Notch pathway genes are expressed in mammalian ovarian follicles

Gene expression pattern

Notch pathway genes are expressed in mammalian ovarian follicles

Joshua Johnsona, Tamara Espinozaa, Robert W. McGaugheya,b,Alan Rawlsa,b, Jeanne Wilson-Rawlsa,b,*

aDepartment of Biology, Arizona State University, Tempe, AZ 85287-1501, USAbGraduate Program in Molecular and Cellular Biology, Arizona State University, Tempe, AZ 85287-1501, USA

Received 9 March 2001; received in revised form 26 July 2001; accepted 13 August 2001

Abstract

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation.

This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle

growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are

beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to

regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed

that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was

expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch

signaling pathway in the regulation of mammalian folliculogenesis. q 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Notch; Jagged; Ovary; Follicle; Granulosa

1. Results and discussion

During the reproductive lifespan of mammals there is

continuous follicle development within the ovary. Follicu-

logenesis involves the proliferation of granulosa cells into a

multilaminar epithelium and the development of a fluid

filled antrum. Granulosa cells differentiate into two cell

types, mural granulosa cells that lie close to the basal lamina

and extend to the antrum, and cumulus granulosa cells that

surround the oocyte. The growth and maturation of the

oocyte is tightly coordinated with the proliferation and

differentiation of granulosa cells (Gosden et al., 1997;

Drummond and Findlay, 1999; Rodgers et al., 1999; Erick-

son and Shimasaki, 2000).

The Notch genes encode highly conserved transmem-

brane receptors (Kimble and Simpson 1997; Lewis, 1998;

Artavanis-Tsakonis et al., 1999) that control cell-fate deci-

sions between adjacent cells that may or may not be equiva-

lent. Notch was initially identified in Drosophila (Wharton

et al., 1985), and homologs have been found in C. elegans,

and vertebrates (Notch1-4) (Weinmaster et al., 1991, 1992;

Franco del Amo et al., 1992; Lardelli and Lendahl, 1993;

Lardelli et al., 1994; Uyttendaele et al., 1996). There are two

families of Notch ligands that are also transmembrane

proteins. In vertebrates, they are Deltalike-1, -3, and -4

(Dll-1, 3, 4) (Bettenhausen et al., 1995; Dunwoodie et al.,

1997; Shutter et al., 2000), and Jagged1 and -2 (Lindsell et

al., 1995; Shawber et al., 1996).

As both Notch and its ligands are transmembrane

proteins, signaling only occurs between closely apposed

cells. Activation of Notch by ligand binding triggers clea-

vage of the receptor, releasing the intracellular domain,

which then translocates to the nucleus (Schroeter et al.,

1998; Kopan et al., 1996; Blaumueller et al., 1997; Struhl

and Adachi, 1998). In the nucleus, Notch forms transcrip-

tional complexes with transcription factors of the CSL

family (Weinmaster, 1997; Greenwald, 1998). The expres-

sion of Notch1-4 in the ovary was determined by in situ

hybridization of ovaries from 6 week old mice using

gene-specific digoxygenin-labeled antisense RNA probes.

Gene expression was analyzed using the follicle staging

scheme of Pedersen and Peters (1968). Notch2 transcripts

were initially detected in type 3b follicles (Fig. 1A,B) and

this gene was expressed up to type 8. There was a loss of

Notch2 expression in the cells that are coincident with the

sites of initial antral pocket formation and in periantral gran-

ulosa cells (Fig. 2A, black arrows). Oocytes did not express

Notch2 (Fig. 1B). Notch3 was expressed in an overlapping

Mechanisms of Development 109 (2001) 355–361

0925-4773/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0925-4773(01)00523-8

www.elsevier.com/locate/modo

* Corresponding author. Department of Biology, Box 871501, Tempe,

AZ 85287-1501, USA. Tel.: 11-480-727-6675; fax: 11-480-965-2519.

E-mail address: [email protected] (J. Wilson-Rawls).

pattern with Notch2 in the granulosa cells. Notch3 was

expressed in all granulosa cells of type 4a-type 8 follicles

(Fig. 2A). Again, oocytes did not express this gene. Inter-

estingly, Notch3 was also expressed in corpora lutea.

Notch1 and Notch4 were expressed in the ovary, but were

restricted to the vasculature (data not shown).

The expression patterns of Notch ligands were also deter-

mined. Jagged2 was expressed in all granulosa cells of

type3b-4 follicles (Fig. 3B), but its expression was restricted

in larger follicles. In type 5a and 5b follicles, there was a

loss of Jagged2 expression in the presumptive cumulus

granulosa cells and in the cells that lie in an analogous

position to the site of formation of the antral pockets (Fig.

