MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Nov. 2001, p. 7481–7494Vol. 21, No. 21
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
BENEDIKT VOLLRATH,1† JEFFREY PUDNEY,2SYLVIA ASA,3PHILIP LEDER,1*
AND KEVIN FITZGERALD1‡
Howard Hughes Medical Institute and Department of Genetics, Harvard Medical School,1and Department of Obstetrics,
Gynecology, and Reproductive Biology, Brigham and Women’s Hospital and Harvard Medical School,2Boston, Massachusetts
02115, and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X53
Received 5 June 2001/Returned for modification 11 July 2001/Accepted 31 July 2001
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.
The Drosophila neuralized gene has been identified in screens
for embryonic lethal mutations with defective lateral specifi-
cation (5, 24). Hypomorphic neuralized mutations cause neu-
ronal hyperplasia, which is characteristic of neurogenic genes,
suggesting that neuralized is involved in cell fate specifications
in the neurectoderm. Genetic interaction analysis suggests that
neuralized appears to act upstream of Notch and E(spl) since
increased Notch or E(spl) expression can partially rescue the
neuralized phenotype (9). The Drosophila neuralized gene en-
codes a C3HC4ring finger protein and is expressed throughout
the ectoderm during neuronal cell fate specification, consistent
with its proposed role in this process (6, 33). As with other
neurogenic mutants, mesodermal cell fates also appear to be
defective in Drosophila carrying a neuralized mutation (4, 27).
neuralized mutant files produce an excess number of cells ex-
pressing the MyoD homologue, nautilus, at the expense of
surrounding mesodermal cells, indicating aberrant cell fate
specifications in this tissue (8).
A human homologue of the Drosophila neuralized gene has
been identified in a region of chromosome 10q24-25 which
shows frequent alterations in malignant astrocytomas (28).
Like the Drosophila gene, the human Neuralized gene encodes
a C3HC4ring finger-containing protein of 574 amino acids.
Interestingly, while the expression of human Neuralized is high
in normal human brain tissue, expression is very low or absent
in astrocytomas and multiple glioma cell lines. It has been
postulated that human Neuralized, like the Drosophila gene, is
involved in cell fate specifications or maintenance in the cen-
tral nervous system and that loss of Neuralized expression is an
important step in the development of malignant transforma-
tion in the central nervous system (CNS).
The Notch signaling pathway has been implicated in cell fate
decisions in a variety of developmental contexts in Drosophila,
Caenorhabditis elegans, and vertebrates (2, 16, 44). Notch en-
codes a transmembrane receptor that binds to the membrane-
bound ligands Delta and Serrate. On ligand binding, Notch is
cleaved by a Presenilin-dependent mechanism (13, 40) and an
intracellular portion of the molecule translocates, together
with the Suppressor of Hairless [Su(H)] gene product, to the
nucleus, where the protein complex acts as a transcriptional
activator (22, 39). As in Drosophila, Notch signaling in verte-
brates is involved in specifying cell fates in a variety of devel-
opmental processes. Targeted disruption of Notch expression
or disruption of its downstream targets in mice leads to em-
bryonic death by about midgestation. Notch null embryos have
a variety of developmental defects including severe disruption
of development of the CNS and somites (7, 10, 41). Constitu-
tive activation of Notch signaling by expression of hypermor-
phic Notch alleles or downstream targets of Notch interferes
with cell fate specifications during neurogenesis and myogen-
esis in vitro as well as in vivo (30, 31, 37, 43). In addition to its
well-established role in cell fate specification during develop-
ment, recent evidence suggests that Notch signaling might be
involved in the maintenance and homeostasis of differentiated
cells. Neurite outgrowth of cortical neurons appears to be
regulated by Notch signaling, indicating that Notch might be
involved in maintenance or plasticity in the CNS (36).
In this study, we report the isolation and characterization of
* Corresponding author. Mailing address: Howard Hughes Medical
Institute and Department of Genetics, Harvard Medical School, 200
Longwood Ave., Boston, MA 02115. Phone: (617) 432-7667. Fax: (617)
432-7944. E-mail: email@example.com.
† Present address: Merck Research Laboratories, West Point, PA
‡ Present address: Bristol Myers Squibb, Pennington, NJ 08530.
a murine homologue of the Drosophila neuralized gene. Our
analysis indicates that expression of Neuralized is not essential
for development and survival in vertebrates since Neuralized
knockout mice are fully viable. Neuralized null mice, however,
have defects in mammary gland development leading to defi-
cient lactation. In addition, defects in the axonemes of sper-
matozoa isolated from Neuralized null mice result in immotile
spermatozoa and male sterility. The axonemal and spermatid
abnormalities seen in Neuralized null mice in part mimic the de-
fects seen in many human spermatogenic disorders (32, 45, 46).
