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. 2003 Apr 15;23(8):3343-52.
doi: 10.1523/JNEUROSCI.23-08-03343.2003.

Characterization of the WAVE1 knock-out mouse: implications for CNS development

John P Dahl  1 , Jeanne Wang-Dunlop, Cathleen Gonzales, Mary E P Goad, Robert J Mark, Seung P Kwak
Affiliations

Characterization of the WAVE1 knock-out mouse: implications for CNS development

John P Dahl et al. J Neurosci. .

Abstract

Developing neurons must respond to a wide range of extracellular signals during the process of brain morphogenesis. One mechanism through which immature neurons respond to such signals is by altering cellular actin dynamics. A recently discovered link between extracellular signaling events and the actin cytoskeleton is the WASP/WAVE (Wiscott-Aldrich Syndrome protein/WASP-family verprolin-homologous protein) family of proteins. Through a direct interaction with the Arp2/3 (actin-related protein) complex, this family functions to regulate the actin cytoskeleton by mediating signals from cdc42 as well as other small GTPases. To evaluate the role of WASP/WAVE proteins in the process of neuronal morphogenesis, we used a retroviral gene trap to generate a line of mice bearing a disruption in the WAVE1 gene. Using a heterologous reporter gene, we found that WAVE1 expression becomes increasingly restricted to the CNS over the course of development. Homozygous disruption of the WAVE1 gene results in postnatal lethality. In addition, these animals have severe limb weakness, a resting tremor, and notable neuroanatomical malformations without overt histopathology of peripheral organs. We did not detect any alterations in neuronal morphology in vivo or the ability of embryonic neurons to form processes in vitro. Our data indicate that WAVE1, although important for the general development of the CNS, is not essential for the formation and extension of neuritic processes.

