Rho1: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - Rho1

Synonyms - DrhoA, Rho and RhoA

Cytological map position - 52E3--52E6

Function - GTP-binding protein

Keywords - gastrulation, cytoskeleton, tissue polarity

Symbol - Rho1

FlyBase ID: FBgn0014020

Genetic map position - 2-

Classification - rho-subfamily GTPase

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene

Recent literature
Ma, X., Chen, Y., Xu, W., Wu, N., Li, M., Cao, Y., Wu, S., Li, Q. and Xue, L. (2015). Impaired Hippo signaling promotes Rho1-JNK-dependent growth.Proc Natl Acad Sci U S A 112: 1065-1070. PubMed ID: 25583514
Summary:
The Hippo and c-Jun N-terminal kinase (JNK) pathways both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. This study shows that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppresses Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promotes tissue growth when apoptosis is prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. Thus, these results have identified a novel molecular link between the Hippo and JNK pathways and implicated the essential role of the JNK pathway in Hippo signaling-related tumorigenesis.

Petsakou, A., Sapsis, T.P. and Blau, J. (2015). Circadian rhythms in Rho1 activity regulate neuronal plasticity and network hierarchy. Cell [Epub ahead of print]. PubMed ID: 26234154
Summary:
Neuronal plasticity helps animals learn from their environment. However, it is challenging to link specific changes in defined neurons to altered behavior. This study focuses on circadian rhythms in the structure of the principal s-LNv clock neurons in Drosophila. By quantifying neuronal architecture, it was observed that s-LNv structural plasticity changes the amount of axonal material in addition to cycles of fasciculation and defasciculation. It was found that this is controlled by rhythmic Rho1 activity that retracts s-LNv axonal termini by increasing myosin phosphorylation and simultaneously changes the balance of pre-synaptic and dendritic markers. This plasticity is required to change clock network hierarchy and allow seasonal adaptation. Rhythms in Rho1 activity are controlled by clock-regulated transcription of Puratrophin-1-like (Pura), a Rho1 GEF. Since spinocerebellar ataxia is associated with mutations in human Puratrophin-1, these data support the idea that defective actin-related plasticity underlies this ataxia.

Petsakou, A., Sapsis, T. P. and Blau, J. (2015). Circadian rhythms in Rho1 activity regulate neuronal plasticity and network hierarchy. Cell 162: 823-835. PubMed ID: 26234154
Summary:
Neuronal plasticity helps animals learn from their environment. However, it is challenging to link specific changes in defined neurons to altered behavior. This study focused on circadian rhythms in the structure of the principal s-LNv clock neurons in Drosophila. By quantifying neuronal architecture, s-LNv structural plasticity was found to change the amount of axonal material in addition to cycles of fasciculation and defasciculation. This is controlled by rhythmic Rho1 activity that retracts s-LNv axonal termini by increasing myosin phosphorylation and simultaneously changes the balance of pre-synaptic and dendritic markers. This plasticity is required to change clock network hierarchy and allow seasonal adaptation. Rhythms in Rho1 activity are controlled by clock-regulated transcription of Puratrophin-1-like (Pura), a Rho1 GEF. Since spinocerebellar ataxia is associated with mutations in human Puratrophin-1, these data support the idea that defective actin-related plasticity underlies this ataxia.

