Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos.

Homozygosity for the Egfr(tm1Mag) null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta were quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfr(tm1Mag) homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation.

Comparative genomic sequence analysis and isolation of human and mouse alternative EGFR transcripts encoding truncated receptor isoforms.

This study presents the annotated genomic sequence and exon-intron organization of the human and mouse epidermal growth factor receptor (EGFR) genes located on chromosomes 7p11.2 and 11, respectively. We report that the EGFR gene spans nearly 200 kb and that the full-length 170-kDa EGFR is encoded by 28 exons. In addition, we have identified two human and two mouse alternative EGFR transcripts of 2.4-3.0 kb using both computational and experimental methods. The human 3.0-kb and mouse 2.8-kb EGFR mRNAs are predominantly expressed in placenta and liver, respectively, and both transcripts encode 110-kDa truncated receptor isoforms containing only the extracellular ligand-binding domain. We also have demonstrated that the aberrant 2.8-kb EGFR transcript produced by the human A431 carcinoma cell line is generated by splicing to a recombinant 3'-terminal exon located in EGFR intron 16, which apparently was formed as a result of a chromosomal translocation. Finally, we have shown that the human, mouse, rat, and chicken 1.8- to 3.0-kb alternative EGFR transcripts are generated by distinct splicing mechanisms and that each of these mRNAs contains unique 3' sequences that are not evolutionarily conserved. The presence of truncated receptor isoforms in diverse species suggests that these proteins may have important functional roles in regulating EGFR activity.

Distinct signal/response mechanisms regulate pax1 and QmyoD activation in sclerotomal and myotomal lineages of quail somites.

Pax1 and QmyoD are early sclerotome and myotome-specific genes that are activated in epithelial somites of quail embryos in response to axial notochord/neural tube signals. In situ hybridization experiments reveal that the developmental kinetics of activation of pax1 and QmyoD differ greatly, suggesting that myotome and sclerotome specification are controlled by distinct developmental mechanisms. pax1 activation always occurs in somite IV throughout development, indicating that pax1 regulation is tightly coordinated with early steps in somite maturation. In contrast, QmyoD is delayed and does not occur until embryos have 12-14 somites. At this time, QmyoD is the first of the myogenic regulatory factor (MRF) genes to be activated in preexisting somites in a rapid, anterior to posterior progression until the 22 somite stage, after which time QmyoD is activated in somite I immediately following somite formation. Experiments involving transplantation of newly formed somites to ectopic sites along the anterior to posterior embryonic axis were performed to distinguish the contributions of axial signals and somite response pathways to the developmental regulation of pax1 and QmyoD. These studies show that pax1 activation is regulated by somite formation and maturation, not by the availability of axial signals, which are expressed prior to somite formation. In contrast, the delayed activation of QmyoD is controlled by developmental regulation of the production of axial signals as well as by the competence of somites to respond to these signals. These somite transplantation studies, therefore, provide a basis for understanding the different developmental kinetics of activation of pax1 and QmyoD during sclerotome and myotome specification, and suggest specific molecular models for the developmental regulation of myotome and sclerotome formation in somites through distinct signal/response pathways.

Notochord signals control the transcriptional cascade of myogenic bHLH genes in somites of quail embryos.

Microsurgical, tissue grafting and in situ hybridization techniques have been used to investigate the role of the neural tube and notochord in the control of the myogenic bHLH genes, QmyoD, Qmyf5, Qmyogenin and the cardiac alpha-actin gene, during somite formation in stage 12 quail embryos. Our results reveal that signals from the axial neural tube/notochord complex control both the activation and the maintenance of expression of QmyoD and Qmyf5 in myotomal progenitor cells during the period immediately following somite formation and prior to myotome differentiation. QmyoD and Qmyf5 expression becomes independent of axial signals during myotome differentiation when somites activate expression of Qmyogenin and alpha-actin. Ablation studies reveal that the notochord controls QmyoD activation and the initiation of the transcriptional cascade of myogenic bHLH genes as epithelial somites condense from segmental plate mesoderm. The dorsal medial neural tube then contributes to the maintenance of myogenic bHLH gene expression in newly formed somites. Notochord grafts can activate ectopic QmyoD expression during somite formation, establishing that the notochord is a necessary and sufficient source of diffusible signals to initiate QmyoD expression. Myogenic bHLH gene expression is localized to dorsal medial cells of the somite by inhibitory signals produced by the lateral plate and ventral neural tube. Signaling models for the activation and maintenance of myogenic gene expression and the determination of myotomal muscle in somites are discussed.