Igf1r Signaling Is Indispensable for Preimplantation Development and Is Activated via a Novel Function of E-cadherin

Insulin-like growth factor I receptor (Igf1r) signaling controls proliferation, differentiation, growth, and cell survival in many tissues; and its deregulated activity is involved in tumorigenesis. Although important during fetal growth and postnatal life, a function for the Igf pathway during preimplantation development has not been described. We show that abrogating Igf1r signaling with specific inhibitors blocks trophectoderm formation and compromises embryo survival during murine blastocyst formation. In normal embryos total Igf1r is present throughout the membrane, whereas the activated form is found exclusively at cell contact sites, colocalizing with E-cadherin. Using genetic domain switching, we show a requirement for E-cadherin to maintain proper activation of Igf1r. Embryos expressing exclusively a cadherin chimera with N-cadherin extracellular and E-cadherin intracellular domains (NcEc) fail to form a trophectoderm and cells die by apoptosis. In contrast, homozygous mutant embryos expressing a reverse-structured chimera (EcNc) show trophectoderm survival and blastocoel cavitation, indicating a crucial and non-substitutable role of the E-cadherin ectodomain for these processes. Strikingly, blastocyst formation can be rescued in homozygous NcEc embryos by restoring Igf1r signaling, which enhances cell survival. Hence, perturbation of E-cadherin extracellular integrity, independent of its cell-adhesion function, blocked Igf1r signaling and induced cell death in the trophectoderm. Our results reveal an important and yet undiscovered function of Igf1r during preimplantation development mediated by a unique physical interaction between Igf1r and E-cadherin indispensable for proper receptor activation and anti-apoptotic signaling. We provide novel insights into how ligand-dependent Igf1r activity is additionally gated to sense developmental potential in utero and into a bifunctional role of adhesion molecules in contact formation and signaling

Hoxa2 downregulates Six2 in the neural crest-derived mesenchyme

The Hoxa2 transcription factor acts during development of the second branchial arch. As for most of the developmental processes controlled by Hox proteins, the mechanism by which Hoxa2 regulates the morphology of second branchial arch derivatives is unclear. We show that Six2, another transcription factor, is genetically downstream of Hoxa2. High levels of Six2 are observed in the Hoxa2 loss-of-function mutant. By using a transgenic approach to overexpress Six2 in the embryonic area controlled by Hoxa2, we observed a phenotype that is reminiscent of the Hoxa2 mutant phenotype. Furthermore, we demonstrate that Hoxa2 regulation of Six2 is confined to a 0.9 kb fragment of the Six2 promoter and that Hoxa2 binds to this promoter region. These results strongly suggest that Six2 is a direct target of Hoxa2

Increased apoptosis is detected in the outer cells of homozygous NcEc embryos and is blocked by iloprost treatment, rescuing blastocyst formation.

<p>(A) Labeling of cleaved and activated Caspase 3 (red) shows only one apoptotic cell in the ICM of control and EcNc ki/ki embryos, whereas the outer cells of NcEc homozygous mutant embryos display a substantial increase in Caspase 3-positive cells. Cleavage of Caspase 3 was not detected upon iloprost treatment (1 µM) (B) Cell blebbing and vacuolization is detected in TROMA-1 positive outer cells of NcEc ki/ki embryos demonstrating induction of PCD in cells destined to become TE (arrow). (C) Treatment of NcEc ki/ki embryos with 1 µM iloprost at the precompacted or compacted morula stage (E2.25–E3.0) observed by time-lapse microscopy rescues the TE formation defect and hatching is initiated (arrow). (D) Treatment with iloprost at a later time-point (E3.5) did not rescue the phenotype. (E) One representative frame of a time-lapse recording of iloprost-treated EcNc ki/ki embryos. The formation of the blastocyst is marginally improved. (F) Treatment of N-cad ki/ki embryos with iloprost resulted in a rescue similar to that for NcEc homozygous mutants. (G) <i>E-cad</i>-null embryos were not rescued upon treatment with iloprost. (H) Percentage of iloprost-treated embryos that formed a proper blastocyst in time-lapse experiments and during <i>in vitro</i> culture for wt (n = 65), NcEc homozygous mutants (n = 33) and <i>E-cad^−/−</i> (n = 15) in >5 independent experiments. (I) With the exception of <i>E-cad^−/−</i> embryos, proper specification of the rescued TE of iloprost-treated homozygous mutants was confirmed by cytokeratin 8 expression, which is restricted to TE cells (TROMA-1, red). Scale bar, 25 µm.</p