Mason, F. M., Xie, S., Vasquez, C. G., Tworoger, M. and Martin, A. C. (2016). RhoA GTPase inhibition organizes contraction during epithelial morphogenesis. J Cell Biol 214: 603-617. PubMed ID: 27551058
Summary:
During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. This study identified a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. These data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding.
Nakamura, M., Verboon, J. M. and Parkhurst, S. M. (2017). Prepatterning by RhoGEFs governs Rho GTPase spatiotemporal dynamics during wound repair. J Cell Biol [Epub ahead of print]. PubMed ID: 28923977
Summary:
Like tissues, single cells are subjected to continual stresses and damage. As such, cells have a robust wound repair mechanism comprised of dynamic membrane resealing and cortical cytoskeletal remodeling. One group of proteins, the Rho family of small guanosine triphosphatases (GTPases), is critical for this actin and myosin cytoskeletal response in which they form distinct dynamic spatial and temporal patterns/arrays surrounding the wound. A key mechanistic question, then, is how these GTPase arrays are formed. This study shows that in the Drosophila melanogaster cell wound repair model Rho GTPase arrays form in response to prepatterning by Rho guanine nucleotide exchange factors (RhoGEFs), a family of proteins involved in the activation of small GTPases. Furthermore, Annexin B9, a member of a class of proteins associated with the membrane resealing, was shown to be involved in an early, Rho family-independent, actin stabilization that is integral to the formation of one RhoGEF array. Thus, Annexin proteins may link membrane resealing to cytoskeletal remodeling processes in single cell wound repair.
Segal, D., Zaritsky, A., Schejter, E. D. and Shilo, B. Z. (2018). Feedback inhibition of actin on Rho mediates content release from large secretory vesicles. J Cell Biol [Epub ahead of print]. PubMed ID: 29496739
Summary:
Secretion of adhesive glycoproteins to the lumen of Drosophila melanogaster larval salivary glands is performed by contraction of an actomyosin network assembled around large secretory vesicles, after their fusion to the apical membranes. This study has identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and actin nucleation. This leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. When contraction of vesicles is blocked, the strict temporal order of the recruited elements generates repeated oscillations of actin coat formation and disassembly. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be widely used to coordinate a succession of morphogenetic events or maintain homeostasis.
Jha, A., van Zanten, T. S., Philippe, J. M., Mayor, S. and Lecuit, T. (2018). Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis. Curr Biol 28(10): 1570-1584.e1576. PubMed ID: 29731302
Summary:
Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. The GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, the surface of the plasma membrane was probed, and it was shown that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and beta-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, this study found higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. Dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.
Izquierdo, E., Quinkler, T. and De Renzis, S. (2018). Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis. Nat Commun 9(1): 2366. PubMed ID: 29915285
Summary:
During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. This study used optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. Precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.
Cordoba, S. and Estella, C. (2018). The transcription factor Dysfusion promotes fold and joint morphogenesis through regulation of Rho1. PLoS Genet 14(8): e1007584. PubMed ID: 30080872
Summary:
The mechanisms that control tissue patterning and cell behavior are extensively studied separately, but much less is known about how these two processes are coordinated. This study shows that the Drosophila transcription factor Dysfusion (Dysf) directs leg epithelial folding and joint formation through the regulation of Rho1 activity. Dysf-induced Rho1 activity promotes apical constriction specifically in folding epithelial cells. This study shows that downregulation of Rho1 or its downstream effectors cause defects in fold and joint formation. In addition, Rho1 and its effectors are sufficient to induce the formation of epithelial folds when misexpressed in a flat epithelium. Furthermore, as apoptotic cells can actively control tissue remodeling, the role of cell death in the formation of tarsal folds and its relation to Rho1 activity was analyzed. Surprisingly, no defects were found in this process when apoptosis is inhibited. These results highlight the coordination between a patterning transcription factor and the cellular processes that cause the cell shape changes necessary to sculpt a flat epithelium into a three dimensional structure.
Zandvakili, A., Uhl, J. D., Campbell, I., Salomone, J., Song, Y. C. and Gebelein, B. (2018). The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila. Dev Biol. PubMed ID: 30468713
Summary:
During development diverse transcription factor inputs are integrated by cis-regulatory modules (CRMs) to yield cell-specific gene expression. Defining how CRMs recruit the appropriate combinations of factors to either activate or repress gene expression remains a challenge. This study compares and contrasts the ability of two CRMs within the Drosophila embryo to recruit functional Hox transcription factor complexes. The Distal-less DCRE CRM recruits Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox complexes that include the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the Distal-less leg selector gene, whereas the RhoA CRM selectively recruits Abd-A/Exd/Hth complexes to activate rhomboid and stimulate Epidermal Growth Factor secretion in sensory cell precursors. By swapping binding sites between these elements, it was found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE. It was further shown that the orientation and spacing of Hox sites relative to additional binding sites within the RhoA and DCRE is critical to mediate cell- and segment-specific output. These results indicate that the configuration of Exd, Hth, and Hox site within RhoA is neither Abd-A specific nor activation specific. Instead Hox specific output is largely dependent upon the presence of appropriately spaced and oriented binding sites for additional TF inputs. Taken together, these studies provide insight into the cis-regulatory logic used to generate cell-specific outputs via recruiting Hox transcription factor complexes.
Ong, K., Collier, C. and DiNardo, S. (2019). Multiple feedback mechanisms fine-tune Rho signaling to regulate morphogenetic outcomes. J Cell Sci. PubMed ID: 30872456
Summary:
Rho signaling is a conserved mechanism for generating forces through activation of contractile actomyosin. How this pathway can produce different cell morphologies is poorly understood. In the Drosophila embryonic epithelium, this study investigated how Rho signaling controls force asymmetry to drive morphogenesis. A distinct morphogenetic process termed "alignment" was studied. This process results in striking columns of rectilinear cells connected by aligned cell-cell contacts. This was found to be driven by contractile actomyosin cables that elevate tension along aligning interfaces. The data show that polarization of Rho effectors, ROK and Dia, direct formation of these cables. Constitutive activation of these effectors causes aligning cells to instead invaginate. This suggests that moderating Rho signaling is essential to producing the aligned geometry. Therefore, tests were performed for feedback that could fine-tune Rho signaling. It was discovered that F-actin exerts negative feedback on multiple nodes in the pathway. Further, evidence is presented that suggests that ROK in part mediates feedback from F-actin to Rho in a Myo-II-independent manner. Collectively, this work suggests that multiple feedback mechanisms regulate Rho signaling, which may account for diverse morphological outcomes.
BIOLOGICAL OVERVIEW

Rho is a member of the Ras GTPase superfamily; it is a GTP-binding protein that regulates cell shape and motility through modulation of the actin cytoskeleton. A general review of this Rho function and of similar functions for two other Ras GTPase superfamily members, Rac and Cdc42, is provided at the Rac site. This overview will deal with the role of Rho in gastrulation. For general information about Rho activation and inactivation and about the downstream targets of Rho, see the Rho Evolutionary homologs section. In Drosophila, Rho is involved in tissue polarity, and this Rho function is dealt with in the Effects of mutation section.

