Isolation of a murine homologue of the Drosophila neuralized gene, a gene required for axonemal integrity in spermatozoa and terminal maturation of the mammary gland - PubMed (original) (raw)

Isolation of a murine homologue of the Drosophila neuralized gene, a gene required for axonemal integrity in spermatozoa and terminal maturation of the mammary gland

B Vollrath et al. Mol Cell Biol. 2001 Nov.

Abstract

The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.

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Figures

FIG. 1

FIG. 1

(A) Alignment between Neuralized proteins from Drosophila, mouse, and human. NHR-1 and NHR-2 are marked by a dashed line above the amino acid sequence, and identical residues are boxed. The Drosophila neuralized gene encodes a protein of 754 amino acids, while the human and mouse gene encode proteins of 574 and 557 amino acids, respectively. Neuralized proteins from all three organisms show significant sequence homology in the two NHR domains and in the ring finger (solid line). (B) Alignment of the Neuralized ring finger domain with ring finger domains of the IAP protein family. Sequences from human (h-IAP 1 and h-IAP 2), mouse (m-IAP 1 and m-IAP 2), and rat (r-IAP 2) are shown.

FIG. 2

FIG. 2

Expression pattern and subcellular localization of mouse Neuralized. (A) Poly(A) RNA Northern blot analysis of mouse tissues with a mouse Neuralized cDNA probe detects a transcript of approximately 4 kbp in adult brain and a slightly smaller transcript in skeletal muscle. (B) In situ hybridization on day 10.5 p.c. mouse embryos using a mouse Neuralized probe shows expression of mouse Neuralized in the somites. (C) Dorsal view of the same embryo as shown in panel B at higher magnification. (D) Littermates were hybridized to a mouse myogenin probe as a positive control. Mouse Neuralized is expressed in the dermomyotomes of the somites in a pattern highly similar to myogenin. (E) Subcellular localization of a human Neuralized-GFP fusion protein in differentiating C2C12 cells. C2C12 myoblasts were transfected with a CMV-_Neuralized_-GFP construct and induced to differentiate by serum starvation. An equivalent subcellular localization was observed in a variety of different human and murine cell lines.

FIG. 2

FIG. 2

Expression pattern and subcellular localization of mouse Neuralized. (A) Poly(A) RNA Northern blot analysis of mouse tissues with a mouse Neuralized cDNA probe detects a transcript of approximately 4 kbp in adult brain and a slightly smaller transcript in skeletal muscle. (B) In situ hybridization on day 10.5 p.c. mouse embryos using a mouse Neuralized probe shows expression of mouse Neuralized in the somites. (C) Dorsal view of the same embryo as shown in panel B at higher magnification. (D) Littermates were hybridized to a mouse myogenin probe as a positive control. Mouse Neuralized is expressed in the dermomyotomes of the somites in a pattern highly similar to myogenin. (E) Subcellular localization of a human Neuralized-GFP fusion protein in differentiating C2C12 cells. C2C12 myoblasts were transfected with a CMV-_Neuralized_-GFP construct and induced to differentiate by serum starvation. An equivalent subcellular localization was observed in a variety of different human and murine cell lines.

FIG. 3

FIG. 3

(A) Targeting strategy for disruption of the mouse Neuralized locus. A targeting cassette containing an IRES-GFP construct and a PGK-neo cassette for positive selection of transfected clones was inserted into the exon encoding the NHR-2 region of the protein. A thymidine kinase cassette (PGK-TK) was used for negative selection with FIAU. (B) Genotyping was performed by Southern blot analysis using a 5′ external probe and genomic DNA digested with _Bam_HI. The wild-type allele yields a 14-kbp fragment, and the mutant allele yields a 9-kbp band. The Southern blot shows a typical result from tail DNA of offspring from a heterozygous intercross and the appearance of all three expected genotypes. (C) PCR genotyping assay on tail DNA from offspring of a heterozygous intercross using a three-primer setup. The wild-type allele yields a PCR product of 367 bp, the mutant allele yields a product of 190 bp, and all three genotypes are readily detectable.