MATERIALS AND METHODS
Isolation of the murine Neuralized gene. Degenerate PCR primers were de-
signed based on the alignment of Drosophila melanogaster and D. virilis neuralized
sequence. Primers were designed to the amino acid sequence AITFS and
FWAKA, respectively, and PCR was performed using a mouse skeletal muscle
cDNA library (Clontech) as template. The resulting 200-bp mouse Neuralized
fragment was used to screen mouse skeletal muscle and brain cDNA libraries
(Stratagene) by standard procedures (3). To obtain a full-length cDNA clone, 3?
rapid amplification of cDNA ends was performed using murine brain and skel-
etal muscle libraries (Marathon-ready cDNA; Clontech) and Advantage cDNA
polymerase mix (Clontech). The gene-specific primer used in this reaction was
5?-GCT GTC CTT CGG GGT CAC CAC GTG TGA GGC-3?.
Northern blot analysis. Total cytoplasmic RNA was isolated from murine
tissues using RNA STAT (Tel-Test Inc.). For the expression analysis in adult
mouse tissues, 2 ?g of poly(A) RNA was loaded on a 1% agarose gel containing
formaldehyde, transferred onto GeneScreen Plus membranes (NEN), and hy-
bridized to a mouse Neuralized cDNA fragment using standard procedures. For
the expression analysis in Neuralized null animals, 10 ?g of total cytoplasmic
RNA isolated from brain and skeletal muscle of Neuralized null adult mice and
wild-type littermate controls was used. An 820-bp Neuralized cDNA fragment
corresponding to amino acids 1 to 209 was used as a 5? probe, a 1-kbp Neuralized
cDNA fragment corresponding to amino acids 210 to 557 was used as a 3? probe.
A murine glyceraldehyde 3-phosphate dehydrogenase full-length cDNA probe
was used for the loading control. For the expression analysis in human tissues, a
human RNA master blot (Clontech) was hybridized with a full-length human
Neuralized cDNA probe.
Expression constructs. The full-length human Neuralized coding sequence was
PCR amplified from a cloned human Neuralized cDNA using nrzF1 (GAA GCT
TCC GAA GAT GGG GGG ACA GAT CAC CCG G) and nrzR1 (CGG TGG
ATC CCG GGA GCT GCG GTA GGT CTT GAT GAT). The PCR product
was cloned into pCMV-EGFP using HindIII and BamHI restriction sites creating
an in-frame fusion between human Neuralized and enhanced green fluorescent
protein. This construct was used to monitor subcellular localization in various
cell lines by fluorescence microscopy.
The retroviral Neuralized vector pLIA-Neuralized was constructed by cloning
the full-length human Neuralized cDNA coding sequence into the SmaI site of
pLIA (generously donated by C. Cepko, Harvard Medical School). This con-
struct places the human Neuralized coding sequence under the transcriptional
control of the Moloney murine leukemia virus long terminal repeat promoter
and contains an internal ribosome entry site (IRES)-alkaline phosphatase cas-
sette that allows staining of infected cells by established methods (3).
The constitutive active allele of Notch contained the intracellular domain of
Notch 1 under the control of the cytomegalovirus CMV promoter. Functional
activity of the construct used was assessed by cotransfection with a Notch sig-
naling reporter construct containing four Cbf-1 binding repeats and a minimal
simian virus 40 promoter driving luciferase transcription. Notch signaling activity
was assessed after 24 h using a luciferase assay (Boehringer Mannheim).
Cell lines and tissue culture. PC-12 (mouse pheochromocytoma) cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) con-
taining 10% fetal calf serum, 3 mM L-glutamine, 100 U of penicillin per ml, and
100 ?g of streptomycin per ml. PC-12 cells were differentiated on polylysine-
treated tissue culture dishes by adding 50 ng of nerve growth factor (NGF) (2.5
S NGF; Promega) per ml For infection of PC-12 cells with retrovirus, the cells
were allowed to reach 60 to 70% confluency. They were fed prior to infection,
and Polybrene was added to a final concentration of 80 ?g/ml. C2C12 cells
(mouse myoblast cells; American Type Culture Collection) were cultured in
DMEM containing 20% fetal calf serum, 3 mM L-glutamine, 100 U of penicillin
per ml, and 100 ?g of streptomycin per ml. C2C12 cells were differentiated into
myocytes by cultivating cells DMEM containing 10% horse serum, 3 mM L-
glutamine, 100 U of penicillin per ml, and 100 ?g of streptomycin per ml.
Retroviral stocks were prepared in Phoenix cells by a procedure adapted from
that of Cepko and Pear (3).
Targeted disruption of mouse Neuralized in ES cells and generation of Neu-
ralized null mice. A murine genomic fragment was isolated by screening a mouse
129/SvEv genomic BAC library (Genome Systems) using the 200-bp partial
cDNA fragment described above. From the isolated BAC clone containing
murine Neuralized sequence, two NotI fragments of 15 kbp (G) and 6 kbp (K)
were subcloned into pBluescript. A NotI-XbaI 1.4-kbp Neuralized genomic frag-
ment isolated from K was inserted into pOSdupdel (a kind gift from Oliver
Smithies) digested with NotI and XbaI. The resulting plasmid was digested with
PmlI and an 8.8-kbp NotI genomic fragment isolated from G was inserted. An
IRES-green fluorescent protein (GFP) cassette was constructed by digesting
pIRES-neo (Gibco BRL) with XbaI and SmaI, which removes the neo cassette.
