Search

Sprouty proteins function as negative regulators of the receptor tyrosine kinase (RTK)-mediated Ras/Raf/MAPK pathway in many varied physiological and developmental processes, inhibiting growth factor-induced cellular proliferation, migration and differentiation. Like other negative regulators, Sprouty proteins are expressed in various organs during development, including the eye; ubiquitously expressed in the optic vesicle, lens pit, optic cup and lens vesicle. Given the synexpression of different antagonists (e.g, Sprouty, Sef, Spred) in the developing lens, to gain a better understanding of their specific role, in particular, their ability to regulate ocular growth factor signaling in lens cells, we characterized transgenic mice overexpressing Sprouty1 or Sprouty2 in the eye. Overexpression of Sprouty in the lens resulted in reduced lens and eye size during ocular morphogenesis, influenced by changes to the lens epithelium, aberrant fiber cell differentiation and compromised de novo maintenance of the lens capsule. Here we demonstrate an important inhibitory role for Sprouty in the regulation of lens cell proliferation and fiber differentiation in situ, potentially through its ability to modulate FGF- (and even EGF-) mediated MAPK/ERK1/2 signaling in lens cells. Whilst growth factor regulation of lens cell proliferation and fiber differentiation are required for orchestrating lens morphogenesis and growth, in turn, antagonists such as Sprouty are just as important for regulating the intracellular signaling pathways driving lens cellular processes.

The development of stem cell daughters into the differentiated state normally requires a cascade of proliferation and differentiation steps that are typically regulated by external signals. The germline cells of most animals, in specific, are associated with somatic support cells and depend on them for normal development. In the male gonad of Drosophila melanogaster, germline cells are completely enclosed by cytoplasmic extensions of somatic cyst cells, and these cysts form a functional unit. Signaling from the germline to the cyst cells via the Epidermal Growth Factor Receptor (EGFR) is required for germline enclosure and has been proposed to provide a temporal signature promoting early steps of differentiation. A temperature-sensitive allele of the EGFR ligand Spitz (Spi) provides a powerful tool for probing the function of the EGRF pathway in this context and for identifying other pathways regulating cyst differentiation via genetic interaction studies. Using this tool, we show that signaling via the Ecdysone Receptor (EcR), a known regulator of developmental timing during larval and pupal development, opposes EGF signaling in testes. In spi mutant animals, reducing either Ecdysone synthesis or the expression of Ecdysone signal transducers or targets in the cyst cells resulted in a rescue of cyst formation and cyst differentiation. Despite of this striking effect in the spi mutant background and the expression of EcR signaling components within the cyst cells, activity of the EcR pathway appears to be dispensable in a wildtype background. We propose that EcR signaling modulates the effects of EGFR signaling by promoting an undifferentiated state in early stage cyst cells.

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.

The atonal (ato) proneural gene specifies different numbers of sensory organ precursor (SOP) cells within distinct regions of the Drosophila embryo in an epidermal growth factor-dependent manner through the activation of the rhomboid (rho) protease. How ato activates rho, and why it does so in only a limited number of sensory cells remains unclear. We previously identified a rho enhancer (RhoBAD) that is active within a subset of abdominal SOP cells to induce larval oenocytes and showed that RhoBAD is regulated by an Abdominal-A (Abd-A) Hox complex and the Senseless (Sens) transcription factor. Here, we show that ato is also required for proper RhoBAD activity and oenocyte formation. Transgenic reporter assays reveal RhoBAD contains two conserved regions that drive SOP gene expression: RhoD mediates low levels of expression in both thoracic and abdominal SOP cells, whereas RhoA drives strong expression within abdominal SOP cells. Ato indirectly stimulates both elements and enhances RhoA reporter activity by interfering with the ability of the Sens repressor to bind DNA. As RhoA is also directly regulated by Abd-A, we propose a model for how the Ato and Sens proneural factors are integrated with an abdominal Hox factor to regulate region-specific SOP gene expression.