Rho's role in gastrulation

Ras superfamily proteins function as regulated molecular switches that alternate between active GTP-bound and inactive GDP-bound states. The GTP/GDP balance of these small GTPases is dictated by the net rates of guanine nucleotide exchange and GTP hydrolysis; these rates are tightly controlled by three classes of regulatory molecules: (1) guanine nucleotide exchange factors (GEFs) act as positive regulators that promote the release of GDP and consequent formation of the active GTP-bound state; (2) GTPase activating proteins (GAPs) act as negative regulators that stimulate the intrinsic GTPase activity to cycle them back to the inactive GDP-bound form, and (3) guanine nucleotide dissociation inhibitors (GDIs), associate with GTPases to maintain the existing nucleotide-bound state. Rho, acting through other proteins, mediates actin rearrangements that are likely to be required for the numerous cell shape changes in a developing embryo.

DRhoGEF2 was identified in a genetic screen for Rho signaling pathway components in Drosophila. DRhoGEF2 was found to encode a Rho-specific guanine nucleotide exchange factor (Barrett, 1997). The same gene, DRhoGEF2 was similarly identified in an independent screen carried out by Hacker (1998), a screen designed to characterize the maternal effects of zygotic lethal mutations. The gene was independently cloned by Werner (1997). DRhoGEF2 encodes a protein that contains a PDZ domain near the amino terminus, and a cystine-rich butterfly motif in the central region that is present in isoforms of protein kinase C and the mouse Dbl family oncoprotein Lfc. The C-terminal region of DRhoGEF2 contains an extensive region of homology with two separate protein motifs characteristic of the Dbl family of oncoproteins. The first motif, termed the Dbl homology domain promotes the exchange of guanine nucleotides within Rho family GTPases. The second domain, juxtaposed to the Dbl homology domain and C-terminal to it, is a Pleckstrin homology domain (Hacker, 1998).

Embryos lacking DRhoGEF2 fail to gastrulate due to a defect in cell shape changes required for tissue invagination. Expression of a dominant-negative Rho GTPase in early embryos results in similar defects. Of the DRhoGEF2 homozygotes, 100% die as late embryos or early larvae in the absence of obvious abnormalities. DRhoGEF2 mRNA is expressed uniformly at high levels in the syncitial blastoderm; levels decrease until, by the time gastrulation is initiated, no expression is detected. This apparent maternal contribution of DRhoGEF2 mRNA suggests a likely role for the encoded protein in early embryogenesis. Embryos lacking maternal DRhoGEF2 exhibit normal dorso-ventral and anterior-posterior patterning, as well as mesoderm. However, germband extension and posterior midgut invagination appear to be defective. In addition, the cells of the mesectoderm fail to intercalate at the ventral midline, indicating a defect in ventral furrow formation. In embryos lacking maternal DRhoGEF2, the process of gastrulation is highly disorganized and ventral furrow formation never occurs. In such embryos it appears that random cells within an approximately 20-cell width spanning the ventral midline undergo apical membrane constrictions. There is also a substantially reduced number of addition constrictions in neighboring cells, resulting in a pitted ventral surface. In addition to the defects in ventral furrow formation and invagination of the posterior midgut, DRhoGEF2 mutant embryos are defective in invagination of the anterior midgut, a closely related gastrulation event (Barrett, 1997 and Hacker, 1998).

In addition to the DRhoGEF2 defects, embryos expressing a dominant negative Rho1 exhibit obvious defects in gastrulation. While the furrow in dominant negative Rho1 embryos does form, it fails to extend at the posterior end, resembling the ventral furrow defects in folded gastrulation and concertina mutants. The T-shaped invagination of the anterior midgut does not form normally in these embryos and they also exhibit defects in posterior midgut invagination and germband extension. However, the cephalic furrow forms normally in both DRhoGEF2 and dominant negative Rho1 embryos (Barrett, 1997)

Evidence is also presented that DRhoGEF2 mediates these specific cell shape changes in response to the extracellular ligand, Folded gastrulation. fog was expressed ectopically from a huckebein promoter, which is normally active in a subset of cells at the anterior and posterior ends of the embryo. In all the hkb-fog expressing embryos a characteristic transient depression in the dorsal head region can be seen. The surfaces of cells in this depression exhibit membrane blebbing and constrictions closely resembling those normally seen in cells along the ventral furrow in wild-type embryos. In addition, the nuclei of these cells have migrated from an apical to a basal position. In the absence of DRhoGEF2, ectopic Fog expression fails to induce any detectable cell shape changes, despite equivalent levels of fog transgene expression. Together, these results establish a Rho-mediated signaling pathway that is essential for the major morphogenetic events in Drosophila gastrulation (Barrett, 1997).