Into this vector, a GFP cDNA fragment isolated by PCR from the vector pLan-
tern GFP (Gibco BRL) was cloned to yield pIRES-GFP. pIRES-GFP was di-
gested with SalI and XhoI, and the IRES-GFP cassette was cloned into the
targeting vector at the XhoI site, yielding pNRZKO-1.
TC1 embryonic stem cells (ES cells) derived from 129/SvEv mice (11) were
electroporated with NotI-linearized pNRZKO-1 and selected with G418 and
1-(2-deoxy-2-fluoro-1-?-D-abino-furanosyl)-5-iodouracil as described previously
(12). Genomic DNA from G418- and FIAU-resistant clones was isolated as
described previously (12) and screened for targeting by digestion with BamHI
followed by Southern blot analysis. The blots were hybridized with an 800-bp
BamHI-EcoRV genomic fragment located 5? to the genomic region used for
targeting vector construction and isolated from a BAC subclone. Two positive ES
cell clones were microinjected into C57BL/J6 blastocysts, which were transferred
into pseudopregnant Swiss Webster foster mothers (Taconic). High-grade chi-
meras judged by agouti coat color of the offspring were mated to 129/SvEv mice
(Taconic), and germ line transmission was confirmed by Southern blot analysis.
Heterozygous offspring from this F1cross were intercrossed to derive the mouse
PCR genotyping was performed on genomic DNA using primers nrzF (5?-
GAC AGC GAG CTG GTG CTG CCC GAC TG-3?), nrzR (5?-GAA GAT
GGT TTC GGC CAC GCG CAC AGG CCG-3?), and nrzIRES (5?-GGA CGC
GGC CAC CCT CAA AGG CAT C-3?). The wild-type allele (product nrzF-
nrzR) was expected to give a PCR product of 367 bp, and the mutant allele
(product nrzF-nrzIRES) was expected to give a PCR product of 190 bp.
In situ hybridizations. Embryos were dissected from pregnant wild-type ani-
mals (FVB, Taconic) at various time points of pregnancy (8.5, 9.5, 10.5, and 12.5
days postcoitum (p.c.) and fixed overnight in 4% paraformaldehyde at 4°C. After
incubation overnight in methanol, the embryos were rehydrated in a series of
methanol–Tris-buffered saline with Tween 20 (PBT), bleached with 6% hydro-
gen peroxide, treated with 10 ?g of proteinase K (Boehringer Mannheim) per ml
for 15 min at room temperature, and washed with 2 mg of glycine per ml in PBT
for 10 min at room temperature. The embryos were then postfixed with 4%
paraformaldehyde–0.2% glutaraldehyde in PBT for 10 min and prehybridized in
50% formamide–5? SSC (pH 4.5) (1? SSC in 0.15 NaCl plus 0.015 sodium
citrate)–1% sodium dodecyl sulfate (SDS)–50 ?g of yeast RNA (Boehringer
Mannheim) per ml–50 ?g of heparin per ml for at least 1 h at 70°C.
Mouse Neuralized riboprobes (a 1,326-bp partial mouse Neuralized cDNA
fragment; 1 ?g per reaction) were digoxigenin DIG-labeled using T7 and T3
RNA polymerases (DIG RNA labeling kit; Boehringer Mannheim) and purified
using ethanol precipitation. The embryos were then hybridized in 50% form-
amide–5? SSC (pH 4.5)–1% SDS–50 ?g of yeast RNA per ml–50 ?g of heparin
per ml overnight at 70°C.
After hybridization, the embryos were washed in 50% formamide–5?SSC (pH
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.
7482 VOLLRATH ET AL.MOL. CELL. BIOL.
VOL. 21, 2001TARGETED DELETION OF MURINE Neuralized7483
4.5)–1% SDS and blocked in 10% sheep serum–TBST, and the transcript was
detected using an anti-DIG antibody (Boehringer Mannheim) and nitroblue
tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) staining.
Histology. For histological analysis, tissues were fixed in Omnifix (Zymed),
dehydrated in a graded alcohol series, and embedded in paraffin. Sections 4 to 6
?m thick were stained with hematoxylin and eosin using standard procedures.
For analysis of brain and pituitary, tissues were fixed in 4% paraformaldehyde
in phosphate-buffered saline (PBS) and embedded in paraffin. Sections 4 to 5 ?m
thick were stained with hematoxylin and eosin as well as by the Gordon-Sweet
silver method to demonstrate the reticulin fiber network. Immunocytochemical
stains to localize adenohypophysial hormones were performed using the avidin-
biotin-peroxidase complex method. Primary antibodies against the following
antigens were used at the specific dilutions: adrenocorticotropin (ACTH), 1:15;
growth hormone, 1:2,500; prolactin, 1:2,500; ?-thyroid-stimulating hormone,
1:3,000; ?-follicle-stimulating hormone (?-FSH) 1:6,000; and luteinizing hor-
mone (LH) 1:2,500. All antibodies were donated by the National Hormone and
Pituitary Program (NHPP), National Institute of Diabetes and Digestive and
Kidney Diseases, National Institute of Child Health and Human Development
(Bethesda, Md.) except for the ACTH antibody which was purchased from Dako
Corp. (Carpinteria, Calif.). Primary antibodies were incubated at 4°C for 24 h
prior to detection.