The neural crest gives rise to glial cells in the peripheral nervous system. Among the peripheral glia, Schwann cells form the myelin often wrapping the peripheral axons. Compared to other crest-derived cell lineages such as neurons, the analysis of fate determination and subsequent differentiation of Schwann cells is not well advanced, partly due to the lack of early markers of this phenotype. In this study, we have identified a gene, uniquely expressed in avian embryo Schwann cell precursors, which encodes a novel secreted factor, designated Seraf (Schwann cell-specific EGF-like repeat autocrine factor). Expression of Seraf and P0 delineates the earliest phase of Schwann cell differentiation. Seraf binds to neural crest cells and Schwann cells, and affects the distribution of Schwann cells, when introduced to chicken embryos during neural crest migration. Our results suggest an autocrine function of Seraf and provide a significant step to understand the developmental processes of Schwann cell lineage.

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-a converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-a (TNF-a). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a “sheddase” during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-a or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development. © 2001 Academic Press

The macromolecules mediating species-specific events during fertilization and early development and their molecular evolution are only beginning to be understood. We screened sea urchin ovary mRNA for species-specific gene products using representational differential analysis to identify unique transcripts in Strongylocentrotus franciscanus that are absent or divergent from a closely related species, S. purpuratus. One of the transcripts identified by this screening process is SfEGF-II, which contains four EGF repeats. SfEGF-II is orthologous to the previously reported genes S. purpuratus SpEGF-II and Anthocidaris crassispina AcEGF-II, encoding exogastrulation-inducing peptides (EGIP). EGF peptides derived from EGIP induce exogastrulation, a classical developmental defect, when added to embryos prior to gastrulation. The first three EGF repeats (EGF1–3) share 50 to 60% identity among the three species, but the fourth repeat (EGF4) is more divergent, displaying only 30% identity. Analysis of the sequence divergence indicates that the EGF-II genes display a relatively high nonsynonymous-to-synonymous ratio, a significant excess of radical compared to conservative amino acid substitutions, and a lack of polymorphism within SfEGF-II, indicating that these genes have been subjected to positive Darwinian selection. Recombinant EGF3 from S. franciscanus induces exogastrulation in both S. franciscanus and S. purpuratus. In contrast, recombinant EGF4 from both S. franciscanus and S. purpuratus induces exogastrula in a species-specific manner. In hybrid embryos, both species of EGF4 induce exogastrulation, suggesting that the receptor for this EGF molecule is expressed from both parental genomes during development. Both EGF3 and EGF4 induce the phosphorylation of membrane proteins of the blastula stage embryos, but EGF4 stimulates phosphorylation of proteins only in membranes prepared from homologous embryos, suggesting that it utilizes a unique pathway involving a species-specific receptor for EGF4. Thus, species-specific events of gastrulation and early development may be controlled by these rapidly diverging EGF molecules, through a novel species-specific signal transduction pathway.

Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed.

How is cell fate diversity reliably achieved during development? Insect sensory organs have been a favorable model system for investigating this question for over 100 years. They are constructed using defined cell lineages that generate a maximum of cell diversity with a minimum number of cell divisions, and display tremendous variety in their morphologies, constituent cell types, and functions. An unexpected realization of the past 5 years is that very diverse sensory organs in Drosophila are produced by astonishingly similar cell lineages, and that their diversity can be largely attributed to only a small repertoire of developmental processes. These include changes in terminal cell differentiation, cell death, cell proliferation, cell recruitment, cell–cell interactions, and asymmetric segregation of cell fate determinants during mitosis. We propose that most Drosophila sensory organs are built from an archetypal lineage, and we speculate about how this stereotyped pattern of cell divisions may have been built during evolution.