The central domain of DRhoGEF2 contains a likely phorbol ester-response motif. The homologous domain in Protein kinase C mediates kinase activation in response to diacylglycerol, which is generate by phospholipase C (PLC). Thus is it possible that the GEF activity of DRhoGEF2 is responsive to diacylglycerol. Since PLC-mediated production of diacylglycerol can be promoted by both receptor tyrosine kinase activation and by activation of receptor-coupled heterotrimeric G proteins, it is possible that the nucleotide-exchange activity of DRhoGEF2 is stimulated by signals transduced by both of these types of receptors. The presence of a PDZ domain in DRhoGEF2, suggests that it may interact with additional signaling proteins. Therefore, it appears that the GEF activity of DRhoGEF2 may be regulated by multiple upstream signals (Barrett, 1997).

It is postulated that Fog acts via the G alpha protein Concertina (see G protein salpha 60A: Evolutionary homologs section for more information about Concertina) to activate DRhoGEF2, thereby promoting Rho1 activation and consequent actin rearrangements. Significantly, the Drosophila G alpha subunit, Concertina, exhibits the strongest sequence similarity to the mammalian Galpha12 and Galpha13 proteins, which mediate the activation of Rho by LPA. Thus, it appears likely that a Rho-mediated signaling pathway linked to heterotrimeric G proteins has been evolutionarily conserved (Barrett, 1997 and references).

The obsevation that DRhoGEF2 is required for the cell shape changes induced by ectopic Fog expression strongly supports the model that a signal from Folded gastrulation via Concertina activates DRhoGEF2 (Barrett, 1997). There is, however, one case of a significant difference between the mutant phenotypes of fog and cta and those of DRhoGEF2: in contrast to DRhoGEF2, fog and cta are not essential for ventral furrow formation. For this reason DRhoGEF2 appears to be activated (at least to some extent) independently of either Fog or Cta. In fact, because of the non-essential function of fog and cta in the mesoderm, a second pathway instructing cells to undergo shape changes has been postulated (Costa, 1994). It is proposed that DRhoGEF2 identifies this pathway as a G-protein-coupled signaling cascade involving the GTPase Rho1. Whether this pathway is also required to transduce additional signals besides that of Fog is presently unclear and this question will require further attention in the future (Hacker, 1998).

Rho's role in dorsal closure

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoAN19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoAN19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoAN19-expressing cells (Bloor, 2002).

Previous studies on RhoA function during dorsal closure have focused on its role in the formation of the leading edge actomyosin purse-string. This study shows that RhoA is required for proper organization of actin and nonmuscle myosin II throughout the epidermis. In addition, inhibition of RhoA causes misregulation of the JNK transcription activation pathway, loss of DE-cadherin from the cell surface and disruption of the apicolateral distribution of ßHeavy-spectrin. These changes in cell physiology have differential effects on cell behavior that depend upon the position of the cell within the dorsal-ventral axis. In particular, cell-cell adhesion in the ventral and lateral epidermis is severely compromised, but at the leading edge RhoAN19-expressing cells form new, ectopic cell-cell adhesions (Bloor, 2002).

Tension generated in the amnioserosa and the leading edge of the lateral epidermis independently contributes to the forces that drive dorsal closure. It has been proposed that nonmuscle myosin II activation generates tension in the leading edge and that this causes a leading edge intracellular actomyosin purse-string to shorten. Signaling downstream of RhoGTPase activates nonmuscle myosin II by modulating the level of myosin regulatory light chain phosphorylation. As such, expression of RhoAN19 in epidermal stripes might disrupt contraction of the leading edge purse-string. Defects in actin and nonmuscle myosin II organization caused by RhoAN19 expression are first observed at germband extension, up to 2 hours before purse-string formation. Thus, while actin and nonmuscle myosin II are localized at the leading edge in wild-type tissue, a purse-string structure is never formed in leading edge cells that express RhoAN19. RhoAN19 expression therefore effectively cuts the leading edge purse-string at multiple sites. This does not necessarily prevent progression of dorsal closure, confirming previous experiments which demonstrate that the integrity of the leading edge is not required for dorsal closure to continue to completion. It is concluded that small independent regions of leading edge in wild-type epidermal stripes can, in conjunction with contraction of the amnioserosa, migrate dorsally with relative normalcy (Bloor, 2002).

