Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration.

RNA-binding proteins are emerging as key regulators of transitions in cell morphology. The RNA-binding motif protein 3 (RBM3) is a cold-inducible RNA-binding protein with broadly relevant roles in cellular protection, and putative functions in cancer and development. Several findings suggest that RBM3 has morphoregulatory functions germane to its roles in these contexts. For example, RBM3 helps maintain the morphological integrity of cell protrusions during cell stress and disease. Moreover, it is highly expressed in migrating neurons of the developing brain and in cancer invadopodia, suggesting roles in migration. We here show that RBM3 regulates cell polarity, spreading and migration. RBM3 was present in spreading initiation centers, filopodia and blebs that formed during cell spreading in cell lines and primary myoblasts. Reducing RBM3 triggered exaggerated spreading, increased RhoA expression, and a loss of polarity that was rescued by Rho kinase inhibition and overexpression of CRMP2. High RBM3 expression enhanced the motility of cells migrating by a mesenchymal mode involving extension of long protrusions, whereas RBM3 knockdown slowed migration, greatly reducing the ability of cells to extend protrusions and impairing multiple processes that require directional migration. These data establish novel functions of RBM3 of potential significance to tissue repair, metastasis and development.

The plasminogen receptor, Plg-RKT, is essential for mammary lobuloalveolar development and lactation.

Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. SUMMARY: Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

The upstream ectoderm enhancer in Pax6 has an important role in lens induction.

The Pax6 gene has a central role in development of the eye. We show, through targeted deletion in the mouse, that an ectoderm enhancer in the Pax6 gene is required for normal lens formation. Ectoderm enhancer-deficient embryos exhibit distinctive defects at every stage of lens development. These include a thinner lens placode, reduced placodal cell proliferation, and a small lens pit and lens vesicle. In addition, the lens vesicle fails to separate from the surface ectoderm and the maturing lens is smaller and shows a delay in fiber cell differentiation. Interestingly, deletion of the ectoderm enhancer does not eliminate Pax6 production in the lens placode but results in a diminished level that, in central sections, is apparent primarily on the nasal side. This argues that Pax6 expression in the lens placode is controlled by the ectoderm enhancer and at least one other transcriptional control element. It also suggests that Pax6 enhancers active in the lens placode drive expression in distinct subdomains, an assertion that is supported by the expression pattern of a lacZ reporter transgene driven by the ectoderm enhancer. Interestingly, deletion of the ectoderm enhancer causes loss of expression of Foxe3, a transcription factor gene mutated in the dysgenetic lens mouse. When combined, these data and previously published work allow us to assemble a more complete genetic pathway describing lens induction. This pathway features (1) a pre-placodal phase of Pax6 expression that is required for the activity of multiple, downstream Pax6 enhancers; (2) a later, placodal phase of Pax6 expression regulated by multiple enhancers; and (3) the Foxe3 gene in a downstream position. This pathway forms a basis for future analysis of lens induction mechanism.

Fgf receptor signaling plays a role in lens induction.

We describe experiments showing that fibroblast growth factor receptor (Fgfr) signaling plays a role in lens induction. Three distinct experimental strategies were used: (1) using small-molecule inhibitors of Fgfr kinase activity, we showed that both the transcription level and protein expression of Pax6, a transcription factor critical for lens development, was diminished in the presumptive lens ectoderm; (2) transgenic mice (designated Tfr7) that expressed a dominant-negative Fgf receptor exclusively in the presumptive lens ectoderm showed defects in formation of the lens placode at E9.5 but in addition, showed reduced levels of expression for Pax6, Sox2 and Foxe3, all markers of lens induction; (3) by performing crosses between Tfr7 transgenic and Bmp7-null mice, we showed that there is a genetic interaction between Fgfr and Bmp7 signaling at the induction phases of lens development. This manifested as exacerbated lens development defects and lower levels of Pax6 and Foxe3 expression in Tfr7/Tfr7, Bmp7(+/-) mice when compared with Tfr7/Tfr7 mice alone. As Bmp7 is an established lens induction signal, this provides further evidence that Fgfr activity is important for lens induction. This analysis establishes a role for Fgfr signaling in lens induction and defines a genetic pathway in which Fgfr and Bmp7 signaling converge on Pax6 expression in the lens placode with the Foxe3 and Sox2 genes lying downstream.

Misexpression of IGF-I in the mouse lens expands the transitional zone and perturbs lens polarization.

Insulin-like growth factor-I (IGF-I) has been implicated as a regulator of lens development. Experiments performed in the chick have indicated that IGF-I can stimulate lens fiber cell differentiation and may be involved in controlling lens polarization. To assess IGF-I activity on mammalian lens cells in vivo, we generated transgenic mice in which this factor was overexpressed from the alphaA-crystallin promoter. Interestingly, we observed no premature differentiation of lens epithelial cells. The pattern of lens polarization was perturbed, with an apparent expansion of the epithelial compartment towards the posterior lens pole. The distribution of immunoreactivity for MIP26 and p57(KIP2) and a modified pattern of proliferation suggested that this morphological change was best described as an expansion of the germinative and transitional zones. The expression of IGF-I signaling components in the normal transitional zone and expansion of the transitional zone in the transgenic lens both suggest that endogenous IGF-I may provide a spatial cue that helps to control the normal location of this domain.

