Interactive Fly, Drosophila

Novel forms of EGF

A novel murine gene encodes a secreted molecule containing a variant epidermal growth factor-like (EGF) motif. This gene is named Cryptic, based on its predicted protein sequence similarity with Cripto, which encodes an EGF-related growth factor. Based on their strong sequence similarities, it is proposed that Cryptic, Cripto, and the Xenopus FRL-1 gene define a new family of growth factor-like molecules, which has been termed the 'CFC' (Cripto, Frl-1, and Cryptic) family. Although Cryptic is not a structural homolog of Spitz, information on Cryptic is provided because its function is similar to that of Spitz. Analysis of Cryptic expression by in situ hybridization shows that it is expressed during gastrulation in two spatial domains that correspond to the axial and lateral mesoderm. In the first domain of expression, Cryptic expression is progressively localized to the anterior primitive streak, the head process, and the node and notochordal plate. In the second domain, Cryptic expression is initially concentrated in the lateral region of the egg cylinder, and is later found circumferentially in the intermediate and lateral plate mesoderm. Furthermore, Cryptic expression can also be detected at the early head-fold stage in the midline neuroectoderm, and consequently is an early marker for the prospective floor plate of the neural tube. Expression of Cryptic ceases at the end of gastrulation, and has not been observed in later embryonic stages or in adult tissues. Thus, Cryptic encodes a putative signaling molecule whose expression suggests potential roles in mesoderm and/or neural patterning during gastrulation (Shen, 1997).

Development of the vertebrate inner ear begins during gastrulation with induction of the otic placode. Several embryonic tissues, including cephalic mesendoderm, notochord, and hindbrain, have been implicated as potential sources of otic-inducing signals. However, the relative contributions of these tissues have not been determined, nor have any genes affecting placode induction been identified. To address these issues, otic placode induction was analyzed in zebrafish mutants that are deficient in prospective otic-inducing tissues. Otic development was monitored by examining mutant embryos for morphological changes and, in some cases, by visualizing expression patterns of dlx-3 or pax-2.1 in preotic cells several hours before otic placode formation. In cyclops (cyc-) mutants, which develop with a partial deficiency of prechordal mesendoderm, otic induction is delayed by up to 1 h. The cyclops locus encodes the nodal-related protein Ndr2, a member of the transforming growth factor type beta superfamily of factors. In one-eyed pinhead (oep-) mutants, which are more completely deficient in prechordal mesendoderm, otic induction is delayed by 1.5 h, and morphology of the otic vesicles is abnormal. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Expression of marker genes in other regions of the neural plate is normal, suggesting that ablation of prechordal mesendoderm selectively inhibits otic induction. In contrast, the timing and morphology of otic development is not affected by mutations in no tail (ntl) or floating head (flh), either of which prevent notochord differentiation. Similarly, a mutation in valentino (val), which blocks early differentiation of rhombomeres 5 and 6 in the hindbrain, does not delay otic induction, although subsequent patterning of the otic vesicle is impaired. To test whether inductive signals from one tissue can compensate for loss of another, double or triple mutants were generated with various combinations of the above mutations. In none of the multiple mutants do the flh or val mutations exacerbate delays in placode induction, although val does contribute additively to defects in subsequent patterning of the otic vesicle. In contrast, mutants homozygous for both oep and ntl, which interact synergistically to disrupt differentiation of cephalic and axial mesendoderm, show a delay in otic development of about 3 h. These data suggest that cephalic mesendoderm, including prechordal mesendoderm and anterior paraxial mesendoderm, provides the first otic-inducing signals during gastrulation, whereas chordamesoderm plays no discernible role in this process. Because val- mutants are deficient for only a portion of the hindbrain, a role for that tissue in otic placode induction cannot be ruled out. However, if the hindbrain does provide otic-inducing signals, they apparently differ quantitatively or qualitatively from the signals required for vesicle patterning, since val disrupts only the latter (Mendonsa, 1999).

The anterior-posterior axis of the mouse embryo is established by two distinct organizing centers in the anterior visceral endoderm and the distal primitive streak. These organizers induce and pattern the head and trunk respectively, and have been proposed to be localized through coordinate cell movements that rotate a pre-existing proximal-distal axis. Correct localization of both head- and trunk-organizing centers requires Cripto, a putative signaling molecule that is a member of the EGF-CFC gene family. Before gastrulation, Cripto is asymmetrically expressed in a proximal-distal gradient in the epiblast, and subsequently is expressed in the primitive streak and newly formed embryonic mesoderm. A Cripto null mutation generated by targeted gene disruption results in homozygous Cripto-/- embryos that mostly consist of anterior neuroectoderm and lack posterior structures, thus resembling a head without a trunk. Notably, markers of the head organizer are located at the distal end of the embryo, whereas markers of the primitive streak are absent or localized to the proximal side. These results indicate that Cripto signaling is essential for the conversion of a proximal-distal asymmetry into an orthogonal anterior-posterior axis (Ding, 1998).

