View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Developmental Biology 265 (2004) 329–340 www.elsevier.com/locate/ydbio

Developmental regulation of Notch signaling in the embryonic pituitary: Prop1 deficiency affects Notch2 expression

L.T. Raetzman,a,1 S.A. Ross,b,1 S. Cook,b S.L. Dunwoodie,c,d S.A. Camper,a and P.Q. Thomasb,e,*

a Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109-0638, USA b Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia c Victor Chang Cardiac Research Institute, St. Vincent’s Hospital, Darlinghurst, NSW 2010, Australia d Department of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2033, Australia e Department of Paediatrics, University of Melbourne, Victoria 3010, Australia

Received for publication 1 May 2003, revised 19 September 2003, accepted 19 September 2003

Abstract

Normal development of the pituitary gland requires coordination between the maintenance of a progenitor cell pool and the selection of progenitor cells for differentiation. As Notch signaling controls progenitor cell differentiation in many embryonic tissues, we investigated the involvement of this important developmental pathway in the embryonic pituitary. We report that expression of Notch signaling genes is spatially and temporally regulated in pituitary embryogenesis and implicate Notch2 in the differentiation of several cell lineages. Notch2, Notch3, and Dll1 are initially expressed by most cells within the pituitary primordium and become restricted to a subset of the progenitor cell pool as differentiated pituitary cells begin to appear. Mutations in the Prop1 interfere with pituitary growth and cell specification, although the mechanism is unknown. Notch2 expression is nearly absent in the developing pituitaries of Prop1 mutant mice, but unaltered in some other panhypopituitary mutants, revealing that Prop1 is directly or indirectly required for normal Notch2 expression. Transgenic overexpression of Prop1 is not sufficient for enhancement of endogenous Notch2 expression, indicating that there are multiple inputs into this pathway. Dll3 is expressed only in the presumptive corticotrope and melanotrope cells. Analysis of Dll3 null mutants indicates that Dll3 is not required for specification of these two cell types, although there may be functional overlap with Dll1. The spatial and temporal expression patterns of Notch signaling genes in the pituitary suggest overlapping roles in pituitary growth and cell specification. D 2003 Elsevier Inc. All rights reserved.

Keywords: Rathke’s pouch; Pituitary; Prop1; Notch signaling

Introduction et al., 2002; Scully and Rosenfeld, 2002). This information, coupled with the availability of pituitary cell-type-specific The function of the pituitary gland is largely conserved promoters and well-defined cell phenotypes, makes the across vertebrate evolution, and the hormone-secreting cells pituitary an excellent system to investigate critical develop- appear in a spatially and temporally regulated pattern (Japon mental events including progenitor cell commitment and et al., 1994; Kawamura et al., 2002; Simmons et al., 1990; differentiation. Sloop et al., 2000). In recent years, many of the key All hormone-secreting cells of the anterior and interme- signaling molecules and transcriptional regulators that con- diate lobes are derived from a common primordium, trol pituitary organogenesis have been identified (Cushman Rathke’s pouch (RP). The anterior pituitary has five hor- mone-secreting cell types that are defined by their hormone products. These terminally differentiated cell types (and their secreted products) are corticotropes (adrenocorticotro- * Corresponding author. Pituitary Research Unit, Murdoch Childrens phic hormone, ACTH), thyrotropes (thyroid stimulating Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia. Fax: +61-3-9348-1391. hormone, TSH), somatotropes (growth hormone, GH), lac- E-mail address: [email protected] (P.Q. Thomas). totropes (prolactin, PRL), and gonadotropes (follicle-stimu- 1 These authors contributed equally to this publication. lating hormone [FSH] and luteinizing hormone [LH]). An