The question that arises is how do epidermal cells expressing RhoAN19 move dorsally in the absence of a leading edge purse-string? These cells could hitchhike, i.e. they are pulled dorsally by the amnioserosa or dragged along with neighboring wild-type cells. Although spread and disorganized, dorsal RhoAN19-expressing cells do maintain adhesion with wild-type neighbors and this might then allow passive RhoAN19-expressing cells to move dorsally with wild-type tissue. This is consistent both with the inverse correlation between integrity of the ventral epidermis and the extent to which dorsal closure proceeds, as well as with observations on the distribution of tension at the embryo surface during dorsal closure. Thus, during the time that the epidermis lateral to the leading edge opposes dorsal closure, ventral failure of epidermal integrity (and hole formation) would release the tensional restraints on the remaining lateral epidermis, allowing it to move dorsally with more success. Similarly, in the absence of this release (i.e. the ventral epidermis retains its integrity and opposes dorsal movement of the epidermis), the leading edge is presumably no longer capable of generating sufficient force to drive dorsal closure to completion (Bloor, 2002).

RhoAN19 expression causes ectopic activation of the JNK pathway in the lateral epidermis, suggesting that RhoA normally functions to inhibit JNK signaling. JNK activation is antagonized by the protein phosphatase encoded by puc, and in puc mutants JNK signaling is increased at the leading edge and is activated in the lateral epidermis. Thus, JNK signaling in wild-type embryos is not maximal and basal JNK activity in the lateral epidermis is revealed in the absence of either puc or RhoA mediated repression. Interestingly, RhoAN19 expression does not increase JNK signaling in the leading edge. This difference between the effect of puc mutations and RhoAN19 expression could be due to ectopic RhoAN19 suppressing an upstream JNK activator that is itself maximally activated in the leading edge (Bloor, 2002).

Co-expression of RhoAN19 and GMA, an actin marker in which GFP is fused to the Drosophila Moesin actin-binding domain, demonstrates that, in addition to effects on the actin cytoskeleton, inhibition of RhoA has profound effects on the adhesive properties of epidermal cells. In accordance with this, DE-cadherin is lost from the surface of RhoAN19-expressing cells. Rho is required for E-cadherin-mediated epithelial cell-cell adhesion in cultured vertebrate cells: in keratinocytes and MDCK cells, blocking Rho function prevents formation of E-cadherin-based junctions and causes preformed junctions to breakdown. This effect is dependent on cell-cell junction maturity; blocking Rho causes E-cadherin to be lost rapidly (within 1 hour) from immature junctions, but E-cadherin can persist for several hours at mature junctions. This differential affect is also observed in this study, since removal of DE-cadherin from the cell surface is not uniform throughout the RhoAN19-expressing stripe; ventral cells lose surface staining sooner than dorsal cells. This phenomenon most probably reflects regional differences in the maturity of epidermal cell junctions. Cells of the dorsal epidermis form a compact epithelium early in stage 10, while neuroblast delamination in the ventral neurectoderm delays formation of the ventral epidermis proper until well into stage 11, by which time enGAL4- and prdGAL4-driven protein expression is apparent. Alternatively, the differential effect might be due to a dorsoventral gradient in enGAL4- or prdGAL4-driven expression of RhoAN19. This seems unlikely, since no regional differences in fluorescence are observed when these GAL4 lines are used to drive GMA expression (Bloor, 2002).

If the primary defect associated with the epidermal expression of RhoAN19 is loss of DE-cadherin, then the phenotypes induced by RhoAN19 should phenocopy those of shotgun (DE-cadherin) mutants. The defects exhibited by shg embryos are difficult to compare with those shown by embryos expressing RhoAN19 in epidermal stripes. However, genetic analysis of shg demonstrates that the embryonic dorsal epidermis is less sensitive than the presumptive ventral epidermis to a reduction in DE-cadherin levels; embryos mutant for null shg alleles are missing head and ventral cuticle, while the dorsal cuticle appears unaffected. Thus, as with epidermal expression of RhoAN19, shg mutants disrupt ventral epidermal integrity and cells undergo apoptosis, while dorsally epidermal cells remain adherent and secrete cuticle. However, there is at least one clear distinction between the genetic reduction of DE-cadherin and the defects induced by expression of RhoAN19: both null and dominant-negative mutations in shg do not affect epithelial cell polarity, while inhibition of RhoA activity does. It seems likely that this difference reflects the additional function of RhoA in generating cell polarity, possibly through organization of the actin cytoskeleton. It is concluded that the epidermal defects caused by RhoAN19 expression cannot be explained simply on the basis of loss of DE-cadherin mediated adhesion (Bloor, 2002).