For skeletal staining, animals were euthanized with CO2and the skin was
removed. The animals were then eviscerated, fixed in 95% ethanol, and stained
with Alizarin red S and Alcian blue.
Fertility and analysis of sperm motility. Male Neuralized null animals aged
between 12 weeks and 9 months were housed with 129/SvEv (Taconic), NIH
Black Swiss (Taconic), or Neuralized heterozygous females, and the females were
analyzed for the presence of vaginal plugs each morning. Sperm was isolated by
flushing the epididymidis with Tyrode’s solution (0.15 M NaCl, 3 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 1 mM NaHCO3, 5 mM glucose). Sperm motility was
assessed by light microscopy in Tyrode’s solution after incubation at 30°C for at
least 30 min to allow dissociation of spermatozoa.
Electron microscopy. Testes and epididymides were fixed in 2.5% glutaralde-
hyde in cacodylate buffer (0.2 M sodium cacodylate [pH 7.6]) at 4°C for ca. 12 h.
The fixed tissues were dehydrated through a graded series of alcohols and finally
embedded in Spurr’s low-viscosity embedding medium. Thick (1-?m) and ultra-
thin (60 to 80-nm) sections were cut on an LKB MarkIII ultramicrotome. Thick
sections were stained with 1% toluidine blue for routine light microscopic ex-
amination. Contrast was enhanced in ultrathin sections by sequential staining
with a saturated uranyl acetate solution in 50% ethanol–25% methanol for 10
min followed by incubation in lead citrate. Ultrathin sections were examined
under a Zeiss 10 electron microscope. Sperm abnormalities were quantified by
analyzing sections of the cauda epididydimis at a magnification of ?10,000. The
frequency of abnormalities was obtained by counting the incidence of flagellar
structural defects in 100 cross-sections of the flagellum for both Neuralized
knockout and wild-type mice.
Nucleotide sequence accession number. The complete mouse neuralized
cDNA sequence was deposited in GenBank under accession number AF401228.
Isolation of a murine homologue of the Drosophila neuralized
gene. We have identified a murine homologue of the Drosoph-
ila and human Neuralized genes with a cDNA sequence of 3.63
kbp and an open reading frame of 1,674 bp. The gene encodes
a protein of 557 amino acids and a predicted molecular mass of
59.9 kDa (Fig. 1A). The sequence identity between the mouse
and human Neuralized proteins is 93%, and the sequence
identity between the mouse and Drosophila Neuralized pro-
teins is 34% (28, 33). The murine Neuralized gene isolated here
is syntenic to the human Neuralized gene isolated previously
(28) and thus is very likely to be the mouse ortholog. The only
detectable protein motif is a C3HC4-type ring finger at the
carboxy terminus of the protein. In addition to the ring finger,
Neuralized proteins contain two regions of approximately 160
amino acids, each of which appears to be composed of tandem
repeats although the sequence identity between the two re-
peats in each protein is limited (Fig. 1A). These regions have
previously been termed neuralized homology repeats (NHR),
and their function has not yet been established. Sequence
similarity searches in the database using the BLAST algorithm
revealed significant sequence homology in the ring finger do-
main between Neuralized proteins and proteins of the IAP
(inhibitor of apoptosis) family (Fig. 1B). No sequence similar-
ity between Neuralized and IAP proteins was detected outside
the ring finger domain, suggesting that they belong to distinct
Expression analysis of various murine tissues by Northern
blot hybridization revealed a transcript of approximately 4,400
bp in adult brain and a slightly smaller transcript in skeletal
muscle (Fig. 2A). Interestingly, no Neuralized transcript was
detected in heart muscle, indicating a high degree of tissue
specificity in myocytes. The size of the transcript is consistent
with the cDNA sequence described above. The reason for the
size difference in transcripts from brain and skeletal muscle is
so far unknown. However, 3? rapid amplification of cDNA
ends from skeletal muscle and brain libraries yielded identical
clones, suggesting that the presumed alternative splicing event
occurs at the 5? end of the transcript. Using a dot blot analysis
with poly(A) RNA from human tissues and a human Neural-
ized cDNA probe, we also detected transcript in testis, pituitary
gland, pancreas, and bone marrow (data not shown). This
analysis of human tissues also confirmed the expression of
Neuralized in all regions of the brain, including fetal brain and
adult skeletal muscle.
