Male-specific cell migration into the developing gonad is a conserved process involving PDGF signalling

Male-specific migration of cells from the mesonephric kidney into the embryonic gonad is required for testis formation in the mouse. It is unknown, however, whether this process is specific to the mouse embryo or whether it is a fundamental characteristic of testis formation in other vertebrates. The signalling molecule/s underlying the process are also unclear. It has previously been speculated that male-specific cell migration might be limited to mammals. Here, we report that male-specific cell migration is conserved between mammals (mouse) and birds (quail-chicken) and that it involves proper PDGF signalling in both groups. Interspecific co-cultures of embryonic quail mesonephric kidneys together with embryonic chicken gonads showed that quail cells migrated specifically into male chicken gonads at the time of sexual differentiation. The migration process is therefore conserved in birds. Furthermore, this migration involves a conserved signalling pathway/s. When GFP-labelled embryonic mouse mesonephric kidneys were cultured together with embryonic chicken gonads, GFP+ mouse cells migrated specifically into male chicken gonads and not female gonads. The immigrating mouse cells contributed to the interstitial cell population of the developing chicken testis, with most cells expressing the endothelial cell marker, PECAM. The signalling molecule/s released from the embryonic male chicken gonad is therefore recognised by both embryonic quail and mouse mesonephric cells. A candidate signalling molecule mediating the male-specific cell migration is PDGF. We found that PDGF-A and PDGF receptor-alpha are both up-regulated male-specifically in embryonic chicken and mouse gonads. PDGF signalling involves the phosphotidylinositol 3-kinase (PIK3) pathway, an intracellular pathway proposed to be important for mesonephric cell migration in the mammalian gonad. We found that a component of this pathway, PI3KC2alpha, is expressed male-specifically in developing embryonic chicken gonads at the time of sexual differentiation. Treatment of organ cultures with the selective PDGF receptor signalling inhibitor, AG1296 (tyrphostin), blocked or impaired mesonephric cell migration in both the mammalian and avian systems. Taken together, these studies indicate that a key cellular event in gonadal sex differentiation is conserved among higher vertebrates, that it involves PDGF signalling, and that in mammals is an indirect effect of Sry expression.     

Endothelial and steroidogenic cell migration are regulated by WNT4 in the developing mammalian gonad

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. Here we show that the role of WNT4 in gonad development is to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. We show that WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis did not inhibit coelomic vessel formation but vascular pattern was affected. Leydig cell differentiation was not affected in these transgenic animals and our data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development.     

Neural crest cell migration in the developing embryo

In vertebrate embryos, neural crest cells migrate extensively to defined sites where they differentiate into a complex array of derivatives, ranging from neurons to pigment cells. Neural crest cells emerge uniformly from the neural tube but their subsequent migratory pattern is segmented along much of the body axis. What factors control this segmental migration? At trunk levels, it is imposed by the intrinsic segmentation of the neighbouring somitic mesoderm, while in the head, intrinsic information within the neural tube as well as extrinsic influences from the ectoderm are involved. A variety of cell-cell and cell-extracellular matrix interactions are thought to influence initiation and movement of neural crest cells. This review summarizes recent progress from both experimental embryology and cell biology approaches in uncovering the mechanisms underlying neural crest cell migration.     

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.     

Sexually dimorphic gene expression in the developing mouse gonad

Over the course of a few days, the bipotential embryonic mouse gonad differentiates into either a testis or an ovary. Though a few gene expression differences that underlie gonadal sex differentiation have been identified, additional components of the testicular and ovarian developmental pathways must be identified to understand this process. Here we report the use of a PCR-based cDNA subtraction to investigate expression differences that arise during gonadal sex differentiation. Subtraction of embryonic day 12.5 (E12.5) XY gonadal cDNA with E12.5 XX gonadal cDNA yielded 19 genes that are expressed at significantly higher levels in XY gonads. These genes display a variety of expression patterns within the embryonic testis and encode a broad range of proteins. A reciprocal subtraction (of E12.5 XX gonadal cDNA with E12.5 XY gonadal cDNA) yielded two genes, follistatin and Adamts19, that are expressed at higher levels in XX gonads. Follistatin is a well-known antagonist of TGFbeta family members while Adamts19 encodes a new member of the ADAMTS family of secreted metalloproteases.     

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.     