0012-1606/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2003.09.033 330 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 additional hormone-secreting cell type is the melanotrope, Prop1 controls progenitor cell commitment and differentia- which secretes alpha melanocyte-stimulating hormone tion remains to be determined. Mutant pituitaries also (aMSH) and resides within the intermediate lobe. In the contain cells that are not clearly fated to become anterior murine embryo, RP forms at approximately 9.5 days post- or intermediate lobe cells, suggesting a problem with coitum (dpc) from an invagination of oral ectoderm at the establishing borders of expression. anterior ventral midline. During 9.5–12.5 dpc, RP expands Insight into the genetic basis of the Prop1df/df mutant and extends dorsally to contact the infundibular recess, and phenotype has come from the identification of target then detaches from the oral ectoderm to become a ‘‘flat- genes for this transcription factor. Prop1df/df mice do not tened’’ ball of cells. Loss-of-function, ectopic expression activate Pit1 expression, and subsequently fail to and explant culture experiments suggest that signaling generate somatotropes, lactotropes, and thyrotropes, re- molecules from neighboring tissues, which include BMP4, vealing that Prop1 is required for activation of Pit1 (Gage FGF8, FGF10, SHH, BMP2, WNT4, WNT5A, and chordin, et al., 1996; Sornson et al., 1996). Furthermore, there is a specify cell fate within the pituitary primordium during this temporal extension of Hesx1 expression in Prop1df/df initial phase (Ericson et al., 1998; De Moerlooze et al., mice, revealing that Prop1 is required for the extinction 2000; Ohuchi et al., 2000; Treier et al., 1998, 2001). The of Hesx1. Prop1 is also essential for repressing the overall effect of these signaling molecules is to induce expression of the gonadotrope marker SF1 (Nr5a1)in regionally restricted expression of transcription factors that the dorsal region of the anterior lobe (Raetzman et al., specify cell fate through activation of specific target genes. 2002). The identification of additional target genes for The best characterized example of this inductive interaction Prop1 is essential for a complete understanding of the is the localized expression of the LIM domain transcription complex phenotype that results from Prop1 mutation in factors Lhx3 and Isl1 in response to FGF8 signaling from mice and man. the infundibulum and opposing BMP signaling from the The Notch signaling pathway is an evolutionarily con- ventral mesenchyme (Ericson et al., 1998). This process is served mechanism that controls cellular differentiation, critical for the determination of corticotropes and the rostral proliferation, and death in a broad spectrum of develop- tip thyrotropes (a minor thyrotrope population that expresses mental systems (Artavanis-Tsakonas et al., 1999; Beatus and aGSU and TSHh, but differs from most thyrotropes by Lendahl, 1998). The core pathway components were orig- lacking the transcription factor PIT1). The remaining cell inally identified in Drosophila and include a transmembrane types emerge over the next few days, in response to (Notch), two transmembrane ligands (Delta and activation of additional transcription factors that are sensi- Serrate), and an activator of transcription (Suppressor of tive to positional information, such as GATA2 (Dasen et al., Hairless, Su(H)). Notch signaling is initiated by interaction 1999). The mechanism for establishing the border between between the extracellular domains of the ligand and recep- the differentiation of the anterior lobe and the intermediate tor. This results in cleavage of the intracellular domain of lobe is unknown. the receptor and translocation to the nucleus, where through One of the key components of the genetic program that interaction with Su(H), target genes are activated. One of the controls progenitor cell differentiation in the developing best characterized developmental events that is controlled pituitary is Prop1. A hypomorphic mutation of Prop1, by Notch signaling is the generation of neuroblasts in which was originally identified in the Ames dwarf mutant Drosophila by a process termed lateral inhibition. In this (Prop1df/df), causes severe pituitary hypoplasia and a mas- system, neuroblasts that emerge from a progenitor cell pool sive reduction in somatotropes, lactotropes, and thyrotropes, (equivalence group) express high levels of Delta, which and reduced gonadotrope function (Gage et al., 1996; interacts with Notch receptors on neighboring cells to Sornson et al., 1996; Tang et al., 1993). Interestingly, prevent them from adopting a neural fate. Many studies several PROP1 mutations have been identified in humans have also shown that Notch signaling plays a major role in with multiple pituitary hormone deficiency (Agarwal et al., inhibition of progenitor cell differentiation, particularly 2000; Wu et al., 1998), underlining the conserved role of within the vertebrate CNS, although it can also promote this homeoprotein in the pituitary. Prop1 expression is differentiation of progenitors to glial cell fates (reviewed by pituitary-specific and is initially enriched in the rapidly Gaiano and Fishell, 2002; Lewis, 1998). In addition, Notch proliferating progenitor cells that line the lumen of RP signaling is required to direct cell fate during differentiation (Ikeda and Yoshimoto, 1991; Sornson et al., 1996).At in a wide range of developmental contexts such as glomer- 14.5 dpc, mutant pituitaries are dysmorphic and contain ular differentiation and patterning in mammals (McCright et an excess of peri-luminal progenitor cells (Raetzman et al., al., 2001). 2002). The presumptive anterior lobe, which lies immedi- Mammals have multiple homologs for the Notch receptor ately ventral to the peri-luminal cells, is severely hypocel- (Notch 1, 2, 3, 4) and ligands (Delta-like 1, 3, 4, Jagged1 lular in Prop1 mutant pituitaries. These data indicate that and Jagged2). Expression of these genes is spatially and Prop1 is essential for the exit of peri-luminal progenitor temporally restricted during embryogenesis, indicating that cells from the high-proliferation zone and their subsequent Notch signaling is used to control cellular differentiation in differentiation. However, the precise mechanism by which many tissues. The developmental role of these genes has L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 331 been confirmed through the generation of mouse mutants Mice and the identification of mutations in patients with congen- ital abnormalities. However, their role in the developing Ames dwarf mice (DF/B-Prop1df/df) were obtained from pituitary has not been explored. To investigate this, we Dr. A. Bartke (Southern Illinios University, Carbondale, examined the expression of Notch signaling genes during IL) and have been maintained at the University of Mich- the commitment and differentiation of pituitary cell line- igan. Transgenic mice with constitutive Prop1 expression ages. Notch2, Notch3, and Delta-like1 (Dll1) are expressed were created with the aGSU promoter and Prop1 genomic in the peri-luminal progenitor cells of RP. In contrast, Delta- sequences as previously described, TgN(Cga-Prop1)D6 like 3 (Dll3) expression is restricted to the melanotropes and (Cushman et al., 2001). Mice carrying a null allele for early corticotropes, and may be functionally redundant with Dll3 were maintained at the Victor Chang Cardiac Re- Dll1 during differentiation of these lineages. We examined search Institute (Sydney) on a C57BL/6 genetic back- Notch family in Prop1df/df mutant mice to ground. Embryos were collected from timed pregnant determine whether the cell specification failure correlates Ames dwarf and TgN(Cga-Prop1)D6 transgenic founder with alterations in pattern or level of Notch gene expression. mice, with the day the vaginal plug was detected desig- Notch2 mRNA and is dramatically reduced at 12.5 nated as 0.5 dpc. TgN(Cga-Prop1)D6, Prop1df/df, and Dll3 dpc and absent at 14.5 dpc in these mutants, indicating that embryos were genotyped from genomic DNA as described Notch2 may be involved in the emergence of terminally previously (Cushman et al., 2001; Dunwoodie et al., differentiated cell types during pituitary development. These 1997). Embryos used in Figs. 1 and 5 were HSDola-Swiss data suggest that the Notch signaling pathway may play a strain from the breeding colony at Melbourne University, role in the commitment and lineage-specific differentiation Australia. of progenitor cells in the embryonic pituitary, in particular, Prop1-dependent cell lineages. Immunohistochemistry