Using whole-mount in situ hybridizations on mouse embryos
at various stages of development, we were able to detect Neu-
ralized transcript in the somites from day 9.5 p.c. (Fig. 2B and
C shows an embryo on day 10.5 p.c). The expression pattern of
Neuralized is very similar to the expression found for the myo-
tome marker myogenin (Fig. 2D). myogenin is expressed in the
myotome from day 8.5 p.c. and is maintained in skeletal muscle
throughout embryonic development (35). Control hybridiza-
tions using a sense Neuralized probe did not show staining
above background throughout the embryo’s torso. This whole-
mount analysis did not reveal Neuralized expression in the
developing brain due to high background in this tissue.
Neuralized is not localized to the nucleus. We expressed a
full-length human Neuralized cDNA-GFP fusion construct un-
der the control of the CMV promoter in a variety of human
and mouse cell lines including HeLa, C2C12, COS, and NIH
3T3. In all cell lines, the human Neuralized-GFP fusion was
excluded from the nucleus, with perinuclear, Golgi-like stain-
ing detectable in many cells (Fig. 2E). Our results are consis-
tent with those of Yeh et al. (48), who reported a function of
Drosophila Neuralized outside the nucleus. Cotransfection
with constructs encoding constitutively active forms of Notch 1
did not change this cellular localization (data not shown).
However, these constitutive alleles of Notch 1 did activate
Cbf-1-dependent reporter gene transcription in a Notch re-
porter assay (data not shown), indicating that the Notch con-
structs are functionally active. These data suggest that cellular
localization of Neuralized is not regulated by Notch signaling.
Targeted disruption of Neuralized in ES cells and generation
of Neuralized null mice. A targeting strategy for the mouse
Neuralized locus was designed that inserts PGK-neo and IRES-
GFP cassettes into the exon encoding NHR2 (Fig. 3A). The
IRES-GFP cassette includes transcriptional termination se-
quences 3? to the GFP coding sequence. The insertion of the
7484 VOLLRATH ET AL.MOL. CELL. BIOL.
targeting cassette is therefore expected to disrupt the expres-
sion of all portions of the gene encoding the NHR2 domain
and the ring finger, thus eliminating the majority of the coding
sequence from the transcript.
To confirm that we generated a true targeted deletion of the
murine Neuralized gene, we performed Northern blot RNA
analysis on wild-type controls and knockout animals. Using a
cDNA probe containing sequence 5? to the integration site of
the IRES-GFP-PGK-neo cassette, we were able to detect two
aberrantly migrating transcripts in the mutant animals com-
pared to wild-type control RNA derived from skeletal muscle
and brain (Fig. 4). The sizes of these transcripts are consistent
with the generation of fusion transcripts containing the 5? end
of the murine Neuralized gene and the IRES-GFP cassette.
Using a cDNA probe containing sequence 3? to the integration
site of the IRES-GFP-PGK-neo cassette, we failed to detect
any Neuralized transcripts in the mutant animals, indicating
that the transcripts produced in mutant animals lack sequence
encoding the NHR 2 domain as well as the ring finger. This
deletion is expected to result in a null allele.
In multiple heterozygous intercrosses from F1or later gen-
erations for a total of 444 animals, we observed live Neuralized
null animals at the expected Mendelian frequency of approx-
imately 1:4 (the ratio of Neuralized null to Neuralized heterozy-
gous to wild type is 1.2:2.0:1.1). This indicates full viability of
animals carrying a targeted deletion of the Neuralized gene.
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.
VOL. 21, 2001 TARGETED DELETION OF MURINE Neuralized 7485
The oldest animals in our colony have now reached ages of
over 1 year, showing that murine Neuralized expression is not
required for full development and survival. These data are
consistent with observations on two mouse lines derived from
two independently targeted ES cell clones.
Neuralized null mice do not show aberrant cell fate specifi-
cations in the CNS and somites. Notch null mice die during
embryogenesis with severe defects in neurogenesis. On histo-
logical analysis of the CNS, we did not detect any differences
between brains derived from Neuralized knockout mice and
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 BamHI. 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.
7486 VOLLRATH ET AL.MOL. CELL. BIOL.
wild-type controls (data not shown). All major brain structures
and cell types were present, and the cortex showed correct
Notch signaling has been implicated in cell fate specifica-
tions during somitogenesis, and targeted deletion of Notch or
genes involved in Notch signaling leads to aberrant somite
development (7, 23). Since Neuralized is expressed in develop-
ing somites, we investigated whether loss of Neuralized expres-
sion interferes with skeletal or myocyte differentiation. On
histological analysis, we found that skeletal muscle from Neu-
ralized null animals showed clearly developed myotubes and we
failed to detect any differences between skeletal muscle from
Neuralized null animals and wild-type controls (data not
shown). Likewise, the skeletons of Neuralized null animals ap-
peared to be identical to skeletons derived from wild-type
controls after staining with Alizarin red S and Acian blue (data
not shown). These observations indicate that loss of Neuralized
expression does not interfere with cell fate specifications dur-
Ectopic expression of Neuralized does not interfere with dif-
ferentiation of PC-12 or C2C12 cells in vitro. The PC-12 cell
line has been extensively studied in vitro as a model for neu-
ronal differentiation and neurotropin signaling. Stimulation of
PC-12 cells with NGF induces neuronal differentiation, with
differentiated cells expressing neuronal markers and forming
long neurite extensions from the cell bodies. Activation of
Notch signaling blocks the differentiation of neuronal precur-
sor cells during development of the CNS in vivo and the Notch
target Hes-1 modulates the differentiation of PC-12 cells in
vitro (38). To assess whether ectopic expression of Neuralized
was able to block neuronal differentiation of PC-12 cells in a
similar manner, PC-12 cells were infected with Neuralized-
expressing IRES alkaline phosphatase virus and differentiated
by stimulation with NGF. After treatment of infected cells with
NGF, neurite extensions were clearly detectable in virus-in-
fected cells after staining for the viral marker alkaline phos-
phatase and no difference compared to cells infected with the
empty virus (pLIA) or uninfected cells could be detected (Fig.