3C), similar to that observed with Notch2. In follicles of

types 6–8, there was a band of Jagged2-positive cells in

the central layers of the epithelium granulosum, but not in

the periantral or cumulus granulosa cells. There was much

reduced expression in mural cells closest to the basal lamina

of type 8 follicles (Fig. 3A,B).

Jagged1 was expressed exclusively in oocytes. Jagged1

transcripts were initially detectable in oocytes of type 3b

follicles (Fig. 4A,B). Expression was maintained in follicles

of types 4-5b (Fig. 4C,D), but decreased markedly in type 6

follicles with very small antral pockets (Fig. 4E,F). Jagged1

transcription was not detected in type 7 or 8 follicles. The

overlapping patterns of expression of Notch receptors and

ligands are similar to the expression of this gene family

during vertebrate embryogenesis (Lardelli and Lendahl,

1993; Williams et al., 1995; Lindsell et al., 1995, 1996;

J. Johnson et al. / Mechanisms of Development 109 (2001) 355–361356

Fig. 1. Notch2 mRNA was detected in developing ovarian follicles. Serial

sections from 6 week old CD-1 mouse ovaries were incubated with an

antisense RNA digoxygenin-labeled Notch2 probe. After washing, hybri-

dized probe was detected using an anti-digoxygenin antibody coupled with

alkaline phosphatase and the BM purple substrate. (A) Follicle type is

indicated and was determined by examination of serial adjacent sections.

Notch2 transcripts were detected in follicles beginning at type 3b, a grow-

ing oocyte with a complete ring of cuboidal granulosa cells. Transcripts

continued to be detected in all granulosa cells in growing follicles up to type

5b, the largest preantral follicle type. At this stage, granulosa cells that are

coincident with the site of initial antral pocket formation lose expression

(see follicle 5b with black arrows). Once an antrum has formed there is no

detectable Notch2 expression in periantral granulosa cells (follicle type 6,

black arrows). Corpora lutea (CL) do not express Notch2. Dark field micro-

scopy, 50 £ magnification. (B) Detail of type 3b follicle. Higher magnifi-

cation of a small growing follicle demonstrating expression of Notch2 in

the granulosa cells, not the oocyte or surrounding cells. Dark field micro-

scopy at 100 £ magnification. (C) Detail of type 7 follicle. This follicle

demonstrates expression of Notch2 in cumulus (CG) and mural (MG) gran-

ulosa cells, note the decreased levels of expression near the basal lamina.

The theca (T) does not express Notch2. Bright field Hoffman optics, 100 £

magnification.

Fig. 2. Expression of Notch3 in ovarian follicles. Notch3 mRNA was

detected in the granulosa cells of follicles over a wide developmental

range from type 4 follicles to type 8 follicles (not shown). Notch3 was

the only member of this gene family to be expressed consistently in CL.

Notch3 mRNA was detected in granulosa cells throughout the follicle.

Oocytes do not express Notch3. Panels (A,B) were photographed at 50 £

magnification using dark field illumination. Panel (B) no probe control, all

experiments included this control. 25 £ magnification, dark field illumina-

tion.

Shawber et al., 1996; Guillemot and Joyner, 1993). Neither

Dll1 nor Dll3 were detectable in the mouse ovary.

In response to Notch activation, basic helix-loop-helix

(bHLH) transcription factors of the Drosophila enhancer

of split complex [E(spl)] and their mammalian homologs,

hairy enhancer of split -1 (HES-1) and HES-5, and a related

family of bHLH genes variously referred to as Hesr, HRT,

Hey, CHF, and Gridlock, are up-regulated, and have been

demonstrated to be downstream target genes of Notch

signaling (Jarriault et al., 1995, 1998; Hsieh et al., 1996;

Ohtsuka et al., 1999; Nakagawa et al., 1999, 2000; Kokubo

et al., 1999; Leimeister et al., 1999; Chin et al., 2000; Maier

and Gessler, 2000; Zhong et al., 2000). We examined the

expression pattern of Hesr-1 and Hesr-2 in ovaries from 6

week old CD-1 mice by in situ hybridization. Transcripts for

these downstream target genes were detected in the granu-

losa cells of follicles from types 3b–8 (Fig. 5). Interestingly,

there was increased expression in the cells closest to the

oocyte in preantral follicles (Fig. 5B,D). HES-5 and Hesr-

3 demonstrate identical expression patterns to these genes

(data not shown). These data demonstrate that the Notch

receptor and ligand proteins are translated and acting on

their downstream targets, however their role in folliculogen-

esis remains to be determined.