Epithelial cell proliferation and migration in the developing rat gastric mucosa

To investigate cell proliferation in developing gastric antrum and fundus, proliferating gastric epithelial cells were labeled in fetal rats by intravenously injecting mothers with [³H]thymidine. In addition 14-day postnatal (dPN) rats were given [³H]thymidine intraperitoneally. Tissue was removed 1.5 hr later, and autoradiographs were prepared to identify proliferating cells. Total epithelial labeling indices (L.I.) reached a peak at 20 days gestation (dG), coincident with the appearance of pit/glands, then declined again by 22 dG (gestation end). At 18 dG, labeled cells were distributed throughout all levels of the stratified epithelia. Between 20 and 22 dG, as pit/gland development proceeded, labeled cells became concentrated in the gland bases and were only rarely seen on the surface (L.I. of surface cells <1% at 22 dG). By 14 dPN, proliferating cells were entirely absent from the epithelial surface. Approximately 15% of the endocrine cells were labeled at 22 dG, compared to only 2% at 14 dPN; zymogen cells were labeled (～6%) at 14 dPN; parietal cells did not label at any age studied. In addition, cell migration to the epithelial surface was studied in rats labeled at 22 dG, 14 dPN, or 28 dPN, and killed 1–20 days later. Migration times were slightly shorter in the 28 dPN group and were longer in fundus than antrum in all groups.     

Pathways of avian neural crest cell migration in the developing gut

The NC-1 and E/C8 monoclonal antibodies recognize a similar population of neural crest cells as they migrate from vagal levels of the neural tube and colonize the branchial arch region of 2- to 3-day-old chicken embryos. Some of these immunoreactive cells then appear to enter the gut mesenchyme on the third day of incubation just caudal to the third branchial cleft. After entering the gut, these cells migrate in a rostral-caudal direction, using primarily the superficial splanchnic mesodermal epithelium of the gut as a substratum. The antigen-positive cells remain preferentially associated with the splanchnopleure. Few antigenic cells enter the mesenchyme surrounding the endoderm at anterior levels whereas they are found throughout the mesenchyme when nearing the umbilicus. At postumbilical levels, immunoreactive cells are distributed on both sides of the differentiating muscle layer but not within it. Although fibronectin immunoreactivity can be found throughout the wall of the gut, there is no apparent relationship between the distribution of fibronectin and the location of the immunoreactive cells. These results suggest that a mechanism more complex than a mere interaction with fibronectin may account for migration of crest-derived cells in the gut.     

Cadherin-mediated differential cell adhesion controls slow muscle cell migration in the developing zebrafish myotome

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.     

Retinoic acid inhibits rat XY gonad development by blocking mesonephric cell migration and decreasing the number of gonocytes

Vitamin A (also called retinol) and its derivatives, retinoic acids (RAs), are required for postnatal testicular function. Abnormal spermatogenesis is observed in rodents on vitamin A-deficient diets and in retinoic acid receptor alpha (RARalpha) knockout mice. In contrast, RA has an inhibitory effect on the XY gonad development in embryos. To characterize this inhibitory effect of RA, we investigated the cellular events that are required for the XY gonad development, including cell migration from the adjacent mesonephros into the gonad, fetal Sertoli cell differentiation, and survival of gonocytes. In organ cultures of Embryonic Day 13 (E13) XY gonads from rats, all-trans-retinoic acid (tRA) inhibited mesonephric cell migration into the gonad. Moreover, treatment with tRA decreased the expression of Müllerian-inhibiting substance in Sertoli cells and dramatically reduced the number of gonocytes. Increased apoptosis was detected in the XY gonads cultured with tRA, suggesting that the loss of gonocytes could be due to increased apoptosis. In addition, Am580, a synthetic compound that exhibits RARalpha-specific agonistic properties, mimicked the inhibitory effects of tRA on the XY gonad development including mesonephric cell migration and gonocyte survival. Conversely, a RARalpha-selective antagonist, Ro 41-5253, suppressed the inhibitory ability of tRA on the XY gonad development. These results suggest that retinoic acid acting through RARalpha negatively affects fetal Sertoli cell differentiation and gonocyte survival and blocks the migration of mesonephric cells, thereby leading to inhibition of the XY gonad development.     