Embryos were fixed for 2–24 h in paraformaldehyde (4% Materials and methods w/v in PBS), dehydrated, and embedded in paraffin. Six- micrometer sagittal and coronal sections of these embryos In situ hybridization were prepared and immunostained for NOTCH2. For this purpose, slides were boiled in (10 mM) citric acid, pH 6, for Mouse embryos were fixed for 18 h at 4jC in parafor- 10 min for epitope retrieval and incubated with the mouse maldehyde (4% w/v in PBS) and then placed in sucrose monoclonal NOTCH2 antibody (1:1500, C651.6DbHN De- (20% w/v in PBS) until they sunk. After embedding in OCT velopmental Studies Hybridoma Bank [DSHB], University (TissueTek), 16-Am cryostat frozen sections were prepared of Iowa, Iowa City, IA), which was diluted in PBS contain- and mounted onto Superfrost-Plus slides. Gene expression ing BSA (3%), Tween-20 (0.5%), and normal goat serum was detected by hybridizing digoxigenin-labeled antisense (5% w/v) overnight at 4jC. Antibody detection was carried riboprobes as described previously (Dunwoodie et al., 1997). out with the TSA kit (Perkin-Elmer) according to the The Pomc1 riboprobe spans nucleotides 1–533 bp (Accn. manufacturer’s protocol. Five wild-type and mutant embryo No. NM_008895), the aGSU (Cga) probe spans nucleotides pairs were analyzed at each time point. 42–636 (Accn. No. NM_009889), and the Prop1 probe used in Fig. 1 corresponds to nucleotides 585–985 (Accn. No. NM_008936). Prop1 expression in Fig. 4 was detected using Results an antisense probe as previously described (Cushman et al., 2001). Dll1, Dll3, Serrate1, Hes1, Hes5, Hes6, Notch1, Notch2, Notch3, and Dll1 are expressed in progenitor cells Notch2,andNotch3 probes were prepared as described of the anterior pituitary previously (Akazawa et al., 1992; Dunwoodie et al., 1997; Kusumi et al., 2001; Tomita et al., 1996; Vasiliauskas and To determine whether Notch signaling genes are active Stern, 2000). At least four embryos were examined for each during the commitment and differentiation of anterior pitu- time point and genotype. itary lineages, we examined the expression of Notch recep- tor and ligands at 12.5 and 14.5 dpc using in situ Northern blot analysis hybridization. During this period of development, differen- tiated cell types appear in the ventro-anterior quadrant of the Total RNA was prepared from AtT-20 cells and homog- anterior pituitary primordium. The majority of these cells enized 10.5 dpc embryos using TRIzol. Poly(A) RNA express aGSU, officially named Cga, a marker for thyro- extraction and Northern blot analysis was performed as tropes and gonadotropes (Figs. 1C, D). Proliferating cells described previously (Dunwoodie et al., 1997), and the are detected almost exclusively in the peri-luminal region of filter exposed to X-ray film for 16 h (X-OMAT, Kodak, RP (Ikeda and Yoshimoto, 1991; Raetzman et al., 2002). Australia). These cells are likely progenitors for somatotropes, lacto- 332 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340