5), indicating that ectopic expression of Neuralized does not
interfere with neuronal differentiation in PC12 cells. In addi-
tion, Neuralized-expressing cells still required NGF for neuro-
nal differentiation (data not shown). These observations are
consistent with our observed lack of defects in neuronal dif-
ferentiation in Neuralized null mice, suggesting that, in contrast
to Drosophila, Neuralized is not necessary for the regulation of
neuronal differentiation in vertebrates.
A similar experiment was performed to assess the effect of
Neuralized expression on myocyte differentiation in vitro. The
C2C12 cell line has been studied as an in vitro model for this
process, and, in a manner highly similar to the situation found
in PC-12 cells, ectopic expression of activated alleles of Notch
interferes with the differentiation of C2C12 cells into fused
myocytes (30, 37). Ectopic expression of Neuralized in C2C12
cells did not affect myocyte differentiation, consistent with the
correct formation of myocytes observed in Neuralized null an-
imals (Fig. 2E). This result, together with results obtained with
Neuralized null mice, suggests that Neuralized is not necessary
for regulating myocyte differentiation in vertebrates. However,
ectopic expression of murine Neuralized does induce apoptosis
in various cell lines (data not shown).
Male Neuralized null animals are sterile and have defective
spermatozoa. Male Neuralized null animals failed to fertilize
females in matings with either 129/SvEv wild-type mice, NIH
Black Swiss wild-type mice, or Neuralized heterozygous litter-
mates. Of 10 male homozygote Neuralized animals aged be-
tween 12 weeks and 9 months, none gave rise to a pregnancy in
matings with NIH BI/SW females, while matings of Neuralized
heterozygous males or wild-type control males yielded preg-
nancies and litters of normal sizes.
Surprisingly, standard histological analysis of male repro-
ductive organs failed to reveal significant differences between
Neuralized null animals and wild-type controls. When analyzed
by histological staining, testes derived from Neuralized null
animals showed proper spermatogenesis and the appearance
of mature sperm in the seminiferous tubules (Fig. 6A and B).
In addition, no defect in the histology of the prostate could be
detected (data not shown).
However, a striking difference between Neuralized wild-type
and null animals was detected when sperm motility and mor-
phology of epididymal spermatozoa were analyzed. Spermato-
zoa isolated from wild-type or Neuralized heterozygous animals
showed a homogenous population of intact sperm with high
mobility. In contrast, spermatozoa isolated from Neuralized
null animals showed only marginal motility, with a large ma-
jority of spermatozoa in a sample showing no detectable mo-
tility under standard light microscopic analysis. In addition, we
frequently observed spermatozoa with missing head structures
(Fig. 6D, arrows)
Electron microscopy analysis of cauda epididymal sperma-
FIG. 4. Loss of full-length Neuralized transcript in Neuralized knock-
out 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 aber-
rantly 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.
VOL. 21, 2001TARGETED DELETION OF MURINE Neuralized7487
tozoa revealed the presence of structural defects of the sper-
matozoa involving the flagellum. Cross sections through dif-
ferent regions of the flagellum showed that the middle piece
appeared consistently normal, being composed of the appro-
priate 9?2 configuration of doublets (Fig. 7A). Morphological
aberrations occurring in the flagellum were most commonly
seen in the principal piece and primarily involved the structural
integrity of the axoneme (Fig. 7B to D). These defects were
readily observed in the cross-sectional profiles of the axoneme
and were also noticeably displayed by longitudinal sections
through the flagellum. The most prevalent structural abnor-
mality detected involved missing axonemal doublets that var-
ied from either loss of one doublet (usually no. 7) to loss of half
the axonemal complex (usually no. 4 to 7). Other, less common
morphological defects included translocation of doublets out-
side of the axonemal complex, total disorganization of the
axoneme, and dislocation of dense fibers. In addition, for some
spermatozoa, regions of the fibrous sheath were found to be
malformed or entirely missing, and this was often accompanied
by disruption of the axonemal complex. A large number of
spermatozoa were found to have developed double or some-
times triple axonemes that usually were defective. Moreover,
these axonemal aberrations not only were now confined to the
principal piece but also occurred in the midpiece of the flagel-
lum. Abnormal axonemes were detected in approximately 30%
of flagellar cross sections through the principal piece. This
does not suggest, however, that normal axonemes prevail in the
remaining 70% of the cross sections. Longitudinal sections
through the flagellum clearly showed that structural defects of
the axonemal complex were localized within the principal piece
(Fig. 7F and G). Thus, depending on where the flagellum was
sectioned, the axonemal profile could appear normal even if
the principal piece was structurally compromised elsewhere
along the length of the flagellum. Taken together, these aber-
rations are expected to significantly compromise the integrity
of the flagellum and thus the motility of the spermatozoa.