Diphtheria toxin-mediated cell ablation in developing pollen: Vegetative cell ablation blocks generative cell migration

Summary The technique of genetic cell ablation involves the targeted expression of a cell autonomous cytotoxic protein under the control of cell-specific regulatory sequences. This technique allows the investigation of cell-cell interactions by inducing selective death in a precisely controlled and cell autonomous manner. Here, targeted vegetative cell-specific ablation was used to examine the role of the vegetative cell (VC) in controlling generative cell (GC) behaviour and differentiation during pollen development. The tomatolat 52 late-pollen promoter, which has been shown to be activated specifically in the nascent VC immediately following pollen mitosis I (PMI), was used to direct expression of the cytotoxic diphtheria toxin A chain (DTA) in both transient expression assays using microprojectile bombardment and in transgenic tobacco plants. Transient expression of DTA linked to thelat 52 promoter (lot 52-DTA) in pollen dramatically reduced the expression of a co-transfected reporter gene fusion, demonstrating the cytotoxicity of DTA to pollen. Genetic and phenotypic analysis oflat 52-DTA transformants demonstrated that DTA expression led to a pollen-lethal phenotype, recognisable as small acytoplasmic pollen grains at anthesis, which affected 50% of the pollen population in single locus transformants. Detailed cytological analysis using confocal laser scanning microscopy and vital staining using fluorescein diacetate (FDA), showed that the first sign of cell ablation during pollen development was a loss of vital staining of the VC immediately following PMI. In contrast, the GC retained viability for up to several days following VC ablation, but progressively lost viability in the absence of a functional VC. Of particular interest was the observation that in the absence of VC function the generative cell (GC) failed to undergo normal migration away from the pollen grain wall into the VC cytoplasm. These results directly demonstrate the dependence of the GC on VC cell functions and highlight the importance of VC-GC interactions in controlling GC migration.Abbreviations CaMV cauliflower mosaic virus - nos nopaline synthase - DTA diptheria toxin A chain - lat late anther tomato - VC vegetative cell - GC generative cell - PGM pollen germination medium - EtBr ethidium bromide - FDA fluorescein diacetate - FCR fluorochrome reaction - DAPI 4,6-diamidino-2-phenylindole     

Sry induces cell proliferation in the mouse gonad

Sry is the only gene on the Y chromosome that is required for testis formation in mammals. One of the earliest morphological changes that occurs as a result of Sry expression is a size increase of the rudimentary XY gonad relative to the XX gonad. Using 5'-bromo-2'-deoxyuridine (BrdU) incorporation to label dividing cells, we found that the size increase corresponds with a dramatic increase in somatic cell proliferation in XY gonads, which is not detected in XX gonads. This male-specific proliferation was observed initially in the cells of the coelomic epithelium and occurred in two distinct stages. During the first stage, proliferation in the XY gonad was observed largely in SF1-positive cells and contributed to the Sertoli cell population. During the second stage, proliferation was observed in SF1-negative cells at and below the coelomic epithelium and did not give rise to Sertoli cells. Both stages of proliferation were dependent on Sry and independent of any other genetic differences between male and female gonads, such as X chromosome dosage or other genes on the Y chromosome. The increase in cell proliferation began less than 24 hours after the onset of Sry expression, before the establishment of male-specific gene expression patterns, and before the appearance of any other known male-specific morphological changes in the XY gonad. Therefore, an increase in cell proliferation in the male coelomic epithelium is the earliest identified effect of Sry expression.     

Study of the germ cell migration to the gonad buds of quail embryo after the action of 2,4,5-trichlorophenoxyacetic acid]

At stages 16 and 18, the germ cells counts, in the quail embryos' gonads from control and 2,4,5-T treated eggs, show no significative difference. On the contrary, at stage 20, the gonocyte numbers of the treated embryos are strongly decreased. Actually, this mechanism cannot be stage precisely.     

WASP stings into matrix to lead immune cell migration

WASP is a remodeler of the actin cytoskeleton, but its mechanistic contribution to neutrophil migration is unclear. In this issue, Brunetti et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202104046) show that WASP is recruited to substrate-induced membrane deformations near the cell front, where it induces Arp2/3 complex–mediated local actin assembly to direct migration.