tropes, and thyrotropes, and they express Prop1 (Figs. 1E, F). At 12.5 dpc, Notch2 is expressed in the progenitor cell population and is at highest levels in the peri-luminal cells of RP (Fig. 1G). Notch2 expression is maintained at high levels in the region containing Prop1-positive peri-luminal cells at 14.5 dpc (Fig. 1H and data not shown), but it is not detected in the region that contains differentiated, aGSU- positive cells. At 14.5 dpc, clusters of Notch2-positive cells are also evident in the anterior pituitary in regions that express Prop1. Notch2 is co-expressed with Prop1 and appears to be restricted to a portion of the progenitor cell pool, forming a border between the proliferating precursor cells and the differentiated hormone-producing cells. Notch3 (Fig. 1I) and the ligand Dll1 (Fig. 1K) are also expressed by most progenitor cells at 12.5 dpc, and like Notch2, become restricted to a subset of these cells by 14.5 dpc (Figs. 1J, L), with highest expression in the peri-luminal progenitor cells. Notch1 and Serrate1 expression was not detected in the developing anterior pituitary at 12.5 and 14.5 dpc. Tran- scription of Notch receptors and ligands in the progenitor cell pool support a role for Notch signaling in the develop- ment of the anterior pituitary.

Notch2 expression is dramatically reduced in Prop1 mutants

Prop1 acts as a transcriptional activator and repressor. It is expressed within the progenitor cells of the embry- onic pituitary, and it is essential for differentiation of the PIT1 lineages (thyrotropes, somatotropes, and lactotropes). PROP1 may influence all of the cell types of the anterior lobe, as humans with PROP1 mutations consistently exhibit gonadotropin deficiency and occasionally exhibit acquired ACTH deficiency (Agarwal et al., 2000; Parks and Brown, 1999; Pernasetti et al., 2000; Wu et al., 1998). At 14.5 dpc, mutant pituitaries appear to have an overabundance of peri- luminal progenitor cells and hypoplastic anterior lobes, indicating an inability of Prop1 deficient progenitors to differentiate. The only known target genes of PROP1, namely Hesx1, Pit1, and Nr5a1, do not explain the dysmor- phic, hypoplastic phenotype of Prop1df/df mutants.