Many genes that are important for folliculogenesis

demonstrate cell type restricted expression. For example,

Kit is expressed in oocytes, however, its expression is main-

tained throughout folliculogenesis, and in large antral folli-

cles it is also expressed in theca cells (Manova et al., 1990).

Other oocyte restricted genes include the TGF-b family

members GDF-9 and BMP-15 (Dong et al., 1996; Elvin et

al., 1999a; Dube et al., 1998; Laitinen et al., 1998; Ohtsuka

et al., 1999). Interestingly, Cox-2, which is necessary for

cumulus expansion, becomes restricted in antral follicles

to the cumulus granulosa cells, and is induced by GDF-9

J. Johnson et al. / Mechanisms of Development 109 (2001) 355–361 357

Fig. 3. Jagged2 demonstrated a dynamic expression pattern in granulosa cells. Jagged2 was expressed in the granulosa cells of follicles as assayed by in situ

hybridizations. Serial sections from 6 week old CD-1 ovaries were incubated with antisense digoxygenin-labeled Jagged2 probe. After washing, hybridized

probe was detected using an anti-digoxygenin antibody coupled with alkaline phosphatase and the BM purple substrate. Jagged2, a Notch ligand, was

expressed in granulosa cells of developing follicles. (A) Jagged2 mRNA was detected in type 6 and type 7 follicles in the center layers of mural granulosa cells.

Note the lack of signal in the centermost layers of the epithelium granulosum and near the basal lamina. (B) Type 8 follicles. There was a loss of Jagged2

mRNA in the cumulus granulosa cells closest to the oocyte, and a loss of expression in periantral mural granulosa cells, as indicated by the white arrow. (C) In

preantral follicles, types 5a and 5b, there was a loss of expression in areas coincident with the site of initial antral pocket formation, and in the granulosa cells

nearest the oocyte. Black arrows indicate granulosa cells that have lost expression. Panels (A,B) were photographed at 50 £ and panel (C) at 100 £

magnification using dark field illumination.

Fig. 4. Jagged1 was expressed exclusively in oocytes. Jagged1 was

expressed in the granulosa cells of follicles as assayed by thick section in

situ hybridizations in serial sections from 6 week old CD-1 ovaries. We

have detected strong staining in type 3b follicles (panels A,B), and expres-

sion was maintained in follicles up to 5b (panels C,D). There was weak

staining in early antral or type 6 follicles (panels E,F). Oocytes often

appeared to have some positive signal in dark field, but when viewed in

bright field this was found to be artifactual, so both dark and bright field

illumination is presented here. Panels (A,C,E) were photographed using

dark field illumination, panels (B,D,F) are corresponding DIC photographs.

Panels (A–D) were photographed at 100 £ magnification and panels (E,F)

were photographed at 50 £ magnification.

(Sirois et al., 1992; Sirois, 1995; Elvin et al., 1999b). In the

ovary, kit ligand is expressed only by granulosa cells, and it

becomes restricted to the mural population in antral follicles

(Manova et al., 1993; Joyce et al., 1999). While there are

genes whose expression is restricted to the oocyte or specific

populations of granulosa cells, there are none that have

identical expression patterns to that of Jagged1 or Jagged2.

Nor are there any reports of genes that demonstrate a loss of

expression in areas coincident with the sites of formation of

the antral pockets, as observed for Notch2 and Jagged2.

Atresia in ovarian follicles is marked by the apoptotic

death of the granulosa cells (Hsu and Hsueh, 1998;

McGee et al., 1998). Notch signaling inhibits apoptosis in

hematopoietic cells (Varnum-Finney et al., 1998; Jones et

al., 1998; Deftos et al., 1998; Jehn et al., 1999; Shelly et al.,

1999; Han et al., 2000). Further, a Notch2 mutant that is

signaling defective demonstrated increased apoptosis

during embryogenesis (Hamada et al., 1999). We reasoned

that it was likely that the Notch genes were expressed in

growing, not atretic follicles. Since, treatment with exogen-

ous gonadotropin (PMSG) will induce the proliferation and

differentiation of granulosa cells and increase the number of

developing follicles by rescuing them from atresia, Notch2

expression was compared with TUNEL and BrdU labeling

of ovaries. Mice were given PMSG intraperitoneally and

ovaries were harvested 6 h post-treatment. Serial ovary

sections were alternately placed on slides for in situ hybri-

dization to detect Notch2, or for TUNEL labeling to detect

apoptotic cells. As a comparison, mice were injected with

BrdU to label proliferating cells 1 h before the ovaries were

harvested.