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Figures

Fig. 1.
Fig. 1.
Tissue and brain region expression of WAVE1. Expression analysis was performed using human multitissue (A) and brain region (B) Northern blots probed with a radiolabeled WAVE1 cDNA probe (see Materials and Methods). A single transcript ∼3000 nt in length corresponding to WAVE1 was restricted to the CNS but was widely distributed within the human brain. C, WAVE1 mRNA expression in the mouse brain was analyzed using Taqman quantitative RT-PCR. Data are expressed as the fold change relative to the WAVE1 mRNA levels in the hypothalamus.
Fig. 2.
Fig. 2.
Disruption of the mouse WAVE1 gene. Structure of the mouse WAVE1 gene with boxes representing exons and the shaded areas representing the 5′ and 3′ UTR. Introns are lettered A through H and their approximate lengths are given. The structure of the retroviral gene trap used to disrupt the murine WAVE1 gene is shown with the splice acceptor (SA), the β-galactosidase neomycin resistance gene fusion (β-geo), polyadenylation consensus site (pA), the PGK promoter (PGK), the BTK OST sequence (BTK), and the splice donor site (SD). The site of insertion of the gene trap into the WAVE1 locus is marked with an arrow.
Fig. 3.
Fig. 3.
Analysis of WAVE1, WAVE2, and WAVE3 protein levels. Western analysis was performed on 50 μg of protein extracted from the cerebral cortex and hippocampus of wild-type (Wt) mice as well as from mice heterozygous (Het) and homozygous (KO) for the gene-trap insertion (n = 3). The analysis was performed using an anti-WAVE1 polyclonal primary antibody (A), an anti-WAVE2 polyclonal primary antibody (B), and an anti-WAVE3 polyclonal primary antibody (C). All blots were normalized by reprobing with an anti-actin primary antibody. Error bars indicate SEM.
Fig. 4.
Fig. 4.
β-galactosidase as a surrogate marker for WAVE1.A, β-galactosidase activity in all three genotypes was quantified using o-nitrophenyl β-d-galactopyranoside (ONPG) as a colorimetric substrate. An inverse relationship between WAVE1 protein levels and β-galactosidase activity was observed. B, The CNS expression pattern of WAVE1 in adult mice was determined usingin situ hybridization with a radiolabeled riboprobe directed against the 3′ UTR of the WAVE1 mRNA. WAVE1 mRNA is particularly enriched in the cerebral cortex (Ctx), striatum (Str), and hippocampus (Hipp). C, The CNS expression pattern of β-galactosidase was generated by incubating brain sections from adult mice heterozygous for the gene-trap insertion in a β-galactosidase staining buffer. An overlapping expression pattern was observed between β-galactosidase and WAVE1 mRNA.
Fig. 5.
Fig. 5.
Developmental expression of WAVE1. Embryos heterozygous for the gene-trap insertion were harvested at E9, E12, E15, and E18 and stained using a β-galactosidase staining buffer. Although WAVE1 is expressed throughout the E9 embryo, localization is more restricted in the older embryos. At E12 and E15, the expression of WAVE1 is highest in the brain and spinal cord. In the cortex of both E12 and E15 embryos WAVE1 expression is enriched in the outer cortical layers, including the cortical plate (CP), whereas it almost completely absent from the ventricular zone (VZ). In addition, at both of these ages WAVE1 expression is seen in peripheral organs, including the lung, intestine, heart, and limbs. By E18, WAVE1 expression is restricted to the CNS.
Fig. 6.
Fig. 6.
Morphological analysis of WAVE1 knock-out mice.A, At E17 WAVE1 knock-out embryos appear normal and do not display gross morphological abnormalities compared with wild-type littermates. B, WAVE1 knock-out mice at P20 display a severely runted phenotype compared with wild-type littermates.C, Comparison of brains taken from wild-type mice as well as from mice both heterozygous (Het) and homozygous (KO) for the gene-trap insertion reveals a dramatic reduction in the size of the cerebral cortex in the mice lacking a functional WAVE1 gene. Brains from heterozygous mice were indistinguishable from those of wild-type mice.
Fig. 7.
Fig. 7.
Lifespan and growth rate of WAVE1 knock-out mice.A, Individual lifespans of WAVE1 knock-out mice. The WAVE1 knock-out mice survived 21–26 d after birth, with an average lifespan of 23.6 d (n = 13). B,Average weights for wild-type (n = 10), WAVE1 (+/−) (n = 17), and WAVE1 (−/−) (n = 9) mice at P2, P5, P9, P12, P17, and P22. The average weight of the WAVE1 (−/−) mice is significantly reduced compared with the other two genotypes. *p < 0.001, ANOVA. Within-group comparison reveals a decrease in the weight of WAVE1 (−/−) mice from P17 to P22. †p < 0.001. Error bars indicate SEM.
Fig. 8.
Fig. 8.
Histopathology of WAVE1 knock-out mice. Hematoxylin-and-eosin-stained sections were generated from the kidney, lung, skeletal muscle, liver, and small intestine of P20 wild-type and WAVE1 knock-out mice. These slides were evaluated for the presence and/or severity of macroscopic and microscopic lesions. The muscle fibers, liver, and intestinal villi of WAVE1 knock-out mice appeared different from those of wild-type controls.
Fig. 9.
Fig. 9.
CNS histology of WAVE1 knock-out mice. Brain sections from wild-type and WAVE1 knock-out mice were stained using cresyl violet (middle). Those sections are compared with sections from wild-type mice that have been labeled with a WAVE1-specific probe byin situ hybridization (ISH) (left). WAVE1 knock-out mice have enlarged lateral ventricles accompanied by reductions in the lateral septum, striatum, and corpus callosum (top insets). An abnormal reduction of the cingulate gyrus in the retrosplenial cortex underlying the superior colliculus was evident (bottom insets). All of these areas appear to express WAVE1 at high levels, as determined by ISH. LS, Lateral septum; CC, corpus callosum; Str, striatum; Ctx, cerebral cortex; Cg, cingulate gyrus.
Fig. 10.
Fig. 10.
Morphological analysis of WAVE1 cortical neurons. Primary neuronal cultures were generated from E15 wild-type and WAVE1 knock-out embryos. After 5 d in culture, the cells were fixed and stained. The morphology of the neurons was analyzed at a magnification of 10× using the Cellomics Array Scan II and the accompanying neurite outgrowth software package. Average values are shown +/− the SD. Values generated for wild-type and knock-out neurons were compared using Student's t test (p < 0.05). For all of the parameters analyzed, no significant differences between the two populations of neurons were detected.
Fig. 11.
Fig. 11.
In vivo analysis of neuronal morphology in WAVE1 knock-out mice. Brain sections from wild-type and WAVE1 knock-out mice were processed for Golgi impregnation to assess neuronal morphology in the cortex (Ctx) and the CA1 field of the hippocampus (Hipp). Arrows indicate apical dendrites in the cortex. III, Cortical layer III; IV, cortical layer IV; V, cortical layer V; SO, strata oriens; PC, pyramidal-cell layer; SR, strata radiatum.
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