Fig. 1. Temporal and spatial expression patterns of Notch signaling genes correspond with undifferentiated cells of the pituitary gland. Panels A (12.5 dpc) and B (14.5 dpc) present schematic diagrams of the developing pituitary in sagittal section. Green represents the infundibulum, light blue the diencephalon, yellow the intermediate lobe, red the progenitor cells of the presumptive anterior pituitary, and dark blue the rostral tip thyrotropes. The remaining panels represent the comparison of aGSU, Prop1, Notch2, Notch3, and Dll1 expression at 12.5 dpc (C, E, G, I, K) and 14.5 dpc (D, F, H, J, L) by in situ hybridization. aGSU marks the differentiated cells in the ventro-anterior domain (C, D). Prop1 is expressed by the progenitor cells and is excluded from the aGSU-positive differentiated cells in the ventro- anterior quadrant (E, F). Notch2 expression is restricted to progenitor cells and overlaps extensively with Prop1 expression (G, H). Panels F and H are consecutive serial sections. Notch3 (I, J) and Dll1 (K, L) expression is enriched in progenitor cells. Dll1 is also expressed throughout the entire prospective IL. L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 333

Fig. 2. Prop1 is required for pituitary expression of Notch2. Immunohistochemical detection of NOTCH2 in sagittal sections of wild-type (A, C, E) and Prop1 mutant pituitaries (B, D, F) at 12.5 dpc (A, B), 14.5 dpc (C, D) and 16.5 dpc (E, F). NOTCH2 is present in the ventral aspect of RP and isolated cells in the anterior lobe at 12.5 and 14.5 dpc in wild-type mice, but little or no NOTCH2 was detected in Prop1df/df pituitaries at these ages. By 16.5 dpc, NOTCH2 is restricted to cells near the lumen in wild-type mice and is still not detectable in Prop1df/df pituitaries. Five embryos of each genotype at each age were examined.

The coincident pattern of Prop1 and Notch family gene that Notch2 and Notch3, despite their similar expression, are expression suggested that one or more of the Notch genes regulated differently. might be downstream of Prop1. To assess this possibility, A key feature of lateral inhibition is the ability of we analyzed expression of the Notch genes in Prop1df/df progenitor cells to down-regulate expression of Notch mutant mice. At 12.5 dpc, Notch2 expression decreased ligand in response to Notch receptor activation by neigh- dramatically in the developing pituitary of the Prop1df/df boring cells. The absence of Notch2 expression in Prop1df/df mutants relative to their normal littermates, but expression progenitor cells therefore suggests that there may be an remained unchanged in the surrounding mesenchyme (Figs. inability of progenitor cells to respond to DLL1 ligand 2A, B). By 14.5 dpc, Notch2 protein is completely absent signaling, resulting in increased expression of the ligand. from the Prop1 mutant pituitary (Fig. 2D). The loss of To test this possibility, we examined expression of Dll1 in Notch2 expression is specific to Prop1 deficiency, as Notch2 the Prop1df/df pituitary at 14.5 dpc. The expression of Dll1 is transcripts were detectable in hypoplastic pituitaries that slightly and consistently elevated in pituitaries of Prop1 result from lesions in Hesx1 or Lhx4 (data not shown). mutants (Fig. 3D) relative to wild-type mice (Fig. 3C). This These data suggest that Notch2 is dependent on Prop1 either indicates that decreased Notch2 expression coincides with directly or indirectly, and that Notch2 expression affects the elevated Dll1 expression in the pituitary gland and is emergence of terminally differentiated cell types during consistent with models of lateral inhibition that operate in pituitary development. In contrast, Notch3 expression is other systems (de la Pompa et al., 1997; Heitzler et al., 1996). not altered in the Prop1df/df mutants (Figs. 3A, B). This The Hes genes are known downstream effectors of the indicates that the lack of Notch2 expression in mutant Notch signaling pathway. We examined the expression of pituitaries is not due to an absence of progenitor cells and Hes genes in the pituitary and found that both Hes1 and Hes6 334 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 335