We found that Notch2 was expressed in follicles that were

also TUNEL-positive, however, the level of expression was

greatly reduced (Fig. 6, compare follicles indicated by black

arrows in panels B and D to the follicle with the red arrow).

Further, many follicles were Notch2-positive, and relatively

few were TUNEL-positive. As expected, PMSG induced the

proliferation of granulosa cells in a wide range of follicles at


Fig. 5. Expression of downstream target genes of activated Notch in gran-

ulosa cells. Hesr-1 and Hesr-2 were expressed in the granulosa cells of

follicles as assayed by thick section in situ hybridizations. Serial sections

from 6 week old CD-1 ovaries were incubated with antisense digoxygenin-

labeled gene-specific probes. (A) Hesr-1 was expressed in the granulosa

cells of follicles from type 3b up to the preovulatory stage (type 8, not

shown). This target gene was expressed in all granulosa cells. 50 £ magni-

fication. (B) Higher magnification to show lack of staining in the oocyte and

the positive signal in the surrounding cuboidal granulosa cells. Note the

increased intensity of signal close to the oocyte in preantral follicles (black

arrows in A,B). 100 £ magnification. (C,D) Hesr-2 was expressed in gran-

ulosa cells of type 3b to type 8 follicles. (C) is 50 £ and (D) is 100 £

magnification. All photographs were taken using dark field illumination.

Fig. 6. Notch gene expression is induced in growing follicles. CD-1 mice

were stimulated with PMSG and ovaries were harvested at 6 h post-PMSG

administration and serial sections were alternately placed on slides. These

slides were either processed for in situ hybridization using a probe to detect

Notch2 expression, or were processed for terminal deoxyribonucleotidyl

transferase end labeling (TUNEL) detection of apoptosis. Alternatively,

the mice were given PMSG and BrdU was administered at 5 h post -

PMSG intraperitoneally. After harvesting ovaries, an anti-BrdU antibody

was used to detect proliferating cells immunohistochemically. (A) In situ

hybridization to detect Notch2 mRNA. Note the many positively stained

follicles from type 5a through type 8 in this section. There are also two

corpora lutea that do not express Notch2. (B) Higher magnification of

section in panel (A). The follicles indicated by the black arrows demon-

strate morphologic evidence of atresia. They also have greatly reduced

expression of Notch2 compared to an adjacent similar type of follicle

(see red arrow). (C) TUNEL detection of apoptosis. The brown stain indi-

cates cells undergoing apoptosis. (D) Higher magnification of follicles from

(C), note the same follicles, indicated by black arrows, that had greatly

decreased Notch2 specific signal in (B), have apoptotic granulosa cells as

assayed by TUNEL. In contrast, the follicle indicated by the red arrow, was

negative in this assay. (E) TUNEL control section. No terminal deoxyribo-

nucleotidyl transferase was added to the TUNEL reaction. (F) BrdU prolif-

eration assay. At 6 h post-PMSG there were many proliferating granulosa

cells in a wide range of follicles, as evidenced by the brown stain. Slides

were counter stained with hematoxylin. Panels (A,C,E) were photographed

at 14.5 £ magnification; panels (B,D,F) were photographed at 50 £ magni-

fication.

this same timepoint (Fig. 6F). Since, many follicles were

Notch-positive and few were TUNEL-positive, it is likely

that Notch2 expression is upregulated in developing, not

atretic follicles. However, a role in the induction of apop-

tosis cannot be ruled out.

Our data demonstrate that Notch family genes are

expressed in mammalian ovarian follicles. The Notch signal-

ing pathway has been repeatedly demonstrated to play a

crucial developmental role in tissues where it is expressed,

therefore, we propose that Notch signaling is implicated in

regulating folliculogenesis. Perturbation of this pathway in

developing follicles will be necessary to provide insight into

its functional role(s) during folliculogenesis.

2. Materials and methods

2.1. Animal use and exogenous hormone treatment

CD-1 mice were used for all studies and were 6 weeks old

(42 days) unless otherwise stated. For all in situ hybridiza-

tion experiments, ovaries were immediately explanted into

ice-cold PBS, then processed for individual experiments as

described below. For animals stimulated with pregnant

mare’s serum gonadotropin (PMSG) female mice were

injected intraperitoneally with 5 IU of PMSG (Sigma

Chemical Co., St. Louis, MO) and ovaries were collected

6 h post hormone treatment.