Cripto-1(Cr1) protein, encoded by the teratocarcinoma-derived growth factor1 tdgf1 gene, is a secreted growth factor that is expressed early in embryonic development and is re-expressed in some tumors of the breast and colon. The protein contains a modified EGF-like domain. In Cr1, two of the three bicysteine loops in the EGF-domain are truncated, and it fails to bind to the EGF receptor or other type I receptor tyrosine kinases in the ErbB family. Rather, Cr1 is a member of a different family and interacts with an as yet unidentified receptor and activates intracellular components through the ras/raf/MAPK pathway. During embryonic development, Cr1 is expressed in inner cell mass cells and the primitive streak, and later is restricted to the developing heart. To investigate the role of Cr1 during mouse development, mice were generated that contain a null mutation of both Cr1 genes, derived from homologous recombination in embryonic stem cells. No homozygous Cr1-/- mice are born, indicating that Cr1 is necessary for embryonic development. Embryos initiate gastrulation and up to day E7.5 some embryos produce mesoderm. Increasingly aberrant morphogenesis gives rise to disordered neuroepithelium that fails to produce either a recognizable neural tube or head-fold. Although some biochemical markers of differentiating ectoderm, mesoderm and endoderm are expressed, all the cardiac-specific markers are absent from day E8.7 embryos (alphaMHC, betaMHC, MLC2A, MLC2V and ANF), whereas they are expressed in wild-type embryos. The yolk sac and placental tissues continue development in the absence of the embryo until day E9.5 but lack large yolk sac blood vessels. Chimeric mice were constructed by microinjection of double targeted Cr1(-/-) embryonic stem cells into normal C57BL/6 blastocysts. The Cr1 produced by the normal C57BL/6 cells fully rescues the phenotype of Cr1(-/-) cells, indicating that Cr1 protein acted in a paracrine manner. Cells derived from the embryo proliferate and migrate poorly and have different adhesion properties compared to wild type. Therefore, lethality in the absence of Cr1, likely results largely from defective precardiac mesoderm that was unable to differentiate into functional cardiomyocytes (Xu, 1999).

The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation (Zhang, 1998).

The molecular events of cardiac lineage specification and differentiation are largely unknown. The involvement of a growth factor with an EGF-like domain, Cripto-1 (Cr-1), is described in cardiac differentiation. During embryonic development, Cr-1 is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-1, Cr-1-negative embryonic stem (ES) cell lines were generated by homologous recombination. The resulting double "knockout" ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing the Cr-1 gene back into the Cr(-/-) cells. Furthermore, the lack of functional Cr-1 is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during differentiation. Differentiation into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-1 is essential for contractile cardiomyocyte formation in vitro (Xu, 1998).

Evidence from in vivo lineage analysis and in vitro cell culture experiments has revealed that the rodent telencephalic germinal zone (GZ) at embryonic day 14 is composed of a heterogeneous population of multipotential and committed precuresor cells. Neural stem cells, exhibiting the fundamental stem cell properties of multipotentiality and self-renewal, have been shown to make up a relatively small percentage of this heterogeneous E14 GZ population. In the adult forebrain, neural stem cells are present as a relatively quiescent subpopulation in the subependyma, a remnant of the embryonic GZ, and this population persists into senescence (Tropepe, 1999 and references).

Using an in vitro neurosphere assay for neural stem cell proliferation, it has been demonstrated 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. Limiting dilution analyses reveal that the minimal frequency of neural stem cells in the E14.5 GZ is 0.6% in the presence of EGF; 1.3% in the presence of FGF2, and 2% in the combination of EGF and FGF2. This indicates that stem cells are differentially recruited on the basis of which growth factor is present. Single primary neurospheres generated in culture were dissociated and replated in the identical growth factor concentration conditions to determine the self-renewal capacity of the neurosphere forming cells. When either FGF2 or EGF is provided to primary cultures the number of secondary neurospheres is dependent on the level of growth factor in the primary culture, varying from 6.6 or 3.0 for low levels (0.6 ng/ml) of FGF2 or EGF2 respectively, to 73.9 and 47.4 for high levels (80 ng/ml) of FGF2 or EGF2 respectively (Tropepe, 1999).

Double-labeling reveals that virtually all cells derived from neural stem cell clones (isolated in either EGF or FGF2) express both EGFR and FGFR. However, there are varying levels of expression of these receptors in the individual cells and there are a few single cells within each neurosphere which express mostly EGFR or mostly FGFR. The presence of these few cells dominated by the expression of one receptor suggests the possibility that there may be maintenance of a small number of differentially sensitive neural stem cells that remain undifferentiated and capable of self-renewal. Given that cells in the neurospheres generated in either EGF or FGF2 express both growth factor receptors and that neurospheres can be passaged in either EGF or FGF2, regardless of initial growth factor conditions, it is concluded that the stem cell initially responsive to either EGF or FGF2 can subsequently generate more stem cells that are responsive to both mitogens. By separately blocking EGF and FGF2 signaling, it has been shown that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation; 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 FGF2-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 FGF2-responsive neural stem cells are present in the embryonic telencephalic germinal zone (Tropepe, 1999).

EGF and carcinoma

The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena^INV. This study shows that Mena and Mena^INV promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena^INV potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena^INV is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena^INV could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels (Philippar, 2008).

Table of contents

spitz: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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