We also performed an ultrastructural analysis of the testes of
Neuralized null mice to establish whether abnormal flagellar
development could be detected in germ cells during spermio-
genesis. In the lumen of seminiferous tubules, cross sections
through the middle and principal piece of testicular sperma-
tozoa mostly showed a normal organization of the axoneme.
Longitudinal and grazing planes of sections through the
flagellum, however, provided evidence that regions of the
axonemal complex were in fact clearly disorganized (Fig.
7H). Fine-structure analysis of spermatids during late stages of
maturation showed that although flagellar development ap-
peared morphologically normal in some spermatids, other
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-hNeuralized are indistinguishable from control-infected cells.
7488VOLLRATH ET AL.MOL. CELL. BIOL.
spermatids showed distinct aberrations in the organization and
structure of the burgeoning neck piece.
Neuralized null females fail to lactate and successfully nurse
their pups and have defective mammary gland development
during lactation. In analyzing the fertility of Neuralized female
mice, we observed that pups born from Neuralized null females
in matings with either wild-type control or Neuralized heterozy-
gous males failed to survive beyond day 3 after birth. In addi-
tion, litters from Neuralized null females were often scattered
throughout the cage and we failed to detect milk in the stom-
achs of the pups, whereas pups in control litters were clearly
nursing and had milk in their stomachs. This suggests that
Neuralized female mice were fully fertile and able to support
pregnancies to full term but had defects in lactation or mater-
nal behavior, leading to defective nursing. Since pups of all
genotypes are able to actively nurse and survive until adult-
hood when born from heterozygous or wild-type mothers, this
observed nursing defect was clearly maternal. We therefore
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).
VOL. 21, 2001 TARGETED DELETION OF MURINE Neuralized7489
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
7490 VOLLRATH ET AL.MOL. CELL. BIOL.
analyzed the mammary glands of Neuralized null females on
day 1 of lactation (L1). Mammary glands from Neuralized null
females were clearly defective on L1, with significantly less
alveolar structures penetrating the mammary fat pad com-
pared to the glands from wild-type control animals at the same
stage of lactation (Fig. 8B and C). In addition, some samples
from Neuralized null females showed defective lipid production
in the ducts (Fig. 8D). This result shows that the failure to
properly nurse their pups and the observed death early after
birth is clearly related to a severely underdeveloped mammary
gland at lactation in Neuralized female animals.
The defective maturation of mammary glands during preg-
nancy in Neuralized null animals is consistent with expression
of Neuralized in this tissue. Using Northern blot hybridization
of mRNA derived from mammary glands at various stages of
maturation and development, we could detect Neuralized tran-
scripts in virgin mammary glands as well as during the earlier
stages of pregnancy (Fig. 8A). We failed to detect Neuralized
transcripts at very late stages of pregnancy and during lacta-
tion. However, this is a frequently observed phenomenon since
mammary glands at these stages produce vast amounts of milk
proteins, resulting in lactation-related transcripts outcompet-
ing any other transcripts that might be present.
Neuralized heterozygous and null animals do not develop
malignancies at a significantly higher frequency than wild-
type controls. Human Neuralized is localized on chromosome
10q24–25, a region showing frequent deletion in malignant as-
trocytomas, leading to the hypothesis that the Neuralized gene
is involved in the formation of malignant tumors of the CNS in
humans (28). In addition, loss of Neuralized transcript has been
found in astrocytoma tissue derived from human patients and
in glioblastoma cell lines, suggesting that loss of Neuralized
transcription might be associated with malignant transforma-
tion (28). Therefore, we analyzed Neuralized null animals for
the development of tumors, particularly in the CNS. The oldest
animals in our colony are now older than 1 year. Neuralized
null animals did not appear to become moribund at a rate
significantly higher than their wild-type littermates, and we
failed to detect CNS malignancies at a rate above background.
Of 15 Neuralized null animals analyzed by histological exami-
nation, 1 showed a pituitary adenoma that invaded upwards
from the sella turcica into the base of the brain. This tumor was
characterized as a gonadotroph adenoma with nuclear immu-
noreactivity for steroidogenic factor 1 and cytoplasmic staining
for FSH and LH. Since this is a tumor occasionally found in
older wild-type animals (34), we are unable to definitively
conclude that Neuralized null animals show a predisposition to
tumor development in the CNS. The proposed link between
loss of Neuralized expression and tumor formation in humans
is therefore not yet supported by our mouse model.
