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First published online 20 October 2004
doi: 10.1242/dev.01425

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Modulation of Erbb2 signaling during development: a threshold level of Erbb2 signaling is required for development

Richard Chan^*, W. Rod Hardy, David Dankort, Michael A. Laing and William J. Muller^*,

Institute for Molecular Biology and Biotechnology, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada

View larger version (29K): [in a new window]   Fig. 1. Generation of knock-in animals expressing Erbb2 tyrosine phosphorylation mutants. The knock-in targeting vector was constructed such that exon 1 of the mouse Erbb2 gene was replaced with either (A) a rat Erbb2 cDNA or (B) a cDNA encoding a mutant Erbb2 receptor harboring the Y1028F mutation. The cDNA is followed by a PGK-Neomycin/SV40-polyA expression cassette and is targeted to the endogenous Erbb2 locus by homologous 5' and 3' flanking arms, placing it directly under the transcriptional control of the endogenous Erbb2 promoter. The targeted allele introduces an additional HindIII site that was used to distinguish between the endogenous wild-type allele (7.5 kb fragment) and the targeted allele (4.0 kb fragment) in Southern blot analyses (A, inset). (C) Schematic representation of the Erbb2 receptor depicted with the five tyrosine autophosphorylation sites in the C-terminal tail and with the corresponding amino acid number. Note that the numbering of the amino acids is based on the rat Erbb2 sequence and will be referred to herein by these designates. Also shown are the three individual tyrosine-to-phenylalanine mutations described in this report: Y1028F, Y1144F and Y1227F. (D) Alignment of the amino acid sequences surrounding tyrosine 1028 in Erbb2 and tyrosine 992 in Egfr. (E) Expression analysis of the knock-in allele. Total protein lysates prepared from E12.5 wild-type and Erbb2 cDNA knock-in embryos were used to detect Erbb2 levels. Detection of Grb2 protein (lower panel) served as an internal loading control.

View larger version (75K): [in a new window]   Fig. 2. Expression of the Y1028F mutant genetically rescues the hypomorphic knock-in allele. Whole-mount diaphragm muscle from E18.5 embryos was stained with neurofilament antibodies to label presynaptic axons and with?bungarotoxin-Alexa594 to label AChRs. (A-C) The main central trunk of the phrenic nerve in diaphragm muscle isolated from (A) Erbb2wt/wt (B) Erbb2Erbb2/Erbb2 and (C) Erbb2Y1028F7ko are shown. The clusters of AChR correspond to the path of the presynaptic axon shown at low power magnification and at higher magnification (G'-I'). These particular genotypes are healthy animals and do not exhibit the acute respiratory distress at birth. (D-E) In contrast, the animals that were unable to inflate their lungs (D) Erbb2Erbb2/ko (E) Erbb2Y1144F/ko and (F) Erbb2Y1227F/ko, had poorly innervated diaphragms where the phrenic nerves were thinned, defasciculated and fragmented. However, the density and shape of the AChR clusters appeared to be unaffected in these animals (J-L and J'-L').

View larger version (74K): [in a new window]   Fig. 3. Development of the primary sympathetic ganglion chain. Whole-mount in situ hybridization staining of the sympathetic nervous system using a Phox2a antisense riboprobe on E12.5 embryos. Mid-sagittal views of (A) Erbb2wt/Erbb2, (B) Erbb2Erbb2/ko, (C) Erbb2wt/Y1028F, (D) Erbb2Y1028F/ko. Black arrowheads point to the superior cervical ganglia; white arrowheads highlight the thoracic sympathetic chain ganglia; green and blue arrowheads indicate the cells that migrate from the caudal portion of the primary sympathetic chain to the mesentery or the anlage of the adrenal gland.

View larger version (46K): [in a new window]   Fig. 4. Comparison of Erbb2 protein and transcript levels in knock-in embryos. For immunoblot analyses, total protein was isolated from the different E12.5 embryos as indicated and subjected to SDS-PAGE. Where possible, control littermates were used for comparison analyses. Membranes were subsequently incubated with an anti-Erbb2 antibody (A,B, upper panel). Detection of Grb2 protein (A,B, lower panel) was used as a sample loading control. (C) Erbb2 protein levels were quantified by using 125I-conjugated secondary antibodies and analyzed using PhosphoImager and ImageQuant software (Amersham Biosciences). The absolute levels of Erbb2 detected were normalized to Grb2 levels. The graph depicts relative levels of Erbb2 expressed as a percentage of Erbb2 levels in wild type embryos. (D) Erbb2 transcript levels (upper panel) were detected by RNase protection assays on total RNA isolated from E12.5 embryos. The mouse phosphoglycerate kinase (pgk) riboprobe (lower panel) was used as an internal control for equal sample loading. (E) The protein levels of all four ErbB family members (Egfr, Erbb2, ErbB3, and ErbB4) were also detected as described above for E12.5 embryos.

View larger version (40K): [in a new window]   Fig. 5. Tyrosine 1028 promotes the downregulation of Erbb2 in Rat-1 cells. The stability of mutant Erbb2 receptors in the presence or absence of Y1028 was examined by pulse-chase analyses using 35S-methionine labeled Rat-1 stable cell lines expressing the oncogenic versions (V664E mutation) of the Erbb2* phosphorylation mutants. (A) Erbb2* versus Erbb2*-Y1028F. (B) Erbb2*-Y1144 add-back mutant versus Erbb2*-Y1028/Y1144 double add-back mutant. Representative gels are shown for each and the average of multiple experiments is depicted graphically as a percentage of the original Erbb2 levels remaining. (C) The subcellular localization of Erbb2 was determined by immunofluorescent staining using an anti-Erbb2 antibody (Ab4, Oncogene Science) on Rat-1 cells expressing the non-oncogenic versions of Erbb2. The image was taken on a Zeiss LSM510 confocal microscope and is representative of comparable z-plane sections through the cell. Scale bar: 10 µm. (D) The ubiquitylation status of Erbb2* mutants expressed transiently in 293T cells were examined by immunoprecipitation using an anti-Erbb2 antibody and then blotting the top half of the membrane with an anti-ubiquitin antibody (Santa Cruz). The blots were then stripped and blotted with an anti-Erbb2 antibody to check for equal levels of Erbb2. The bottom half of the membrane was incubated with anti-Cbl antibodies (Santa Cruz) to examine Erbb2-Cbl association. The phosphorylation status of c-Cbl when co-expressed with different Erbb2* phosphorylation site mutants was examined by immunoprecipitation of c-Cbl followed by blotting with an anti-phosphotyrosine antibody (PY20, Transduction Labs). Erbb2*, constitutively activated (oncogenic) Erbb2; Y1028F is the point mutation; NYPD is the tyrosine-deficient Erbb2 mutant; Y1028 is the Y1028 add-back mutant.

View larger version (154K): [in a new window]   Fig. 6. Sensory nerve defects in Y1227F knock-in mutants. Homozygous E12.5 knock-in embryos were subjected to whole-mount immunostaining using anti-neurofilament antibodies. The morphologic appearance of the sensory cutaneous nerves in the thoracic cavity wall region is shown for (A) Erbb2wt/wt (B) Erbb2Y1028F/Y1028F (C) Erbb2Y1144F/Y1144F and (D) Erbb2Y1227F/Y1227F. Note that the nerves in D are disorganized and defasciculated.