Border-cell migration: the race is on

The conversion of stationary epithelial cells into migratory, invasive cells is important for normal embryonic development and tumour metastasis. Border-cell migration in the ovary of Drosophila melanogaster has emerged as a simple, genetically tractable model for studying this process. Three distinct signals, which are also upregulated in cancer, control border-cell migration, so identifying further genes that are involved in border-cell migration could provide new insights into tumour invasion.     

Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however, their role in a developing organism is poorly understood. Here, we identify the Drosophila Ral homologue RalA as a new key regulator of polar-cell differentiation during oogenesis. Polar cells have a crucial role in patterning the egg chamber and in recruiting border cells, which undergo collective and guided migration. We show that RalA function is essential for the maintenance of anterior and posterior polar-cell fate and survival. RalA is required cell autonomously to control the expression of polar-cell-specific markers, including the Jak/Stat ligand Unpaired. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result, border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus, RalA is required in organizing centres to control proper patterning and migration in vivo.     

Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK)

We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene. Nik(-/-) mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik(-/-) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik(-/- )embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.     

The Ste20-like kinase misshapen functions together with Bicaudal-D and dynein in driving nuclear migration in the developing drosophila eye

Nuclear translocation, driven by the motility apparatus consisting of the cytoplasmic dynein motor and microtubules, is essential for cell migration during embryonic development. Bicaudal-D (Bic-D), an evolutionarily conserved dynein-interacting protein, is required for developmental control of nuclear migration in Drosophila. Nothing is known about the signaling events that coordinate the function of Bic-D and dynein during development. Here, we show that Misshapen (Msn), the fly homolog of the vertebrate Nck-interacting kinase is a component of a novel signaling pathway that regulates photoreceptor (R-cell) nuclear migration in the developing Drosophila compound eye. Msn, like Bic-D, is required for the apical migration of differentiating R-cell precursor nuclei. msn displays strong genetic interaction with Bic-D. Biochemical studies demonstrate that Msn increases the phosphorylation of Bic-D, which appears to be necessary for the apical accumulation of both Bic-D and dynein in developing R-cell precursor cells. We propose that Msn functions together with Bic-D to regulate the apical localization of dynein in generating directed nuclear migration within differentiating R-cell precursor cells.     

The Ste20 kinase misshapen regulates both photoreceptor axon targeting and dorsal closure, acting downstream of distinct signals

We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.     

Drosophila spoonbill encodes a dual-specificity A-kinase anchor protein essential for oogenesis

spoonbill is a Drosophila female-sterile mutation, which interferes with normal egg patterning during oogenesis. Previous analyzes linked the mutation to a number of seemingly unrelated pathways, including GRK/EGFR and DPP, two major pathways essential for Drosophila and vertebrate development. Further work suggested that spoonbill may also function in actin polymerization and border-cell migration. Here we describe the molecular cloning of the spoonbill gene and characterize new mutant alleles, further demonstrating that spoonbill's function is essential during oogenesis. We found spoonbill to be allelic to CG3249 (also known as yu), which encodes the only known dual-specificity A-kinase anchor protein in Drosophila. Our data indicate that similar to mammalian AKAPs, Spoonbill protein contains a number of potential kinase and phosphatase binding motifs, and is targeted, in the ovary, to mitochondria and Golgi. Finally, we address some of spoonbill's mutant phenotypes from the perspective of the published data on the AKAP protein family.     

JNK signaling controls border cell cluster integrity and collective cell migration

Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors [1]. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster [2]. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms [3]. However, how border cells are held together while they migrate is not known. Here, we show that a negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. We anticipate a role for the JNK pathway in controlling collective cell movements in other morphogenetic and clinical models.     

Initiating and guiding migration: lessons from border cells

Cell migration occurs in many different contexts. Amoebae and other isolated cells migrate in culture. In animals, 'professional' migratory cells of the immune system constantly survey the body for intruders, whereas other cell types perform specific developmentally regulated migrations. One simple model for the latter type of event is migration of border cells during Drosophila oogenesis. Recent findings have shed light on how border cell fate is induced and on how the migration is guided. This article discusses the implications of these studies and compares (invasive) migration through a tissue with what is known about cells crawling on a flat substratum.     