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
Sequence similarity searches in the database using the
BLAST algorithm revealed significant sequence homology in
the ring finger domain between Neuralized proteins and pro-
teins 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 ap-
optosis by various stimuli in vitro (25, 42). 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 Neu-
ralized 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 ubiq-
uitin-conjugating enzyme-mediated ubiquitination of receptor
protein tyrosine kinases, leading to termination of signaling
through protein degradation (20, 21, 26). Interestingly, IAP
proteins have now also been shown to catalyze their own ubiq-
uitination 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 ubiq-
uitin protein ligase activity.
Targeted deletion of the murine Neuralized gene reveals that
expression of murine Neuralized is not essential for develop-
ment 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, 17, 18, 41). 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. How-
ever, two other possibilities must be considered. (i) Neuralized
function is essential for Notch signaling, as suggested from
genetic evidence in Drosophila, but other vertebrate Neural-
ized homologues or unrelated proteins compensate for loss of
the Neuralized gene isolated here. A search of sequence data-
bases 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
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 ap-
parent from grazing planes of sections that in localized regions of the flagellum, the axonemal complex is disorganized (arrowheads). Bars, 0.2 ?m.
VOL. 21, 2001TARGETED DELETION OF MURINE Neuralized 7491
complete, this possibility cannot be ruled out. (ii) The Neural-
ized 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 do-
mains 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 Neu-
ralized transcript, we think that this possibility is highly un-
The human Neuralized homologue was isolated from a re-
gion at chromosome 10q24–25 that shows frequent alterations
in malignant astrocytomas. In addition, loss of Neuralized tran-
scription 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 develop-
ment of CNS tumors. Thus far, our Neuralized null mice have
failed to develop any gliablastomas or astrocytomas. This ob-
servation questions the proposed link between loss of Neural-
ized transcription and malignant transformation in the CNS.
Neuralized null females failed to nurse their pups and sup-
port 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 dif-
ferences 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 preg-
nancy. 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. In-
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.
7492 VOLLRATH ET AL.MOL. CELL. BIOL.
terestingly, transgenic mice expressing constitutive active al-
leles of the Notch receptor also fail to lactate, with lobular
development being retarded during late stages of pregnancy
(15, 19). 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 fla-
gella 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 ??-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. Interest-
ingly, results from C. elegans indicate that the presenilin family
member spe-4 is involved in tubulin localization during sper-
matogenesis (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 micro-
tubule 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. Anal-
ysis 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 orien-
tation of the centrioles with proper migration to the posterior
pole of the nucleus appeared to be normal during initial de-
velopment of the flagellum. It is therefore likely that the ob-
served defects in axonemal structure occurred during subse-
quent growth and formation of the nascent flagellum. A
striking feature of the morphology of spermatozoa from Neu-
ralized 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 sper-
miogenesis 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 ab-
normalities seen in Neuralized null mice in part mimic the
defects seen in many human spermatogenic disorders (32, 45,
46). Thus, Neuralized null mice may thus be a valid model to
study human infertility syndromes and to gain a better under-
standing of male infertility.
We thank H. Nakamura for providing the full-length human Neu-
ralized cDNA clone, Charles Murtaugh and Andrew Lassar for pro-
viding the constitutive active Notch allele, Diana Hayward for provid-
ing the CBF1-luciferase reporter constructs, and Rachel Neve for
providing the PC-12 cell line. We also thank Jan Pinkas for help with
the analysis of the mammary gland phenotype, Frank Kuo for advice
on the initial analysis of the pituitary glands, Kelvin So (Toronto) for
histological analysis of the pituitaries, Richard V. Pierce for advice in
the early stages of spermatozoa analysis, and members of the Leder
laboratory and the HMS Department of Genetics for helpful com-
ments and suggestions throughout the project.
ADDENDUM IN PROOF
While this paper was in proof, Ruan et al. reported the
isolation of a mouse Neuralized homolog and described the
phenotype of the knockout mouse (Y. Ruan, L. Tecott, M. M.
Jiang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA
98:9907–9912, 2001). The gene described in this report is iden-
tical to the gene described in our study with the exception of
the amino-terminal 28 amino acids. We postulated in our study
the existence of two splice variants of the mouse Neuralized
gene, and comparison of the two sequences clearly suggests
that this is the case. Two differences between our observations
and those of Ruan et al. are noteworthy. First, Ruan et al.
failed to detect any expression of mouse Neuralized in adult
skeletal muscle and did not show convincingly expression in the
developing somites. We believe that this is due to the fact that
Ruan et al. used a probe from the very 5? end of the cDNA in
these experiments, thus presumably detecting only one splice
variant, while our probe is expected to detect both splice vari-
ants. Second, Ruan et al. report normal reproductive behavior
in both male and female mice. This is clearly distinct from our
observations, and the cause for this discrepancy is currently
unknown. However, strain differences or differences in the
targeting strategy could account for these different observa-
tions. Further analysis is required to clarify these issues.
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