2.2. Staging of follicles

Follicles were staged using the scheme of Pedersen and

Peters (1968). This scheme sets out eight types of follicles

based on the size of the oocyte and the number of granulosa

cells in the widest cross-section. Type 1 is a small oocyte with

no attached follicle cells. Type 3a is a small oocyte with a

complete ring of cells, but not more than 20 cells are present

in the largest cross-section. Type 3b is a growing oocyte with

21–60 follicle cells, the largest preantral follicle is type 5b,

this stage has a fully grown oocyte (70 mm diameter) and up

to 4000 follicle cells. Early antral follicles are types 6 and 7,

and Graafian follicles, are type 8. All follicles were staged

from serial adjacent sections using this scheme.

2.3. Thick section in situ hybridization

Sections for in situ hybridization were prepared after Xu

and Wilkinson (1995). Ovaries were fixed overnight in 4%

paraformaldehyde in phosphate buffered saline (PBS) at

48C, then were embedded in paraffin after stepwise dehydra-

tion in ethanol and Hemo-DE tissue clearing agent (Fisher

Biotech, Pittsburgh, PA). Twenty to thirty micron (mm)

sections were cut perpendicular to the axis of entry of the

ovarian blood vessels. Sections were then dewaxed, rehy-

drated, and in situ hybridizations were done as described in

Wilson-Rawls et al. (1999). The sections were hybridized

with antisense digoxygenin-11-UTP-labeled (Roche Mole-

cular Biochemicals, Indianapolis, IN) RNA probes specific

to each of the Notch signaling pathway genes, at 628C.

Detection of the hybridized probe was done using an alka-

line-phosphatase conjugated anti-digoxygenin antibody to

recognize hybridized probe after washing (Roche Molecular

Biochemicals, Indianapolis, IN). The color reaction used to

visualize gene expression was done using the BM purple

alkaline phosphatase substrate (Roche Molecular Biochem-

icals, Indianapolis, IN). Control samples were prepared to

determine non-specific background labeling by omitting

probe from the hybridization buffer. Further controls

included testing sense probes on ovary sections and all anti-

sense probes were tested on embryo controls for specificity.

2.4. Terminal deoxynucleotidyl transferase-mediated

dUTP-fluorescein end labeling (TUNEL) detection of

apoptosis

TUNEL detection of apoptosis in ovary sections was

performed as per Li et al. (1998), with the following modi-

fications: 10 nM fluorescein-labeled UTP (Roche Molecular

Biochemicals, Indianapolis, IN) was used in the end-label-

ing reaction mixture, and horseradish-peroxidase-conju-

gated anti-fluorescein antibody (Roche Molecular

Biochemicals, Indianapolis, IN) diluted 1:100 in the digox-

ygenin blocking reagent was used to visualize. Negative

control samples were prepared by omitting TdT enzyme

(Promega Corp., Madison, WI) from the labeling reactions.

2.5. BrdU labeling of ovaries

Mice were injected with PMSG as described above. Thirty

minutes before the collection of the ovaries a suspension of

50 mg BrdU and 10 mg FUDR/kg body weight, was injected

intraperitoneally. Ovaries were collected, fixed and paraffin-

embedded as described above, then sectioned to 7 mm.

Detection of BrdU/FUDR incorporation was performed

using a streptavidin-biotin BrdU detection kit (ZYMED,

South San Francisco, CA) per the manufacturer’s instruc-

tions, using the optional heat-induced epitope retrieval

step. Color reactions were done using the horseradish perox-

idase substrate, DAB, resulting in a dark brown stain in

proliferating cells. The slides were counterstained with

hematoxylin.

Acknowledgements

We would like to thank Drs R.L. Johnson and H. Kokubo

for Hesr gene probes. We would like to thank Drs B.

Vanderhyden and P. Hunt for helpful discussions. We

would also like to acknowledge imaging support from the

W.M. Keck Bioimaging and Life Sciences Visualization

laboratories at ASU. A.R. is supported by grants from the

Muscular Dystrophy Association and NSF. J.W.R. is

supported by NIH, T.E. is supported by a MARC program

grant to ASU from the NIH.


References

Artavanis-Tsakonis, S., Rand, M.D., Lake, R.J., 1999. Notch Signaling:

Cell fate control and signal integration in development. Science 284,

770–776.

Bettenhausen, B., Hrabe de Angelis, M., Guenet, J.L., Gosler, A., 1995.

Transient and restricted expression during mouse embryogenesis of

Dll1, a murine gene closely related to Drosophila Delta. Development

21, 2407–2418.

Blaumueller, C.M., Qi, H., Zagouras, P., Artavanis-Tsakonis, S., 1997.