FIG. 4

FIG. 4

Loss of full-length Neuralized transcript in Neuralized knockout mice. Total RNA isolated from adult brain or skeletal muscle was analyzed using a cDNA probe containing sequence 5′ (left panel) or 3′ (right panel) to the integration site of the targeting cassette (see Materials and Methods). Northern hybridization with the 5′ probe yields transcripts of the expected size in wild-type tissues and aberrantly migrating transcripts in the tissues derived from Neuralized null animals. The right panel shows normal transcripts in the wild-type controls but loss of transcription of sequence 3′ to the integration site of the targeting vector in Neuralized null animals.

FIG. 5

FIG. 5

Ectopic expression of human Neuralized in PC-12 cells does not interfere with neuronal differentiation through NGF in vitro. The viral expression contruct expresses a bicistronic Neuralized alkaline phosphatase fusion transcript that allows translation of two independent proteins through an IRES sequence inserted between the two genes. Some of the infected, alkaline phosphatase-positive cells are marked by arrows in both panels. (A) PC-12 cells were infected with pLIA control virus, differentiated 24 h after infection by treatment with NGF for 4 days, and stained for alkaline phosphatase. Extensive neurite outgrowths are easily detectable in cell clones infected with pLIA. (B) PC-12 cells infected with pLIA-h_Neuralized_ are indistinguishable from control-infected cells.

FIG. 6

FIG. 6

Histological analysis of testis and spermatozoa isolated from Neuralized null animals. (A) Wild-type testis. There is normal morphology and active spermatogenesis in the seminiferous tubules. (B) Testis isolated from Neuralized null animals. No difference from wild-type tissues could be detected at this level of resolution. (C) Spermatozoa isolated from wild-type control animals. Spermatozoa were isolated from the epididydimus and showed high mobility. (D) Spermatozoa isolated from Neuralized null animals. Most spermatozoa lacked tail motility. In addition, defective spermatozoa were detected frequently in Neuralized null samples (arrows). (E) A Neuralized transcript of approximately 3,000 bp is detected in wild-type mouse testis by poly(A) RNA Northern blot hybridization (lane 3). The transcript is significantly smaller than the transcript found in skeletal muscle (lane 1). As shown in Fig. 1, the Neuralized transcript is absent in the thymus (lane 2).

FIG. 7

FIG. 7

Electron microscopy analysis of spermatozoa and testis from Neuralized null animals. (A) Cross section of a flagellar midpiece showing normal 9+2 arrangement of axoneme doublets. (B) Cross sections of the principal piece of spermatozoa from Neuralized null animals, showing that although some cross sections appear to be normal with the expected 9+2 configuration (no. 1 to 3), several other cross sections clearly show defects in axonemal organization (no. 4 to 6). (C) Cross section showing a common flagellar defect, i.e., deletion of half the axonemal complex. (D) Cross section of principal piece showing a flagellum where a portion of the fibrous sheath is absent as well as a highly disorganized axonemal complex. (E) Abnormal biflagellate spermatozoa identified in samples from Neuralized null animals. (F and G) Abnormal axonemal complexes occur along the length of the flagellum (arrowheads in panel F), as revealed by various longitudinal and grazing planes of sections. (H) Testicular spermatozoa of Neuralized null animals. Although cross-sections of the principal piece present a normal profile of the axoneme (arrows), it is apparent from grazing planes of sections that in localized regions of the flagellum, the axonemal complex is disorganized (arrowheads). Bars, 0.2 μm.

FIG. 8

FIG. 8

Analysis of mammary glands during lactation in Neuralized null females and wild-type controls. Mammary glands were harvested on the first postpartum day (L1) and analyzed by standard histological hematoxylin and eosin staining. (A) Expression of Neuralized in mammary glands during pregnancy (preg.), lactation (lact.), and regression (regres.). (B) Wild-type control mammary gland on day L1. The mammary fat pad is filled with alveoli, and lipid droplets are easily detected in the ducts. (C) Mammary gland from a Neuralized null female on L1. The mammary gland is significantly underdeveloped, with few alveoli in the fat pad. (D) Mammary gland from a Neuralized null female on L1. Alveoli appear to be more developed than in panel C; however, lipid vacuoles are not detectable in the ducts.

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