Fig. 4. Overexpression of Prop1 does not alter NOTCH2 protein levels. Prop1 expression is readily detected in two independent lines of transgenic mice (Tg8, Tg54) at 18.5 dpc by in situ hybridization, but not in wild-type mice at this age. Identical patterns and levels of NOTCH2 expression are detected in transverse sections of transgenic and wild-type mice at 18.5 dpc by immunohistochemistry. are detectable at 12.5 and 13.5 dpc (Fig. 3). Interestingly, sion begins to wane by 14.5 dpc. Robust transgene expres- Hes1 and Hes6 have distinct patterns of expression. Hes1 is sion was detected in the prospective IL, just outside the peri- localized to RP and is excluded from the anterior lobe in both luminal proliferative zone in the dorsal aspect of the gland, wild-type (Fig. 3E) and Prop1 mutants (Fig. 3F). Hes6 is and in patches throughout the anterior lobe. However, only present in the developing anterior lobe of wild-type Notch2 expression was normal in spite of the elevated (Fig. 3G) and Prop1 mutant (Fig. 3H) pituitaries. Hes5 was expression of Prop1. Therefore, although Prop1 mutants not detectable in the pituitary at the time points examined have greatly diminished Notch2 expression, overexpression (data not shown). These data indicate that Notch signaling in of Prop1 is not sufficient to alter endogenous Notch2 the pituitary is activating Hes genes, but that Hes1 and Hes6 expression at 18.5 dpc. This suggests that Notch2 expres- are not changed in response to the loss of Prop1. sion is regulated by both Prop1-dependent and Prop1- Mice overexpressing Prop1 under the control of the independent pathways. aGSU promoter have delayed gonadotrope development and a propensity for pituitary tumors (Cushman et al., Lineage restricted expression of Dll3 in melanotropes and 2001). To investigate whether overexpression of Prop1 is presumptive corticotropes sufficient to alter endogenous Notch2 expression, we gen- erated transgenic embryos that expressed the Prop1 gene Dll3 transcripts were first detected in the presumptive constitutively, under the control of the 4.6-kb aGSU pro- intermediate lobe (IL) at 13.5 dpc and by 14.5 dpc, and were moter (Fig. 4). This promoter is active in the progenitor cells present in all cells in this region (Figs. 5A1, B1). At this of RP from 9.5 to 12.5 dpc and subsequently activated in the stage of pituitary development, the IL contains a single gonadotrope and thyrotrope lineages (Kendall et al., 1994). hormone-secreting cell type, the melanotropes, which In situ hybridization analysis of Prop1 expression at 18.5 appears at 14.5 dpc and is identified by Pomc1 expression dpc was performed in three independent transgenic embry- (Japon et al., 1994).ComparisonofDll3 and Pomc1 os. As expected, no Prop1 expression was detected in expression in 15.5–18.5 dpc embryonic, neonatal, and adult control embryos at this stage, as endogenous gene expres- pituitary tissue indicated that Dll3 is maintained in the

Fig. 3. Notch3 is expressed in Prop1-deficient pituitaries and Dll1 is elevated. Representative sagittal sections of wild-type (A) and Prop1df/df (B) pituitaries at 12.5 dpc hybridized with the Notch3 riboprobe. Similar levels of expression are seen in pituitaries of each genotype. In contrast, Dll1 expression is elevated in Prop1df/df (D) pituitaries as compared to wild-type (C) at 12.5 dpc. Hes1 is detectable in the dorsal aspect of both wild-type (E) and Prop1df/df (F) pituitaries at 12.5 dpc, whereas Hes6 is present only in the anterior lobe at 13.5 dpc of wild-type (G) and Prop1df/df (H) pituitaries. Five embryos of each genotype were examined at 12.5 and 13.5 dpc with each probe. 336 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340