Maintenance of dorsal epithelial integrity in the absence of detectable surface DE-cadherin suggests that a secondary adhesion system must function in the dorsal epidermis. As in vertebrate cells different classical cadherins exhibit cell type dependent sensitivity to Rho inhibition this could involve another member of the cadherin family. The possibility that two cell-cell adhesion systems function in the dorsal epidermis may explain the behavior of RhoAN19-expressing cells. Embryos that express RhoAN19 in epidermal stripes differ from shg mutants in the uniformity of DE-cadherin loss from the cell surface. In shg mutants, zygotic DE-cadherin is lost from all cells, while striped expression of RhoAN19 results in two populations of dorsal epidermal cells -- those with cell surface DE-cadherin and those without. Differential adhesion properties of cell populations are the molecular basis for the classical phenomenon of cell sorting. Thus, the ectopic cell bridges formed by RhoAN19-expressing cells in these experimental embryos could be due to activation of a cell sorting mechanism between populations of dorsal epidermal cells that associate via different adhesion molecules (Bloor, 2002).

In summary, by expressing the dominant negative RhoAN19 construct in epidermal stripes in the developing Drosophila embryo, it has been shown that the small GTPase Rho has multiple functions throughout this tissue. It is therefore clear that experiments designed to test the function of Rho family GTPases in the epidermis cannot be interpreted simply in terms of their effect on the leading edge. The challenge ahead lies in dissecting the pathways downstream of Rho and determining how these Rho-dependent processes contribute individually to epidermal function and morphogenesis (Bloor, 2002).

Rho GTPase controls Drosophila salivary gland lumen size through regulation of the actin cytoskeleton and Moesin

Generation and maintenance of proper lumen size is important for tubular organ function. This study reports on a novel role for the Drosophila Rho1 GTPase in control of salivary gland lumen size through regulation of cell rearrangement, apical domain elongation and cell shape change. Rho1 controls cell rearrangement and apical domain elongation by promoting actin polymerization and regulating F-actin distribution at the apical and basolateral membranes through Rho kinase. Loss of Rho1 results in reduction of F-actin at the basolateral membrane and enrichment of apical F-actin, the latter accompanied by enrichment of apical phosphorylated Moesin. Reducing cofilin levels in Rho1 mutant salivary gland cells restores proper distribution of F-actin and phosphorylated Moesin and rescues the cell rearrangement and apical domain elongation defects of Rho1 mutant glands. In support of a role for Rho1-dependent actin polymerization in regulation of gland lumen size, loss of profilin (Chickadee) phenocopies the Rho1 lumen size defects to a large extent. Ribbon, a BTB domain-containing transcription factor, functions with Rho1 in limiting apical phosphorylated Moesin for apical domain elongation. These studies reveal a novel mechanism for controlling salivary gland lumen size, namely through Rho1-dependent actin polymerization and distribution and downregulation of apical phosphorylated Moesin (Xu, 2011).

Rho1 acts both in salivary gland cells and in the surrounding mesoderm to maintain apical polarity during gland invagination and to mediate cell shape change during gland migration. This study demonstrates a novel role for Rho1 in controlling salivary gland lumen size through regulation of actin polymerization and distribution and regulation of Moesin activity. By analyzing Rho1 alleles for which salivary gland cells invaginated and formed a gland, it was shown that zygotic loss of function of Rho1 results in shortening and widening of the gland lumen, which is accompanied by defects in cell shape change and cell rearrangement and failure of apical domains to elongate along the Pr-Di axis of the gland. These effects of Rho1 are mediated through Rok; inhibition of Rok completely phenocopies loss of Rho1 in these cellular events. Based on these studies, a model is proposed for Rho1 control of salivary gland lumen size, in particular lumen width, which is determined by cell rearrangement and apical domain elongation. Rho1 and Rok, through inhibition of cofilin, regulate cell rearrangement and apical domain elongation by promoting actin polymerization to localize F-actin at the basolateral membrane and by limiting the apical accumulation of F-actin. In parallel to its role in actin polymerization and distribution, Rho1 acts independently of Rok to limit apical p-Moe with Rib by an unknown mechanism and this function of Rho1 is specific for apical domain elongation. The data on cofilin (Twinstar) are consistent with those in cultured HeLa cells that showed that mammalian ROCK can inhibit cofilin activity indirectly through LIMK-mediated phosphorylation of cofilin (Xu, 2011).

Although manipulating Moe activity through gland-specific expression of MoeT559D was sufficient to completely phenocopy the Rho1 lumen defects, including cell rearrangement, it did so without disrupting actin polymerization or distribution. This is likely to be due to activated Moe strengthening the link between the actin cytoskeleton and the apical plasma membrane (without affecting levels of apical F-actin), which would increase apical membrane stiffness and remove the ability of gland cells to rearrange. Indeed, Moesin has been shown to control cortical rigidity during mitosis of cultured Drosophila S2R+ cells. Thus, Rho1 regulates cell rearrangement and apical domain elongation by controlling the actin cytoskeleton and Moesin activity through distinct mechanisms (Xu, 2011).