Generation of EcNc and NcEc cadherin proteins expressed in the <i>E-cad</i> (<i>Cdh1</i>) locus.

<p>(A) Schematic representation of EcNc and NcEc protein structure in their cadherin-catenin complex (adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002609#pgen.1002609-Stemmler1" target="_blank">[8]</a>). (B) Gene targeting strategy and the resultant knock-in allele, representatively shown for NcEc; TK, <i>HSV</i>::<i>tk</i> negative selection cassette; HA, haemagglutinin tag; pA, SV40 polyadenylation signal; Neo, neomycin resistance cassette, flanked by loxP sites (black triangles). (C) Southern blot analysis of obtained ES cell clones using the 5′ probe. (D) Expression of the knock-in alleles in ES cells after removal of the neomycin cassette reveals equal expression of both HA-tagged proteins. (E) Immunofluorescence labeling of EcNc (upper) and NcEc (lower panel) showing complete overlap of anti-HA and anti-E-cad staining in heterozygous E3.5 blastocysts in confocal optical sections. Scale bar, 25 µm.</p

A model of the molecular pathways that are involved in TE survival but are blocked in homozygous NcEc and N-cad ki/ki mutants.

<p>In the presence of full-length E-cad or of the E-cad extracellular domain in EcNc embryos interaction of cadherins with Igf1r is occurring. This enables proper activation of Igf1r upon Igf1 signaling (phosphorylation), which supplies survival signals and blocks PCD (left panel). In the absence of E-cad cell adhesion is maintained in presence of NcEc or N-cad, but both proteins are incapable of interacting with Igf1r. As a consequence of the uncoupled interaction, Igf1r is not fully activated, prosurvival signals are lacking and apoptotic pathways reach the threshold levels for PCD induction (right panel). In the presence of cadherin-mediated adhesion (in homozygous NcEc and N-cad ki/ki, but not in <i>E-cad</i>-null embryos), apoptotic pathways can be blocked only by external cues (red boxes), which inhibit PCD at different levels and thereby rescue TE formation. According to this model E-cad is required for providing survival cues via the extracellular domain in addition to its role in cell adhesion.</p

Markers for lineage specification, cell polarity, and vectorial fluid flow are correctly expressed and localized in NcEc homozygous mutants.

<p>(A) Proper segregation of outer TE cells is shown by anti-Cdx2 labeling in wt, EcNc and NcEc homozygous mutants at E3.5. (B) Wildtype, EcNc and NcEc homozygous mutant embryos labeled for the ICM cell marker Sox2 in inner cells, showing ICM cell specification and its localization inside NcEc homozygous embryos. (C) Ezrin and Na⁺/K⁺-ATPase staining to verify apical-basal polarity with same distribution in wt, EcNc and NcEc homozygous mutants at the apical and basolateral membrane. Correct sealing of the TE layer is indicated by the presence and proper localization of the tight junctional component ZO-1 at apical sites of lateral TE membranes in wt, EcNc and NcEc homozygous embryos. Key components required for vectorial fluid transport are shown by the presence of Na⁺/K⁺-ATPase and Aqp3. In embryos of all genotypes, this expression is detected in the outer cells, without any obvious differences in expression between the embryos. Hence, the first lineage segregation is specified correctly, and proteins that are essential for the TE formation process and its function are present and properly localized in NcEc homozygous mutants. Nuclei were labeled with DAPI (blue). Scale bar, 25 µm.</p