Fig. 5. Dll3 marks the prospective intermediate lobe and anterior lobe corticotropes. Dll3 expression is detected in the prospective intermediate lobe melanotropes by in situ hybridization of sagittal sections of 14.5 dpc embryo (A1). Transverse sections of P1 neonates showing corresponding expression of Dll3 (A2) and Pomc1 in the intermediate lobe (A3) detected by in situ hybridization. Dll3 is expressed in most of the melanotrope cells that line the residual lumen of RP. (A2) and (A3) are consecutive serial sections. Dll3 (B1) and Pomc1 (B2) expression domains coincide in 13.5 dpc consecutive serial sagittal pituitary sections. Low level Dll3 expression is evident in the ventro-anterior domain that contains Pomc1-positive corticotropes. Northern blot analysis of Dll3 expression using 5 Ag of poly(A) selected mRNA from AtT-20 corticotrope cell line and 10.5 dpc embryo (B3). AtT-20 cells express Dll3 at high levels, as indicated by the 2.2 kb transcript and less abundant 0.8 kb transcript (Dunwoodie et al., 1997). a, anterior lobe; i, intermediate lobe; p, posterior lobe. Arrows and arrowheads indicate corticotropes and melanotropes, respectively. melanotrope lineage throughout embryogenesis and postna- were compared to either wild-type or heterozygous litter- tal stages (Figs. 5A2, A3 and data not shown). However, in mates. Analysis of 13.5–17.5 dpc Dll3 null embryos contrast to the embryonic stages, only a subset of the revealed no differences in timing or extent of melanotrope melanotrope population expresses Dll3 after birth (Figs. differentiation (Figs. 6A, B). Corticotrope differentiation 5A2, A3), which may correspond to a residual progenitor also appeared to be unaffected in Dll3 homozygous embryos cell population. Proliferating cells that express hormones are (Figs. 6A, B). Analysis of anterior pituitary lineages using readily detectable after birth, but not prenatally (Taniguchi aGSU (thyrotrope and gonadotrope) and Pit-1 (thyrotrope, et al., 2000, 2001). somatotrope, and lactotrope) probes revealed normal com- Within the developing anterior lobe, low level Dll3 mitment of these lineages in the Dll3 mutant embryos (data expression was detected at 13.5 dpc in a few scattered cells not shown). Thus, Dll3 is not necessary for specification of in the ventro-anterior domain (Fig. 5B1). These cells also any of the hormone-producing pituitary cell types. Investi- appear to express Pomc1 indicating that they are cortico- gation of a role of Dll3 in maintenance or function of tropes (Fig. 5B2). Dll3 expression in early stage cortico- melanotropes or other pituitary cell types is complicated tropes is supported by the high level of Dll3 expression in by the poor viability of the Dll3 mutants (Dunwoodie et al., the corticotrope cell line AtT-20 (Fig. 5B3). In contrast to 2002; Kusumi et al., 1998). melanotropes, Dll3 expression is not maintained in the Normal differentiation of the melanotropes in the absence corticotrope lineage (Fig. 5A3). of Dll3 indicates that another member of the Notch receptor family may compensate for the loss of Dll3. At 12.5 dpc, Functional redundancy of Dll3 and Dll1 during melanotrope Dll1 is expressed in most cells within the presumptive differentiation anterior lobe and therefore precedes Dll3 expression in this region by at least 1 day (Fig. 1K). At 14.5 dpc, Dll1 is The restricted expression of Dll3 to the melanotrope expressed throughout the presumptive IL in a domain that lineage in the IL suggested that this gene might play a role encompasses the Dll3-positive cells and extends to the in the differentiation and/or maintenance of this cell type. To margins of the IL (compare Figs. 1L and 5A1). These data explore this possibility, we investigated melanotrope differ- confirm that Dll3 and Dll1 are co-expressed in the presump- entiation in Dll3 null mutant embryos (Dunwoodie et al., tive IL and may therefore be functionally redundant during 2002) using the Pomc1 marker. Homozygous Dll3 embryos melanotrope differentiation. In situ hybridization analysis L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 337

