a Rac1 mutant cell is 7.4° (compared with 6.0° in the control), in the RhoA mutant (Fig. 3 E and F, red profile, apex). Although

meaning that when combined in an epithelium, they will generate the absence of RhoA reduced the level of basal myosin II, there BIOLOGY

tighter curvature than average, consistent with our observation was a very limited reduction in the level of basal phospho-MRLC DEVELOPMENTAL (Fig. 1 C and D). Combined, this analysis of lens pit cell shape in (Fig. 3 E and F, red profile, base). These observations are con- the Rac1 and RhoA mutants suggested that each GTPase can sistent with the established model for RhoA function where regulate both the height and apical width of a cell. ROCK activation by RhoA leads to the formation of phospho- MRLC (36, 37). They further suggest that a RhoA–phospho- RhoA and Rac1 Show Both Apical and Basal Antagonism for Pathway MRLC pathway is active at both the cell apex and base. When Markers. To investigate possible mechanisms of integration of combined with the observation that RhoA mutant lens pit cells Rac1 and RhoA activities, we labeled control and mutant lens are less apically constricted, these data suggests that RhoA-de- pits for molecular markers of activity in Rac1- and RhoA-dependent formation of apical phospho-MRLC is required for the pendent pathways. It is well established that RhoA generates contractile actin that mediates apical constriction. contractile actin via a pathway requiring RhoA activation of Rac1 somatic mutants show a myosin IIB labeling intensity ROCK, ROCK phosphorylation-mediated activation of MRLC profile that is very similar to the control but shows a slightly (35, 36) and suppression of myosin light chain phosphatase higher, broader peak at the apex and a slightly lower peak at the (MLCP) (37). Thus, phosphorylated MRLC is an activity sur- base (Fig. 3 C and D, blue profile). By contrast, the phospho- rogate for this pathway. For Rac1-dependent pathways, we MRLC labeling profile in the Rac1 mutant shows dramatic measured the labeling intensity for the c2 subunit of the actin increases right across the profile but most obviously at the apex nucleator Arp2/3 as this is required for the generation of pro- and base (Fig. 3 E and F, blue profile). Because the quantifica- trusive actin in lamellipodia (15). We also measured the level of tion of myosin IIB and phospho-MRLC in the RhoA mutant

Chauhan et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18291 Downloaded by guest on October 1, 2021 Fig. 3. Mutual antagonism of RhoA and Rac1 in regulating the cytoskeletal machinery during lens pit invagination. (A, C, E, G, I, and K) Labeling for F-actin (A), myosin IIB (C), phospho-MRLC (E), Arpc2 (G), cortactin (I), and Rac1 (K) in control and Le-cre; RhoAfl/fl and Le-cre; Rac1fl/fl (except Rac1) lens pit cells at E10.5. (B, D, F, H, J, and L) Quantification of the labeling shown in (A, C, E, G, I, and K) for lens pits over an apical–basal line interval (n = 5 eyes, n = 85 line fl fl intervals). The gray line shows quantification of the indicated marker in control cells and the red and blue lines, the quantification in Le-cre; RhoA / and Le- fl fl cre; Rac1 / lens pit cells, respectively. The red and blue arrowheads in A, C, E, G, I, and K (only red) indicate the apical and basal marker labeling, and where numbered, cross-reference a peak on the quantification profiles.