Neutrophils are the most abundant type of white blood cells in humans and constitute an important first line of defense of the innate immune system. Neutrophils exit the blood stream in response to chemoattractants that signal danger in the form of damage, infection, or inflammation, ultimately removing dangerous particles by phagocytosis. Efficient migration is crucial to properly execute such functions, but how precisely the coordination of polarity establishment and complex shape changes needed for migratory processes occurs in these cells is just beginning to be elucidated. The dynamic remodeling of actin filaments, which can differentially assemble and disassemble at both ends, is key to the development of pulling and pushing forces below the plasma membrane of most eukaryotic cells or on the surfaces of their intracellular organelles (1).Actin filaments are organized into either bundles or networks, with networks most commonly generated and maintained by the continuous filament branching activity of the actin-related protein 2/3 (Arp2/3) complex (2). Arp2/3 complex–driven branching in actin networks has often been associated with the development of pushing forces, even in a mechano-responsive fashion (3, 4), and less so linked with pulling or traction forces as developed, for instance, by adhesions. The heteropentameric Arp2/3 complex, which catalyzes the formation of daughter filaments off the sides of mother filaments, is intrinsically inactive, but can be activated by nucleation promoting factors (NPFs). The canonical NPFs include the Wiskott-Aldrich syndrome protein (WASP), its ubiquitous orthologue neural WASP (N-WASP), three WASP family verprolin-homologous protein (WAVE) isoforms as well as WASH, WHAMM, and JMY (5). The C-termini of all these factors physically interact with the Arp2/3 complex, driving it into an active conformation, and aid daughter filament polymerization by adding actin monomers onto Arp-2 and -3, forming the base of the branch. Coordinating the branching of daughter filaments by Arp2/3 complex and their elongation appears to be a major function of NPFs such as WASP. The role of WASP in stimulating actin assembly and the observation that patients with Wiskott-Aldrich syndrome harboring mutations in this gene have white blood cells that are unable to reach the sites of infections (6) points to WASP as a regulator of guided migration of immune cells. In this issue, Brunetti et al. establish the precise cellular function by which WASP controls neutrophil migration: WASP is recruited to sites of inward membrane deformations to stimulate the formation of spot-like actin structures that aid in the adhesion to or in the grabbing onto extracellular structures of high diversity and flexibility such as extracellular matrix fibrils, thus linking substrate topology, cell polarity, and migration (7).The authors first determined the spatiotemporal dynamics of EGFP-tagged, endogenous WASP in the human neutrophil–like cell line HL-60 using total internal reflection fluorescence microscopy. WASP formed puncta that were largely devoid of clathrin accumulation, thus likely not embodying endocytic pits, and exogenously triggered by submicron-sized beads, microfabricated ridges, or collagen fibers. Substratum-associated WASP puncta exhibited adhesive functions, as integrin inactivation by ion chelation largely dissociated them, stimulating their sliding and centripetal displacement. Surprisingly, the prominent WASP accumulation to beads below the plasma membrane was biased to the front half of the cell, and coincided with a gradient of activity of its major activator, the Rho GTPase Cdc42 from front to rear. This observation led Brunetti et al. to postulate WASP as a factor linking substrate topology to cell polarization and effective migration.A closer look at the recruitment of WASP to inward plasma membrane invaginations (positive membrane curvature) stimulated by submicron-sized beads revealed two patterns of association: either following the bead bodies as U-shaped accumulations or to the necks of invaginations reminiscent of endocytic pits, which could reflect later stages of bead–plasma membrane interactions. Although the mechanistic significance of these distinct association patterns remains elusive, it was clear that WASP favored associations at the plasma membrane with sites of comparably high, positive curvature (small, 100-nm-diameter beads more attractive than larger beads, for instance) and in a punctate fashion (even along ridges). In our view, this much diverges from WAVE isoforms exhibiting a prominent, more linear association with the rapidly protruding membranes of negative curvature in lamellipodia (8). Finally, the authors showed that Cdc42 loss-of-function diminished WASP puncta at the cell front, and that lack of WASP reduced actin puncta formation and the frequency of Arp2/3 complex accumulation at beads. WASP-null cells migrated less in the direction of ridges, although their perpendicular movement across the nanopatterns was not affected—indeed, it was increased. These observations connect the role of WASP in reading substrate topology to Arp2/3-dependent actin filament branching and network formation. The findings of Brunetti et al. appear to be of broader impact and relevance than just being a human neutrophil-specific phenomenon—a separate study has recently shown that sliding, WASP-dependent, punctate adhesion sites in murine dendritic cells and T cells, which are triggered in response to mechanical load, polymerize actin networks orthogonally to the plasma membrane, and aid in squeezing and dragging these cells through dense tissues (9).Taking all this together, the study by Brunetti et al. highlights that at least immune cells primarily respond to mechanical impact and indentations of their plasma membranes with rapid accumulation of WASP and Arp2/3 complex–dependent actin assembly, thereby pushing back or grabbing extracellular material to push and pull themselves forward during migration through the complex, dense, and three-dimensional environments on their way through tissues (Fig. 1).Open in a separate windowFigure 1.Neutrophils use WASP puncta to crawl like mountain climbers. Punctate accumulation of WASP and consequently F-actin brings about friction points that support pushing and pulling during cell translocation. In the front half of the cell, formation of these points strictly depends on Cdc42 mediating WASP activation and focal actin assembly. Even on substrates that would allow continuous adhesion (ridges or collagen fibers), WASP is recruited in a punctate fashion and preferentially to the front half of the cell that is dominated by Cdc42 activity signaling to cell polarity. Upon contact, movable objects like beads lead to a strong WASP/actin response potentially culminating in object envelopment.Interestingly, according to Brunetti et al., mechanisms of WASP/N-WASP recruitment to inward membrane deformations might even be conserved during early stages of formation of podosomes and invadopodia, which are WASP- and N-WASP–dependent, both protrusive and adhesive structures operating in matrix degradation of hematopoietic and cancer cells, respectively (10). This hypothesis, however, remains to be experimentally validated. Furthermore, additional questions remain unsolved, including the precise signals, aside from Cdc42, contributing to WASP accumulation and turnover at the plasma membrane. Where does the bias for focal, punctate WASP accumulation come from? WASP family NPFs are unlikely to themselves harbor curvature-sensing activities, so which are the decisive factors for interaction with positively curved membranes? How is specificity brought about? The F-BAR domain-containing TOCA family proteins would have been potential candidates, but surprisingly, disruption of two of their most prominent members, FBP17 and CIP4, did not cause severe defects, as Brunetti et al. showed in this study. Future work will surely improve our insights into the differential sorting and subcellular functions of distinct NPFs in both immune and other cells.     