Intracellular cleavage of Notch leads to a heterodimeric receptor on

the plasma membrane. Cell 90, 281–291.

Chin, M.T., Maemura, K., Fukumoto, S., Jain, M.K., Layne, M.D., Wata-

nabe, M., Hsieh, C.M., Lee, M.E., 2000. Cardiovascular basic helix

loop helix factor 1, a novel transcriptional repressor expressed prefer-

entially in the developing and adult cardiovascular system. J. Biol.

Chem. 275, 6381–6387.

Deftos, M., He, Y.W., Ojala, E.W., Bevan, M.J., 1998. Correlating notch

signaling with thymocyte maturation. Immunity 9, 777–786.

Dong, J.D., Albertini, F., Nishimori, K., Kumar, T.R., Lu, N., Matzuk,

M.M., 1996. Growth differentiation factor-9 is required during early

ovarian folliculogenesis. Nature 383, 531–535.

Drummond, A.E., Findlay, J.K., 1999. The role of estrogen in folliculogen-

esis. Mol. Cell Endocrinol. 151, 57–64.

Dube, J.L., Wang, P., Elvin, J., Lyons, K.M., Celeste, A.J., Matzuk, M.M.,

1998. The bone morphogenetic protein 15 gene is X-linked and

expressed in oocytes. Mol. Endocrinol. 12, 1809–1817.

Dunwoodie, S.L., Henrique, D., Harrison, S.M., Beddington, R.S., 1997.

Mouse Dll3: a novel divergent Delta gene which may complement the

function of other Delta homologues during early pattern formation on

the mouse embryo. Development 16, 3065–3076.

Elvin, J.A., Yan, C., Wang, P., Nishimori, K., Matzuk, M.M., 1999a. Mole-

cular characterization of the follicle defects in the growth differentiation

factor 9-deficient ovary. Mol. Endocrinol. 13, 1018–1034.

Elvin, J.A., Clark, A.T., Wang, P., Wolfman, N.M., Matzuk, M.M., 1999b.

Paracrine actions of growth factor-9 in the mammalian ovary. Mol.

Endocrinol. 13, 1035–1048.

Erickson, G.F., Shimasaki, S., 2000. The role of the oocyte in folliculogen-

esis. Trends Endocrinol. Metab. 11, 193–198.

Franco del Amo, F., Smith, D.E., Swiatek, P.J., Gendron-Maguire, M.,

Greenspan, R.J., McMahon, A.P., Gridley, T., 1992. Expression pattern

of Motch, a mouse homolog of Drosophila Notch, suggests an important

role in early postimplantation mouse development. Development 115,

737–744.

Gosden, R., Krapez, J., Briggs, D., 1997. Growth and development of the

mammalian oocyte. Bioessays 19, 875–882.

Greenwald, I., 1998. Lin-12/Notch signaling: lessons from worms and flies.

Genes Dev. 12, 1751–1762.

Guillemot, F., Joyner, A.L., 1993. Dynamic expression of the murine

Achaete-Scute homologue Mash-1 in the developing nervous system.

Mech. Dev. 42, 171–185.

Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J.R., Tsuji-

moto, Y., 1999. Mutation in ankyrin repeats of the mouse Notch2 gene

induces early embryonic lethality. Development 126, 3415–3424.

Han, W., Ye, Q., Moore, M.A., 2000. A soluble form of human Delta-like -

1 inhibits differentiation of hematopoietic progenitor cells. Blood 95,

1616–1625.

Hsieh, J.J.-D., Henkel, T., Salmon, P., Robey, E., Peterson, M.G., Hayward,

S.D., 1996. Truncated mammalian Notch1 activates CBF1/RBP-Jk-

repressed genes by a mechanism resembling that of Epstein-Barr

virus EBNA2. Mol. Cell Biol. 16, 952–959.

Hsu, S.Y., Hsueh, A.J., 1998. Intracellular mechanisms of ovarian cell

apoptosis. Mol. Cell Endocrinol. 145, 21–25.

Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., Israel, A.,

1995. Signalling downstrean of activated mammalian Notch. Nature

377, 355–358.

Jarriault, S., Le Bail, O., Hirsinger, E., Pourquie, O., Logeat, F., Strong,

C.F., Brou, C., Seidah, N.G., Israel, A., 1998. Delta-1 activation of

notch-1 signaling results in HES-1 transactivation. Mol. Cell Biol. 18,

7423–7431.

Jehn, B.M., Bilke, W., Pear, W.S., Osborne, B.A., 1999. Protective effects

of notch-1 on TCR-induced apoptosis. J. Immunol. 162, 635–638.