Myosin VI is required for E-cadherin-mediated border cell migration

Myosin VI (MyoVI) is a pointed-end-directed, actin-based motor protein, and mutations in the gene result in disorganization of hair cell stereocilia and cause deafness in mice. MyoVI also localizes to the leading edges of growth-factor-stimulated fibroblast cells and has been suggested to be involved in cell motility. There has been no direct test of this hypothesis, however. Drosophila melanogaster MyoVI is expressed in a small group of migratory follicle cells, known as border cells. Here we show that depletion of MyoVI specifically from border cells severely inhibited their migration. Similar to MyoVI, E-cadherin is required for border cell migration. We found that E-cadherin and Armadillo (Arm, Drosophila beta-catenin) protein levels were specifically reduced in cells lacking MyoVI, whereas other proteins were not. In addition, MyoVI protein levels were reduced in cells lacking DE-cadherin or Arm. MyoVI and Arm co-immunoprecipitated from ovarian protein extracts. These data suggest that MyoVI is required for border cell migration where it stabilizes E-cadherin and Arm. Mutations in MyoVIIA, another unconventional myosin protein, also lead to deafness, and MyoVIIA interacts with E-cadherin through a membrane protein called vezatin. Multiple biochemical mechanisms may exist, therefore, for cadherins to associate with diverse unconventional myosins that are required for normal stereocilium formation or maintenance.     

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.     

A cell-type-specific defect in border cell formation in the Acacia mangium root cap developing an extraordinary sheath of sloughed-off cells

Background and Aims

Root caps release border cells, which play central roles in microbe interaction and root protection against soil stresses. However, the number and connectivity of border cells differ widely among plant species. Better understanding of key border-cell phenotype across species will help define the total function of border cells and associated genes.

Methods

The spatio-temporal detachment of border cells in the leguminous tree Acacia mangium was investigated by using light and fluorescent microscopy with fluorescein diacetate, and their number and structural connectivity compared with that in soybean (Glycine max).

Key Results

Border-like cells with a sheet structure peeled bilaterally from the lateral root cap of A. mangium. Hydroponic root elongation partially facilitated acropetal peeling of border-like cells, which accumulate as a sheath that covers the 0- to 4-mm tip within 1 week. Although root elongation under friction caused basipetal peeling, lateral root caps were minimally trimmed as compared with hydroponic roots. In the meantime, A. mangium columella caps simultaneously released single border cells with a number similar to those in soybean.

Conclusions

These results suggest that cell type-specific inhibitory factors induce a distinct defective phenotype in single border-cell formation in A. mangium lateral root caps.     

Regulation of cofilin phosphorylation and asymmetry in collective cell migration during morphogenesis

During Drosophila oogenesis, two actin dynamics regulators, cofilin and Rac, are required for the collective migration of a coherent cluster of cells called border cells. Cell culture data have shown that Rac and cofilin are both essential for lamellipodium formation, but Rac signaling results in phosphorylation and hence inactivation of cofilin. So it remains unclear whether cofilin phosphorylation plays a promoting or inhibitory role during cell migration. We show here that cofilin is required for F-actin turnover and lamellipodial protrusion in the border cells. Interestingly, reducing the dosage of cofilin by half or expressing a phospho-mimetic mutant form, S3E, partially rescues the migration and protrusion defects of Rac-deficient border cells. Moreover, cofilin exhibits moderate accumulation in border cells at the migratory front of the cluster, whereas phospho-cofilin has a robust and uniform distribution pattern in all the outer border cells. Blocking or overactivating Rac signaling in border cells greatly reduces or increases cofilin phosphorylation, respectively, and each abolishes cell migration. Furthermore, Rac may signal through Pak and LIMK to result in uniform phosphorylation of cofilin in all the outer border cells, whereas the guidance receptor Pvr (PDGF/VEGF receptor) mediates the asymmetric localization of cofilin in the cluster but does not affect its phosphorylation. Our study provides one of the first models of how cofilin functions and is regulated in the collective migration of a group of cells in vivo.