We have examined the distribution of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and the chicken EGF receptor (c-erbB), in embryonic chick limbs. Prior to limb budding, TGF-alpha is present in prospective limb-forming mesoderm and in prospective apical ectodermal ridge (AER)-forming ectoderm, but is not detected in non-limb-forming flank mesoderm or ectoderm, nor in presumptive non-AER-forming limb ectoderm, suggesting possible roles in initial limb formation and AER induction. Consistent with this possibility, TGF-alpha is present in the mesoderm of the wing buds of the amelic chick mutants limbless and wingless, which form and bud normally, but is absent from limbless and wingless ectoderm, which fails to form an AER. TGF-alpha and EGF are present in the AER of the developing limb, and TGF-alpha, EGF, and c-erbB are present in the underlying subridge mesoderm, suggesting possible roles in reciprocal AER/subridge mesoderm interactions required for limb outgrowth. We found that exogenous TGF-alpha and EGF can promote the outgrowth of limb mesoderm in the absence of the AER in vitro and can also promote the outgrowth of limbless and wingless wing bud explants. EGF is present in ventral but not dorsal limb ectoderm, suggesting a role for EGF in specification of ventral ectoderm. TGF-alpha and EGF are not detected in the differentiating cartilaginous elements or muscle primordia of the limb, suggesting that cessation of TGF-alpha and EGF expression may be required for cartilage and muscle formation. We have found that exogenous TGF-alpha and EGF inhibit chondrogenesis and myogenesis of limb mesenchyme in vitro. Together these results indicate that signaling through the EGF receptor via endogenous TGF-alpha and EGF may be important for initial limb formation, AER induction, outgrowth of limb mesoderm, and regulation of limb chondrogenic and myogenic differentiation.

The atonal (ato) proneural gene specifies a stereotypic number of sensory organ precursors (SOP) within each body segment of the Drosophila ectoderm. Surprisingly, the broad expression of Ato within the ectoderm results in only a modest increase in SOP formation, suggesting many cells are incompetent to become SOPs. Here, we show that the SOP promoting activity of Ato can be greatly enhanced by three factors: the Senseless (Sens) zinc finger protein, the Abdominal-A (Abd-A) Hox factor, and the epidermal growth factor (EGF) pathway. First, we show that expression of either Ato alone or with Sens induces twice as many SOPs in the abdomen as in the thorax, and do so at the expense of an abdomen-specific cell fate: the larval oenocytes. Second, we demonstrate that Ato stimulates abdominal SOP formation by synergizing with Abd-A to promote EGF ligand (Spitz) secretion and secondary SOP recruitment. However, we also found that Ato and Sens selectively enhance abdominal SOP development in a Spitz-independent manner, suggesting additional genetic interactions between this proneural pathway and Abd-A. Altogether, these experiments reveal that genetic interactions between EGF-signaling, Abd-A, and Sens enhance the SOP-promoting activity of Ato to stimulate region-specific neurogenesis in the Drosophila abdomen.

Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to amphiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2ʹ-O-dibutyryladenosine 3′:5′ cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.

Multipotent, self-renewing neural stem cells reside in the embryonic mouse telencephalic germinal zone. Using an in vitro neurosphere assay for neural stem cell proliferation, we demonstrate that FGF-responsive neural stem cells are present as early as E8.5 in the anterior neural plate, but EGF-responsive neural stem cells emerge later in development in a temporally and spatially specific manner. By separately blocking EGF and FGF2 signaling, we also show that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation and at relatively high cell densities separate cell nonautonomous effects can substantially enhance the mitogen-induced proliferation. At lower cell densities, neural stem cell proliferation is additive in the presence of EGF and FGF2 combined, revealing two different stem cell populations. However, both FGF-responsive and EGF-responsive neural stem cells retain their self-renewal and multilineage potential, regardless of growth factor conditions. These results support a model in which separate, lineage-related EGF- and FGF-responsive neural stem cells are present in the embryonic telencephalic germinal zone. © 1999 Academic Press