Guidance of glial precursor cell migration by secreted cues in the developing optic nerve

Oligodendrocyte precursors are produced in restricted foci of the germinative neuroepithelium in embryo brains and migrate to their sites of function, while astrocytes are produced in a wider area in the neuroepithelium. We investigated the guidance mechanisms of glial precursor (GP) cell migration in the optic nerve. GP cell migration in newborn rat optic nerve was monitored by the UV-thymine-dimer (TD) method. A double labeling study using NG2 and TD revealed that many of these in vivo migrating cells were NG2 positive, while some of them with large TD-positive nuclei were NG2 negative. An in vitro cell migration study using optic nerve with chiasma and/or eyeball tissue revealed that the GP cells migrated under the guidance of repulsive cues secreted from the optic chiasma. We detected the expression of netrin 1 and Sema3a in the optic chiasma, and that of Unc5h1 and neuropilin 1 in the optic nerve. Co-culture experiments of the optic nerve with cell clusters expressing guidance cues revealed that the migrating GP cells in the optic nerve were heterogeneous. Netrin 1 repelled a subtype of NG2-positive and PLP-positive GP cells with small nuclei. Sema3a repelled a subtype of GP cells with large nuclei.     

Involvement of cell surface HSP90 in cell migration reveals a novel role in the developing nervous system

Heat shock protein HSP90 plays important roles in cellular regulation, primarily as a chaperone for a number of key intracellular proteins. We report here that the two HSP90 isoforms, alpha and beta, also localize on the surface of cells in the nervous system and are involved in their migration. A 94-kDa surface antigen, the 4C5 antigen, which was previously shown to be involved in migration processes during development of the nervous system, is shown to be identical to HSP90alpha using mass spectrometry analysis. This identity is further confirmed by immunoprecipitation experiments and by induction of 4C5 antigen expression in heat shock-treated embryonic rat brain cultures. Moreover, immunocytochemistry on live cerebellar rat cells reveals cell surface localization of both HSP90alpha and -beta. Cell migration from cerebellar and sciatic nerve explants is inhibited by anti-HSP90alpha and anti-HSP90beta antibodies, similarly to the inhibition observed with monoclonal antibody 4C5. Moreover, immunostaining with rhodamine-phalloidin of migrating Schwann cells cultured in the presence of antibodies against both alpha and beta isoforms of HSP90 reveals that HSP90 activity is associated with actin cytoskeletal organization, necessary for lamellipodia formation.