Endogenous and ectopic gland induction by FGF-10.

FGF-10, a member of the fibroblast growth factor family, is expressed in mesodermally derived cell populations during embryogenesis. During normal ocular development, FGF-10 is expressed in the perioptic mesenchyme adjacent to the Harderian and lacrimal gland primordia. In this report, we provide evidence that FGF-10 is both necessary and sufficient to initiate glandular morphogenesis. Lens-specific expression of FGF-10 was sufficient to induce ectopic ocular glands within the cornea. In addition, lacrimal and Harderian glands were not seen in FGF-10 null fetuses. Based on these results we propose that FGF-10 is an inductive signal that initiates ocular gland morphogenesis.

FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.

We investigated the mechanism of tissue induction and specification using the lacrimal gland as a model system. This structure begins its morphogenesis as a bud-like outgrowth of the conjunctival epithelium and ultimately forms a branched structure with secretory function. Using a reporter transgene as a specific marker for gland epithelium, we show that the transcription factor Pax6 is required for normal development of the gland and is probably an important competence factor. In investigating the cell-cell signaling required, we show that fibroblast growth factor (FGF) 10 is sufficient to stimulate ectopic lacrimal bud formation in ocular explants. Expression of FGF10 in the mesenchyme adjacent to the presumptive lacrimal bud and absence of lacrimal gland development in FGF10-null mice strongly suggest that it is an endogenous inducer. This was supported by the observation that inhibition of signaling by a receptor for FGF10 (receptor 2 IIIb) suppressed development of the endogenous lacrimal bud. In explants of mesenchyme-free gland epithelium, FGF10 stimulated growth but not branching morphogenesis. This suggested that its role in induction is to stimulate proliferation and, in turn, that FGF10 combines with other factors to provide the instructive signals required for lacrimal gland development.

The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4.

The development of the feather buds during avian embryogenesis is a classic example of a spacing pattern. The regular arrangement of feather buds is achieved by a process of lateral inhibition whereby one developing feather bud prevents the formation of similar buds in the immediate vicinity. Lateral inhibition during feather formation implicates a role of long range signalling during this process. Recent work has shown that BMPs are able to enforce lateral inhibition during feather bud formation. However these results do not explain how the feather bud escapes the inhibition itself. We show that this could be achieved by the expression of the BMP antagonist, Follistatin. Furthermore we show that local application of Follistatin leads to the development of ectopic feather buds. We suggest that Follistatin locally antagonises the action of the BMPs and so permits the cellular changes associated with feather placode formation. We also provide evidence for the role of short range signalling during feather formation. We have correlated changes in cellular morphology in feather placodes with the expression of the gene Eph-A4 which encodes a receptor tyrosine kinase that requires direct cell-cell contact for activation. We show that the expression of this gene precedes cellular reorganisation required for feather bud formation.

Macrophages kill capillary cells in G1 phase of the cell cycle during programmed vascular regression.

Programmed capillary regression occurs during normal development of the eye and serves as a useful model for assessing the forces that drive vascular involution. Using a combination of S-phase labeling and liposome-mediated macrophage elimination, we show that during regression, macrophages induce apoptosis of both pericytes and endothelial cells in a cell cycle stage-dependent manner. Target cells are signaled to die by macrophages approximately 15 hours after S-phase labeling and this corresponds to a point in mid-G1 phase of the cell cycle. The tight correlation between the restriction point of the cell cycle and the point where the macrophage death signal is received suggests that the mitogen, matrix and cytoskeletal signals essential for cell-cycle progression may be inhibited by macrophages as a means of inducing cell death. Furthermore, these experiments show that cells from two distinct lineages are induced to die as a consequence of macrophage action, and this provides evidence that macrophage-induced cell death may be a general phenomenon during development and homeostasis.

Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach.

Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.

Gap junction signalling mediated through connexin-43 is required for chick limb development.

During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43.

Basic FGF increases communication between cells of the developing neocortex.

We have found that basic fibroblast growth factor (bFGF), applied to cortical progenitor cells in vitro, produces an increase in the expression of the gap junction protein connexin (Cx) 43 and in the mRNA encoding Cx 43. This effect was evident in both proliferating and nonproliferating cells. The elevated levels of mRNA suggest that bFGF is likely to exert its effect by upregulating the rate of transcription of the Cx 43 gene. We have further shown that the increase in Cx 43 expression is mediated through the receptor tyrosine kinase pathway and is associated with enhanced intercellular dye-coupling mediated by gap junctions. These results suggest that gap junction channels provide a direct conduit for mitogens released in response to bFGF to effectively regulate proliferation during corticogenesis.

Fibroblast growth factor 4 directs gap junction expression in the mesenchyme of the vertebrate limb Bud.

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.