The observation that chic mutant glands phenocopy Rho1 mutant glands to a large extent, suggests that Rho1 control of salivary gland lumen size is mainly dependent on a requirement for Rho1 in actin polymerization. However, as the chic and Rho1 gland lumen phenotypes are not identical, with chic mutant glands lacking the apical accumulation of F-actin and p-Moe observed in Rho1 mutant glands, Rho1 probably has an additional function in limiting accumulation of F-actin and p-Moe at the apical membrane. This function of Rho1, at least for limiting apical F-actin, might partly involve Rab5- or Shi-mediated endosome trafficking, because inhibition of Rab5 alone or Shi alone led to accumulation of F-actin at the apical membrane. Although Rab5DN- or ShiDN-expressing salivary gland cells were enriched with apical F-actin, lumen size was not affected. This could be due to Rab5DN and ShiDN affecting a pool of apical F-actin distinct from that affected by Rho1 and/or because Rab5DN-expressing gland cells retain basolateral F-actin and the ratio of apical to basolateral F-actin is not altered sufficiently to cause lumen size defects. In Rho11B mutant gland cells, some early endosomes were not coated with F-actin. Actin is known to contribute to multiple steps of the endocytic pathway, including movement of endocytic vesicles through the cytoplasm and their transport to late endosomes and lysosomes. One possible mechanism by which Rho1 normally limits apical accumulation of F-actin is by promoting its removal from the apical membrane and accumulation on endocytic vesicles (Xu, 2011).

Currently, it is not know how Rho1 limits accumulation of apical p-Moe. Membrane localization and activity of Moesin can be regulated via a number of mechanisms, such as its phosphorylation on a conserved Threonine residue, binding to phosphatidylinositol-(4,5)bisphosphate [PtdIns(4,5)P2] and association with components of the sub-membrane cytoskeleton, such as Crb. Studies in cultured mammalian cells have demonstrated that Rho signaling activates Moe either through phosphorylation of Moe by ROCK or through ROCK-mediated inhibition of myosin phosphatase, which is known to dephosphorylate p-Moe. Although it is possible that Drosophila Rho1 positively regulates Moe activity by one or more of these mechanisms, this study shows that in the developing salivary glands Rho1 in fact negatively regulates Moe activity. In rib mutant embryos, in which p-Moe is enriched apically, salivary gland and tracheal cells showed decreased staining for Rab11 GTPase, which localizes to the apical recycling endosomes and to secretory vesicles destined for the apical membrane. Thus, Rho1, like Rib might limit apical p-Moe through its membrane transport (Xu, 2011).

In Drosophila imaginal disc epithelia, Moe negatively regulates Rho1 activity to maintain epithelial integrity and to promote cell survival. These studies demonstrating that in the developing salivary gland Rho1 antagonizes Moe activity by limiting its localization at the apical membrane, shed novel insight into the functional relationship between Rho1 and Moe. It is possible that in a dynamic epithelium, such as the developing salivary gland, Rho1 contributes to the precise spatial and temporal regulation of Moe activity to fine-tune selective changes in apical domain shape. By contrast, in the imaginal disc epithelium, Rho1 regulation of Moe might not be necessary and, instead, Moe regulation of Rho1 activity is required to maintain epithelial integrity and cell survival. Thus, Rho and Moe can antagonize each other's activities depending on the type of epithelia or cellular event (Xu, 2011).

Rescue studies with Rho1WT demonstrate that Rho1 functions predominantly in the salivary gland cells to control apical domain elongation and cell rearrangement. Interestingly, expression of Rho1WT in the mesoderm with twi-GAL4 has no effect on cell rearrangement and has little effect on apical domain elongation and lumen size, whereas it has been shown that Rho1WT expression in the mesoderm significantly rescues the gland migration defect of Rho11B mutant embryos. This suggests that gland migration and lumen size control are regulated by distinct mechanisms. In support of this conclusion, embryos mutant for multiple edematous wings, encoding the αPS1 integrin subunit, which have defects in gland migration, show no defects in gland lumen width. Identifying the distinct and overlapping mechanisms by which salivary gland lumen width and length are controlled will help to elucidate the mechanisms by which lumen size is controlled in tubular organs (Xu, 2011).

Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis

During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. This study used optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. Precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes (Izquierdo, 2018).

The results presented in this study show that localized activation of Rho signaling at the apical surface of cells, which are otherwise not programmed to invaginate, is sufficient to cause tissue invagination and to recapitulate major cell- and tissue-level behaviors associated with endogenous invagination processes. Mechanisms other than apical constriction control a variety of different forms of invaginations during animal development. The current results do not challenge this view, rather, they argue that if considering a monolayer of epithelial cells, apical constriction is sufficient to fold it into a U-shape invagination. However, apical constriction is not sufficient to drive closure of an invagination into a tube-like structure, as seen for example during ventral furrow formation. Additional pushing forces exerted by lateral non-invaginating cells and/or loss of myosin II from the basal surface and basal relaxation might be required to complete this step (Izquierdo, 2018).

