In this study, we report the isolation and characterization of a murine homologue of the Drosophila neuralized gene. The gene isolated is syntenic to the human neuralized gene isolated recently and encodes a protein which is almost identical to human Neuralized.
Sequence similarity searches in the database using the BLAST algorithm revealed significant sequence homology in the ring finger domain between Neuralized proteins and proteins of the IAP family. IAP proteins were initially identified in baculoviruses, and the related viral and mammalian proteins all contain RING finger domains at their carboxy terminus (14
). Expression of IAP proteins inhibits the induction of apoptosis by various stimuli in vitro (25
). Interestingly, ectopic expression of Neuralized
appears to induce rapid cell death in a variety of different cell lines in vitro, suggesting a role of vertebrate Neuralized
in apoptosis (K. Fitzgerald, unpublished observations). However, the sequence homology between Neuralized and IAP proteins is limited to the ring finger, suggesting that these two groups of proteins are distinct. Recent evidence indicates that ring finger-containing proteins can mediate ubiquitin-conjugating enzyme-mediated ubiquitination of receptor protein tyrosine kinases, leading to termination of signaling through protein degradation (20
). Interestingly, IAP proteins have now also been shown to catalyze their own ubiquitination in response to apoptotic stimuli, an activity that requires the presence of the ring finger domain (47
). Further experiments will be needed to establish if Neuralized has ubiquitin protein ligase activity.
Targeted deletion of the murine Neuralized
gene reveals that expression of murine Neuralized
is not essential for development and survival of the animal. In contrast, Notch
null mice die around midgestation with severe defects in development, as do mice with null mutations in most other components of the Notch signaling pathway (10
). In addition, we failed to detect any aberrant cell fate specifications in Neuralized
null animals during neurogenesis and somitogenesis, two processes where Notch signaling has been shown to be involved. This evidence suggests that the murine Neuralized
isolated in this study is not an essential component of the Notch signaling cascade, at least during most developmental processes. However, two other possibilities must be considered. (i) Neuralized function is essential for Notch signaling, as suggested from genetic evidence in Drosophila
, but other vertebrate Neuralized homologues or unrelated proteins compensate for loss of the Neuralized
gene isolated here. A search of sequence databases using the BLAST algorithm did not reveal sequences showing significant homology to the gene described in this report. However, since the mouse genome sequence is not complete, this possibility cannot be ruled out. (ii) The Neuralized
allele produced by targeting the exon encoding NHR-2 is a hypomorph, but not a complete null. Our targeting strategy removes sequence encoding most of the protein including domains highly conserved between Drosophila
and vertebrates and the ring finger, which is thought to be a crucial component of a functional Neuralized protein. Since we have shown here that our targeting does result in the expected changes in Neuralized
transcript, we think that this possibility is highly unlikely.
The human Neuralized
homologue was isolated from a region at chromosome 10q24–25 that shows frequent alterations in malignant astrocytomas. In addition, loss of Neuralized
transcription has been described in human astrocytoma tissue and glioma cell lines (28
). The postulated link between loss of Neuralized
transcription and neoplastic transformation in the CNS in humans is particularly interesting since Notch signaling is involved in cell fate specification during development of the CNS but has not, at least so far, been linked to the development of CNS tumors. Thus far, our Neuralized
null mice have failed to develop any gliablastomas or astrocytomas. This observation questions the proposed link between loss of Neuralized
transcription and malignant transformation in the CNS.
null females failed to nurse their pups and support a litter. We show here that this is caused by defective lobular development of the mammary gland during pregnancy, leading to an insufficiently developed mammary gland at the end of pregnancy. However, we did not detect any clear differences between virgin Neuralized
null animals and wild-type controls at the end of sexual maturation or during early stages of pregnancy. These observations suggest a role of Neuralized
in later stages of mammary gland maturation during pregnancy. Whether the effect of Neuralized
on mammary gland differentiation is cell autonomous or is caused by a defective hormonal environment during pregnancy in Neuralized
null animals awaits the results of transplantation experiments. Interestingly, transgenic mice expressing constitutive active alleles of the Notch receptor also fail to lactate, with lobular development being retarded during late stages of pregnancy (15
). However, unlike these transgenic mice, Neuralized
null animals do not develop mammary gland tumors.
While male Neuralized
null mice were sterile and appeared to have normal reproductive organs when analyzed by standard histological techniques, spermatozoa isolated from these mice were immobile or displayed only residual motility. Electron microscopy clearly revealed structural abnormalities in the flagella of epididymal spermatozoa from Neuralized
null animals. The most common defect observed was in the axoneme, which displayed missing microtubular doublets. The defects ranged from loss of one doublet to the deletion of up to half of the axonemal complex. These defects in the structural integrity of the axoneme would directly compromise the motility of the affected spermatozoa, leading to the observed infertility of male Neuralized
null mice. Normal 9+2 doublet structures of the axoneme consist mainly of αβ-tubulin polymers as well as microtubule-associated proteins. However, the molecular events required for proper assembly of axonemal microtubule structures have not yet been identified. Indeed, this is the first evidence that Neuralized
functions in this process. Interestingly, results from C. elegans
indicate that the presenilin family member spe-4
is involved in tubulin localization during spermatogenesis (1
). Since presenilins are thought to be regulators of Notch and amyloid precursor protein processing, this result provides a link between signal transduction events and microtubule assembly during spermatogenesis.
The severity of the observed axonemal defects identified in Neuralized null animals suggests that the structural alterations occur during spermiogenesis. This observation is confirmed by the fact that similar flagellar defects were detected in testicular spermatozoa in the lumen of the seminiferous tubules. Analysis of spermatid maturation in testes of Neuralized null mice showed that the structure of both the proximal and distal centrioles appeared normal. In addition, alignment and orientation of the centrioles with proper migration to the posterior pole of the nucleus appeared to be normal during initial development of the flagellum. It is therefore likely that the observed defects in axonemal structure occurred during subsequent growth and formation of the nascent flagellum. A striking feature of the morphology of spermatozoa from Neuralized null mice was the fact that axonemal abnormalities occurred as localized defects along the length of the axoneme. Longitudinal sections of flagella observed under the electron microscope displayed regions containing normal axonemal structures followed by a region where the axonemal complex was clearly disrupted. This observation suggests that proper construction of the 9+2 microtubular structure during spermiogenesis involves the presence of local regulatory factors along the length of the flagellum.
Defects in spermatogenesis account for more than 50% of human male infertility (29
). The axonemal and spermatid abnormalities seen in Neuralized
null mice in part mimic the defects seen in many human spermatogenic disorders (32
). Thus, Neuralized
null mice may thus be a valid model to study human infertility syndromes and to gain a better understanding of male infertility.