Jones, P., May, G., Healy, L., Brown, J., Hoyne, G., Delassus, S., Enver, T.,

1998. Stromal expression of Jagged 1 promotes colony formation by

fetal hematopoietic progenitor cells. Blood 92, 1505–1511.

Joyce, I.M., Pendola, F.L., Wigglesworth, K., Eppig, J.J., 1999. Oocyte

regulation of kit ligand expression in mouse ovarian follicles. Dev.

Biol. 214, 342–353.

Kimble, J., Simpson, P., 1997. The lin-12/Notch signaling pathway and its

regulation. Annu. Rev. Cell. Dev. Biol. 13, 333–361.

Kokubo, H., Lun, Y., Johnson, R.L., 1999. Identification and expression of

a novel family of bHLH cDNAs related to Drosophila Hairy and Enhan-

cer of Split. Biochem. Biophys. Res. Commun. 260, 459–465.

Kopan, R., Schroeter, E.H., Weintraub, H., Nye, J.S., 1996. Signal trans-

duction by activated mNotch: importance of proteolytic processing and

its regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA

93, 1683–1688.

Laitinen, M., Vuojolainen, K., Jaatinen, R., Ketola, I., Aaltonen, J., Lehto-

nen, E., Heikinheimo, M., Ritvos, O., 1998. A novel growth differentia-

tion factor-9 (GDF-9) related factor is co-expressed with GDF-9 in

mouse oocytes during folliculogenesis. Mech. Dev. 78, 135–140.

Lardelli, M., Lendahl, U., 1993. MotchA and MotchB-Two mouse Notch

homologues coexpressed in a wide variety of tissues. Exp. Cell Res.

204, 364–372.

Lardelli, M., Dahlstrand, J., Lendahl, U., 1994. The novel Notch homologue

mouse Notch3 lacks specific epidermal growth factor-repeats and is

expressed in proliferating neuroepithelium. Mech. Dev. 46, 123–136.

Leimeister, C., Externbrink, A., Klamt, B., Gessler, M., 1999. Hey genes: a

novel subfamily of hairy- and Enhancer of split related genes specifi-

cally expressed during mouse embryogenesis. Mech. Dev. 85, 173–177.

Lewis, J., 1998. Notch signalling and the control of cell fate choices in

vertebrates. Semin. Cell Dev. Biol. 9, 584–589.

Li, J., Kim, J.M., Li, M., Miyazaki, T., Mackenzie, A., Korneluk, R.G.,

Tsang, B.K., 1998. Expression of inhibitor of apoptosis proteins (IAPs)

in rat granulosa cells during ovarian follicular development and atresia.

Endocrinology 139, 1321–1328.

Lindsell, C.E., Shawber, C.J., Boulter, J., Weinmaster, G., 1995. Jagged: a

mammalian ligand that activates Notch1. Cell 80, 909–917.

Lindsell, C.E., Boulter, J., diSibio, G., Gossler, A., Weinmaster, G., 1996.

Expression patterns of Jagged, Delta1, Notch1, Notch2, and Notch3

genes identify ligand-receptor pairs that may function in neural devel-

opment. Mol. Cell. Neurosci. 8, 14–27.

Manova, K., Nocka, K., Besmer, P., Bachvarova, R.F., 1990. Gonadal

expression of c-kit encoded at the W locus of the mouse. Development

110, 1057–1069.

Manova, K., Huang, E.J., Angeles, M., De Leon, V., Sanchez, S., Prono-

vost, M., Besmer, P., Bachvarova, R.F., 1993. The expression pattern of

the c-kit ligand in gonads of mice supports a role for the c-kit receptor in

oocyte growth and in proliferation of spermatogonia. Dev. Biol. 57, 85–

99.

Maier, M.M., Gessler, M., 2000. Comparative analysis of the human and

mouse Hey1 promoter: Hey genes are new Notch target genes.

Biochem. Biophys. Res. Commun. 275, 652–660.

McGee, E.A., Hsu, S.Y., Kaipia, A., Hsueh, A.J., 1998. Cell death and

survival during ovarian follicle development. Mol. Cell Endocrinol.

140, 15–18.

Nakagawa, O., Nakagawa, M., Richardson, J.A., Olson, E.N., Srivasatava,

D., 1999. HRT1, HRT2, and HRT3: a new subclass of bHLH transcrip-

tion factors marking specific cardiac, somitic, and pharyngeal arch

segments. Dev. Biol. 26, 72–84.