At the tissue-level, the contractile behavior of individual cells depends on the geometry of photo-activation. While a square box results in isotropic apical constrictions, a rectangular shape causes cells to constrict preferentially along the minor axis of the photo-activated area and to elongate along the major axis. This anisotropic contractile behavior resembles the one of ventral furrow cells, which are also organized in a rectangular pattern and constrict preferentially along the short axis of the tissue. Anisotropy in ventral furrow cells is not genetically determined but arises as a consequence of tissue geometrical and mechanical constraints. Consistent with these studies, the increase in the degree of anisotropic constriction as a function of the rectangularity of the photo-activated area can be explained if considering that it is mechanically less favorable to shrink cells along the major axis of a rectangle than along the short axis. Indeed, the former deformation requires the endpoints of the constricting tissue to move farther, and thus a larger deformation of neighboring tissues along that axis (Izquierdo, 2018).

The results also reveal an interesting correlation between pulsatile constrictions and tissue invagination. During endogenous morphogenetic processes, two different pulsatile behaviors have been described. One is based on cycles of myosin II accumulation at the medio-apical plane of the cell, during the contraction phase, and dissolution during the relaxation phase. This type of pulsatile behavior has been first described during dorsal closure in Drosophila and it is not linked to tissue invagination. Another type of pulsatile behavior is based on an incremental accumulation of myosin II at each contraction, which is followed by a stabilization period of cell shape without an intervening relaxation phase (ratcheted contractions). Ratcheted contractions require the radial polarization of Rho signaling components. However, in certain mutant conditions that interfere with the establishment of radial polarity, contractions become non-ratcheted with myosin II miss-localizing at three-ways junctional vertices. The optogenetic-induced pulsatile contractions described in this study display a non-ratchet behavior, and similarly to dorsal closure contractions, are characterized by myosin II medio-apical accumulation and dissolution in phase with contraction and relaxation of the apical surface. However, differently from dorsal closure, optogenetic-induced pulsatile contractions display a higher degree of synchrony with photo-activated cells constricting and relaxing in concert. This difference could be explained if considering that a light pulse provides a coherent and synchronous input, while activation of signaling in a developing tissue might be more subject to cell-to-cell variability. Lack of tissue internalization upon induction of pulsatile constrictions is likely due to the absence of a stabilization phase after constriction of the apical surface, which might result in a dissipation of the forces that are normally needed to build tension and drive invagination. Consistently, continuous administration of light induced synchronous contractile behavior and invagination, mimicking the activity of signaling molecules such as Fog whose function is to control the transition from stochastic to collective contractile behavior during ventral furrow invagination. Pulsatile behavior could be elicited either by a discontinuous administration of light, or by continuous illumination at a lower laser power, or by a single pulse at a higher laser power. These results are interpreted to suggest that pulsations can be induced by the stimulation of a Rho-dependent mechano-chemical oscillatory system up to a certain threshold, above which cells constrict without pulsing. Stimulation of Rho signaling above a certain threshold could override, for example, the activity of a RhoGAP, which is required to control the normal spatio-temporal dynamics of Rho GDP/GTP cycling. In agreement with this proposal, pulsatile constrictions during ventral furrow invagination require the activity of a specific RhoGAP. However, while ventral cells pulse with a mean period of ~80 s, optogenetic-induced pulsations display a mean period of ~150 s, a limit probably imposed by the reversion kinetics of the CRY2/CIB1 system in the dark (Izquierdo, 2018).

In conclusion, these data illustrate the utility of applying concepts of synthetic biology (e.g., precise orthogonal control over signaling pathways, guided cell behavior) to the field of tissue morphogenesis and in particular of how the nascent field of synthetic morphogenesis can help defining the minimum set of requirements sufficient to drive tissue remodeling. The data argue that while normally tissue differentiation and tissue shape are intimately linked, it is possible to direct tissue shape without interfering with complex layers of gene regulatory network and tissue differentiation programs. This might have important implications also for tissue engineering, where it might be desirable to shape any given tissue of interest without changing its fate (Izquierdo, 2018).


GENE STRUCTURE

Transcript length - 2.2 and 1.3 kb

Bases in 5' UTR - 41

Bases in 3' UTR - 858


PROTEIN STRUCTURE

Amino Acids - 193

Structural Domains

Rho1 is highly related to its mammalian counterpart. Drosophila Rho1 is 86% identical to human rhoA, B and C GTPases at the amino acid level, with the vast majority of the differences occcuring in the C-terminal third of the protein. The Drosophila Rho1 protein also exhibits substantial sequence identity (about 75%) with two yeast rho GTPases that have been reported, Yrho1 and Yrho2, indicating that the rho gene has been well maintained throughout evolution (Hariharan, 1995).


Rho1: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 25 July 2002

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