Generation and characterization of neuregulin-2-deficient mice

Abstract

The neuregulins (NRGs) are a family of four structurally related growth factors that are expressed in the developing and adult brain. NRG-1 is essential for normal heart formation and has been implicated in the development and maintenance of both neurons and glia. NRG-2 was identified on the basis of its homology to NRG-1 and, like NRG-1, is expressed predominantly by neurons in the central nervous system. We have generated mice with the active domain of NRG-2 deleted in an effort to characterize the biological function of NRG-2 in vivo. In contrast to the NRG-1 knockout animals, NRG-2 knockouts have no apparent heart defects and survive embryogenesis. Mutant mice display early growth retardation and reduced reproductive capacity. No obvious histological differences were observed in the major sites of NRG-2 expression. Our results indicate that in vivo NRG-2 activity differs substantially from that of NRG-1 and that it is not essential for normal development in utero.

FIG. 1.

Targeted disruption of the mouse NRG-2 gene. (A) Schematic representation of the genomic structure of the mouse NRG-2 gene. Exons are numbered 1 to 12 and are represented by vertical lines. The two alternative start exons are designated 1A and 1B. (B) Numerous transcripts of NRG-2 are generated via alternative splicing events. Exons are numbered as above and labeled according to the structural region that they encode. (C) Targeting strategy. The structures of the wild-type allele and the disrupted allele are shown. Exon numbering is consistent with A. The primers used for routine genotyping are represented by arrows. (D) PCR genotyping with primers for wild-type and recombinant alleles. A 0.75-kb fragment was generated from the wild-type allele, and a 0.6-kb fragment was generated from the mutant allele. (E) Total RNA prepared from adult cerebellum and cortex/hippocampus was reverse transcribed and then used for PCR amplification with primers specific for NRG-2, NRG-1, and NRG-3. No amplification product for NRG-2 was observed in the −/− mice. ATG, start methionine; Ig, immunoglobulin-like domain; EGF, epidermal growth factor-like domain; α and β, alternative epidermal growth factor sequence; V, variable region; TM, transmembrane domain; C1 to C4, cytoplasmic region; +/+, wild-type; +/−, heterozygous; −/−, homozygous knockout; cb, cerebellum; cx, cortex/hippocampus.

FIG. 2.

Growth of NRG-2^−/− mice. (A) The weights of wild-type (open circles, n = 51), heterozygous (triangles, n = 77), and knockout (solid circles, n = 51) mice at various ages were plotted. Each data point is representative of an individual mouse. Lines of best fit were drawn to better represent the growth retardation exhibited by knockout mice. The correlation coefficient of each line was 0.86 or greater. (B) Photograph of wild-type (left) and knockout (right) male littermates at P28, demonstrating the difference in size. (C) The weights of 4-month-old male and female mice revealed no significant difference between knockouts and wild-types or heterozygotes.

FIG. 3.

Gross histology of adult brains. Macroscopic analysis of brains from adult wild-type (A) and knockout (G) mice revealed no major abnormalities. Sagittal sections (stained with cresyl violet) of the cerebellum show normal foliation and cell layering in both wild-type (B and C) and knockout (H and I) mice. Sagittal sections of the olfactory bulb also showed no major abnormality (D and J). Coronal sections through the hippocampus revealed that the characteristic cytoarchitecture was unaltered (E, F, K, and L). Magnification: ×4 (B and H); ×10 (E and K); ×20 (C, D, F, I, J, and L).

FIG. 4.

Immunohistochemistry of P7 brain sections. Immunoperoxidase staining of P7 cerebellum from wild-type (A to C) and knockout (D to F) animals with neuroD (A, B, D, and E) and calbindin (C and F). Note the typical staining patterns of neuroD in granule cells in both the external germinal layer (EGL) and internal granule cell layer (IGL). Calbindin staining was specific to purkinje cells, and no obvious abnormalities were detected in NRG-2-deficient mice at this age. Magnification: ×10 (A and D); ×20 (B and E); ×40 (C and F).

FIG. 5.

Histological analysis of developing heart in NRG-2^−/− mice. Sagittal sections of wild-type (A) and knockout (B) E12.5 embryos illustrate normal ventricular trabeculation (V) and atrium formation (At). EC, endocardial cushion. Magnification, ×10.