18292 | www.pnas.org/cgi/doi/10.1073/pnas.1108993108 Chauhan et al. Downloaded by guest on October 1, 2021 show that their distribution and activity is RhoA dependent, the Our initial observations on the open invagination phenotype Rac1 mutant response reveals that normally, Rac1 suppresses for the RhoA mutants and the opposite for the Rac1 mutants RhoA-dependent formation of phospho-MRLC. The presence (Fig. 1) led us to study their respective average cell shapes. We of increased phospho-MRLC in apical and basal peaks indicates show that Rac1 and RhoA have opposite effects (Fig. 2). Rac1 that Rac1 suppression of RhoA occurs in both locations. promotes cell elongation and suppresses apical constriction, In the Rac1 mutant, the labeling intensity for Arpc2 is sub- whereas RhoA promotes apical constriction and suppresses cell stantially reduced in the lower half of lens pit epithelial cells (Fig. 3 elongation. The resulting changes in the RhoA and Rac1 path- G and H,blueprofile). This effect is most dramatic where the way marker levels (Fig. 3) are consistent with the phenotypic normally intense peak of basal labeling is absent (Fig. 3H,blue data and lead to a model (Fig. 4) describing how balanced ac- arrowhead 3). This is in complete contrast to the Arpc2 quantifi- tivities of RhoA and Rac1 control cell shape. We suggest, on the cation in RhoA mutant cells (Fig. 3 G and H, red profile) where basis of existing data (15) and current observations, that one there were increases in Arpc2 labeling at both the base (Fig. 3 H, activity of RhoA is to produce contractile actin. This is consistent red arrowhead 2) and at the apex (a broad, but modestly increased with the loss of apical phospho-MRLC and reduced apical peak, Fig. 3H, red arrowhead 1). Combined, these data indicate constriction observed in RhoA mutant lens pit epithelial cells. that the level of Arpc2 at the cell base is dependent on Rac1 and in Given the reduced apical level of myosin IIB in the RhoA mu- addition, that RhoA normally suppresses the Rac1-dependent tant, we cannot exclude the possibility that reduced apical basal Arpc2. The distribution of the Arp2/3 enhancer cortactin is phospho-MRLC might in part be a consequence of reduced also changed in the Rac1 mutant and shows a reduced level from apical mysoin IIB availability. The dependence of lens pit epi- the base to a point just below the apex (Fig. 3J,bluearrowhead3). thelial cell apical constriction on Rho kinase (39) suggests that the RhoA-ROCK pathway (35, 36) is also required. Like Arpc2, the basal level of cortactin in the RhoA mutant is Rac1 opposite to that of the Rac1 mutant and shows an increase (Fig. Because somatic mutants show increased apical and basal 3J, red arrowhead 2). Thus, according to two markers of the phospho-MRLC, they are equivalent to a RhoA gain of function Rac1-Arpc2/3/cortactin pathway, RhoA normally suppresses the and this can explain why Rac1 mutants show increased apical assembly of protrusive actin complexes. Finally, with the avail- constriction. Currently, we do not have a good explanation for the ability of validated Rac1 antibodies (Fig. S2 D–I), we examined absence of basal constriction in Rac1 mutants in which the level of the relationship between RhoA mutation and Rac1 distribution. basal phospho-MRLC is increased, but perhaps integrin-mediated anchoring to basal lamina confers rigidity. Similarly, we argue that This showed (Fig. 3 K and L) that when RhoA is mutated, Rac1 the primary activity of Rac1 is to produce protrusive actin (Fig. 2). immunoreactivity is not significantly increased apically (Fig. 3L, This is consistent with the reduced levels of Arpc2 and cortactin at red arrowhead 1) but is significantly increased basally (Fig. 3L, the cell base and with the observation that Rac1 mutant cells are red arrowhead 2). This suggests that one mechanism for RhoA shorter than normal. Because RhoA mutant cells show increased suppression of Rac1 is to suppress its abundance in the base of basal levels of Arpc2 and cortactin, we argue that they are the cell. equivalent to a Rac1 gain of function and this can explain why RhoA Discussion mutant cells are longer than normal. The increased labeling signal intensity for Rac1 at the base of RhoA mutant cells indicates Present models for tissue invagination suggest that increased po- that that one mechanism, by which RhoA suppresses Rac1 activ- lymerization of cadherin- anchored apical contractile actin pro- ity, is to control its level. vides the driving force for curving epithelia (17). Here we present When combined, these observations argue that during in- data showing that both RhoA and Rac1 modulate apical con- vagination of the lens pit epithelium, RhoA and Rac1 are mutually striction and cell length, coordinated by mutual antagonism. In antagonistic and that a balance between their activities fine tunes this model (Fig. 4), the balanced regulation of apical cell width by the apical width and cell height. In turn, the ratio of these two RhoA and cell length by Rac1 determines the angle occupied by dimensions determines the angle that an individual epithelial cell an epithelial cell and in aggregate, the curvature of an epithelium. occupies and in aggregate, the curvature of an epithelium. Because there are many examples where epithelial curvature changes dramatically over just a few cell diameters, it is likely that the ratio of Rac1 to RhoA activity can also be controlled locally, presumably by the signaling pathways known to regulate these GTPases (15). With the ubiquity of the Rho family GTPases, these are likely to be general mechanisms regulating epithelial morphogenesis. The similarity of the mechanisms of cell migration in cultured cells (15) and those uncovered here for epithelial invagination, further suggests that these mechanisms will be generally applicable. BIOLOGY