Nakagawa, O., McFadden, D.G., Nakagawa, M., Yanagisawa, H., Hu, T.,

Srivastava, D., Olson, E.N., 2000. Members of the HRT family of basic


helix-loop-helix proteins act as transcriptional repressors downstream

of Notch signaling. Proc. Natl. Acad. Sci. USA 25, 13655–13660.

Ohtsuka, T., Ishibiashi, M., Gradwohl, F., Nakanishi, S., Guillemot, F.,

Kageyama, R., 1999. Hes1 and Hes5 as Notch effectors in mammalian

neuronal differentiation. EMBO J. 18, 2196–2207.

Pedersen, T., Peters, H., 1968. Proposal for a classification of oocytes and

follicles in the mouse ovary. J. Reprod. Fert. 17, 555–557.

Rodgers, R.J., Lavranos, T.C., van Wezel, H.F., Irving-Rodgers, H.F.,

1999. Development of the ovarian follicular epithelium. Mol. Cell

Endocrinol. 151, 170–171.

Schroeter, E.H., Kisslinger, J.A., Kopan, R., 1998. Notch-1 signaling

requires ligand-induced proteolytic release of intracellular domain.

Nature 393, 382–386.

Shawber, C., Boulter, J., Lindsell, C.E., Weinmaster, G., 1996. Jagged2: a

serrate-like gene expressed during rat embryogenesis. Dev. Biol. 180,

370–376.

Shelly, L., Fuchs, C., Miele, L., 1999. Notch-1 inhibits apoptosis in murine

erythroleukemia cells and is necessary for differentiation induced by

hybrid polar compounds. J. Cell. Biochem. 73, 164–175.

Shutter, J.R., Scully, S., Fan, W., Richards, W.G., Kitajewski, J.,

Deblandre, G.A., Kitner, C.R., Stark, K.L., 2000. DII4, a novel Notch

ligand expressed in arterial endothelium. Genes Dev. 14, 1313–1318.

Sirois, J., Simmons, D.L., Richards, J.S., 1992. Hormonal regulation of

messenger ribonucleic acid encoding a novel isoform of prostaglandin

endoperoxide H synthase in rat preovulatory follicles. J. Biol. Chem.

267, 11586–11592.

Sirois, J., 1995. Induction of prostaglandin endoperoxide H synthase-2 by

human chorionic gonadotropin in bovine preovulatory follicles in vivo.

Endocrinology 135, 406–409.

Struhl, G., Adachi, A., 1998. Nuclear access and action of notch in vivo.

Cell 93, 649–660.

Uyttendaele, H., Marazzi, G., Wu, G., Yan, Q., Sassoon, D., Kitajewski, J.,

1996. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-

specific mammalian Notch gene. Development 122, 2251–2259.

Varnum-Finney, B., Purton, L.E., Yu, M., Brashem-Stein, C., Flowers, D.,

Staats, S., Moore, K.A., Le Roux, I., Mann, R., Gray, G., Artavanis-

Tsakonis, S., 1998. The Notch ligand, Jagged1, influences the devel-

opment of primitive hematopoietic precursor cells. Blood 91, 4084–

4091.

Wharton, K.A., Johansen, K.M., Xu, T., Artavanis-Tsakonis, S., 1985.

Nucleotide sequence from the neurogenic locus Notch implies a gene

product that shares homology with proteins containing EGF-like

repeats. Cell 43, 567–581.

Weinmaster, G., 1997. The ins and outs of notch signaling. Mol. Cell.

Neurosci. 9, 91–102.

Weinmaster, G., Roberts, V.J., Lemke, G., 1991. A homolog of Drosophila

Notch expressed during mammalian development. Development 113,

199–205.

Weinmaster, G., Roberts, V.J., Lemke, G., 1992. Notch2: a second mamma-

lian Notch gene. Development 116, 931–941.

Williams, R., Lendahl, U., Lardelli, M., 1995. Complementary and combi-

natorial patterns of Notch gene family expression during early mouse

development. Mech. Dev. 53, 357–368.

Wilson-Rawls, J., Hurt, C.R., Parsons, S.M., Rawls, A., 1999. Differential

regulation of epaxial and hypaxial muscle development by paraxis.

Development 126, 5217–5229.

Xu, Q., Wilkinson, D., 1995. Whole-mount in situ hybridization of thick

tissue sections. Zebrafish Sci. Monitor 3 (6), 5.

Zhong, T.P., Rosenber, M., Mohideen, M.A., Weinstein, B., Fishman,

M.C., 2000. Gridlock, an HLH gene required for assembly of the

aorta in zebrafish. Science 287, 1820–1824.


Notch pathway genes are expressed in mammalian ovarian follicles

Documents