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
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
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
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
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
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.

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).


Transcript length - 2.2 and 1.3 kb

Bases in 5' UTR - 41

Bases in 3' UTR - 858


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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