Complex Interplay of Rho GTPase Activities Regulates Invagination. DEVELOPMENTAL We have previously shown that another GTPase, Cdc42, has an important function in regulating epithelial invagination in the lens system. In this case, Cdc42-dependent filopodia project from the base of the lens pit, connect to the adjacent optic cup, and appear to serve as physical tethers that assist coordinated invagination. The loss of lens pit filopodia in a Cdc42 somatic mutant results in a relative increase in invagination distance (24). Combined with – Fig. 4. Schematic describing the role of RhoA Rac1 mutual antagonism in the current findings, this indicates that at least three different epithelial bending in the lens pit. From the current analysis, we can infer that GTPase-dependent modulations of the actin cytoskeleton, each of both Rac1 and RhoA have dual functions. Rac1 has a role in elongating cells through Arpc2 and cortactin but also suppresses the production of phospho- which produces only a subtle consequence, function concurrently MRLC and thus the generation of contractile actin. By contrast, RhoA is re- to generate the full process of lens morphogenesis. There is also quired for apical constriction through the production of phospho-MRLC and the very strong possibility given recent data from a culture system contractile actin, but also suppresses the basal Arpc2 and cortactin com- (40) that the presumptive retina and presumptive lens interact in plexes and thus inhibits cell elongation. In this way, a balance between the a dynamic way to refine the shape of the lens pit and optic cup. activities of RhoA and Rac1 controls the apical width and cell length. In turn, There are interesting parallels between these mechanisms and the ratio of these two dimensions controls the angle formed by the cells and, those observed in the fly (17, 27, 41) and sea urchin (25) where in aggregate, the curvature of the epithelium. RhoA (Rho1 in the fly) is required for apical constriction and

Chauhan et al. PNAS | November 8, 2011 | vol. 108 | no. 45 | 18293 Downloaded by guest on October 1, 2021 epithelial invagination. Furthermore, in the sea urchin, epithelial extracellular matrix (ECM), where it has recently been shown that invagination is assisted by contractile filopodia that extend from loss in ECM components leads to lens placode invagination defects secondary mesenchymal cells across the blastocoel (42, 43). (47). Lens invagination defects were previously seen in a heparan Studies so far have focused on the three most studied Rho sulfate mutant (48) and another example of ECM playing a role in GTPases in epithelial invagination and further work is required invagination has been documented for the sea urchin during gas- to unravel the role of other small Rho GTPases in this process. trulation (49). The current study has identified the importance of the small Rho GTPases in the process of epithelial invagination. However, in- Materials and Methods vagination still occurs in the absence of each of the three common B C RhoAfl (SI Materials and Methods) and Rac1fl (50) conditional alleles were GTPases (Fig. 1 and ) (24). Other factors are possibly involved in crossed with the a lens-specific cre driver, Le-cre, to generate conditional the process of epithelial invagination, so that knocking down one of mutants. Immunofluorescence labeling (34) and imaging, via an Apotome-based these activities produces a subtle phenotype. Adhesion as a factor Zeiss fluorescence microscope, was used for visualizing proteins of interest. required for invagination of the lens placode has been shown by For quantitation, curvature analysis was performed by a combination of conditionally knocking out the adherens junctions (AJ) component Matlab 7.1 and Mathematica 7.0 (SI Materials and Methods), average cell β-catenin in the lens ectoderm using the Le-cre driver (34). The shapes were obtained from membrane-labeled β-catenin cryosections and phenotype was more dramatic by reduced adhesion in the lens Axiovision software to measure and average cellular dimensions, and signal ectoderm leading to increased apoptosis, but an attempt at in- intensity profiles were calculated using ImageJ software on immunolabeled vagination was still made. Similar adhesion-deficient phenotypes images and Microsoft Excel processing (SI Materials and Methods). have been shown in Drosophila during gastrulation (44) and in mice during follicular morphogenesis (45). Polarity has been determined ACKNOWLEDGMENTS. We thank Paul Speeg for excellent technical assis- as a factor for invagination as seen by the aberrant invagination of tance. We acknowledge grant support from the National Institutes of Health crumb mutants during Drosophila tracheal placode invagination (R01 EY17848) and from the Abrahamson Pediatric Eye Institute Endowment (46). One of the emerging factors involved in invagination is the at Children’s Hospital Medical Center of Cincinnati (to R.A.L.).

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