Fig. 6. Dll3 is not required for Pomc1 expression. In situ hybridization with a Pomc1 probe reveals similar pattern and level of expression in sagittal sections of 16.5 dpc Dll3 +/ À (A) and À / À (B) embryos. a, anterior lobe; i, intermediate lobe; p, posterior lobe. Arrows and arrowheads indicate corticotropes and melanotropes, respectively. indicated that Dll1 expression is unaltered in the pituitary of can adopt anterior lobe fates, suggesting that the cells in RP Dll3 mutant embryos (data not shown). are an equivalence group (Pulichino et al., 2003). While several components of the genetic program that controls anterior lobe development are known, specific regulators of Discussion melanotrope commitment and differentiation remain to be identified. We have shown that expression of the Notch Notch signaling is an evolutionarily conserved mecha- ligand Dll3 is initiated in melanotropes as they differentiate nism that controls a broad range of developmental processes at 14.5 dpc and is maintained in this lineage into adulthood. including cell fate determination, differentiation, prolifera- Dll3, like other melanotrope markers, such as Pomc1, Tpit, tion, and apoptotic events. Processes for which Notch and NeuroD1, appears to be expressed in corticotropes, signaling is crucial in mammals include left–right asymme- albeit transiently. Dll3 expression is developmentally regu- try, somitogenesis, cell fate determination of the exocrine lated in many developing tissues including the presomitic and endocrine pancreas, neural stem cell proliferation and and somitic mesoderm, the CNS, and the epiblast. Analysis differentiation, and development of the sensory hair cells of of Dll3 null mutant embryos generated by gene targeting the inner ear (Apelqvist et al., 1999; De Bellard et al., 2002; (Dunwoodie et al., 2002) and the spontaneous Dll3 pudgy Hrabe de Angelis et al., 1997; Kiernan et al., 2001; Kim and mouse mutant (Kusumi et al., 1998) revealed the essential Hebrok, 2001; Przemeck et al., 2003). It is not known, role of this gene in somite formation and skeletogenesis. however, whether Notch signaling is involved in pituitary This aspect of Dll3 function is conserved in humans, as gland development. Studies in other organs have suggested DLL3 mutations have been identified in humans with that Notch acts downstream of Shh, Fgf, and TGFh family vertebral malsegmentation syndrome spondylocostal dys- members, influencing expression of transcription factors plasia (SD). There is no difference in the number or location including Pitx2. All of these genes have proven roles in of melanotropes or corticotropes in Dll3 null mice, indicat- pituitary development (Ericson et al., 1998; Suh et al., 2002; ing that Dll3 is not required for the initial differentiation of Treier et al., 1998, 2001), and therefore, we hypothesized these lineages. However, Dll1 is expressed in the melano- that Notch signaling might be involved in pituitary organ- tropes at 14.5 dpc and also in the pre-corticotropes at 12.5 ogenesis. In this study, we show for the first time that Notch dpc, raising the possibility that Dll3 and Dll1 may be signaling components are developmentally regulated in the functionally redundant in melanotrope and corticotrope embryonic pituitary, with temporal and spatial patterns that differentiation. Functional redundancy also appears to occur implicate this pathway in pituitary gland organogenesis. in the developing spinal cord, where Dll3 and Dll1 are Importantly, we demonstrate a correlation between Notch2 expressed in partially overlapping subsets of postmitotic expression and the differentiation of Prop1-dependent cell neural progenitors (Dunwoodie et al., 1997; Kusumi et al., lineages. 2001). This idea is supported by the absence of a neural Little is known about the mechanism whereby different phenotype in patients with SD (Bulman et al., 2000). While parts of RP are committed to anterior lobe versus interme- it would be useful to test the role of Dll1 in pituitary diate lobe fates. The rostral side of RP undergoes expansion development directly by loss-of-function studies, this would to produce the anterior lobe, while the caudal side of the require a conditional mutagenesis approach, as Dll1 null pouch expands very little. Thus, the decision to become embryos die between 10.5 and 11.5 dpc (Hrabe de Angelis intermediate or anterior lobe must involve differential et al., 1997), too early for analysis of pituitary cell specifi- growth regulation. Studies with Tpit-deficient mice reveal cation. The expression patterns of Dll1 and Dll3 suggest that that cells ordinarily committed to become intermediate lobe these genes could have roles in regulation of differential 338 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 growth and differentiation of the anterior versus intermedi- are spatially and temporally restricted in the pituitary, ate lobes. consistent with a developmental role. However, expression Notch2 expression is developmentally regulated in many of Hes1 and Hes6 was unaltered in Prop1 mutants, indicat- tissues including the neuroectoderm, vasculature, branchial ing that these targets of Notch signaling are not downstream arches, and somites (Hamada et al., 1999). Targeted muta- of NOTCH2 in the pituitary. These data are consistent with tions of Notch2 have shown that this gene is essential for the studies of Hamada et al. (1999), who showed that Hes1 normal development of the nervous system, kidney, eye, and expression is not altered in the CNS of 9.5 dpc Notch2 heart (Hamada et al., 1999; McCright et al., 2001, 2002), mutant embryos. Therefore, Hes1 expression does not although the precise role of Notch2 within these tissues is appear to require NOTCH2, at least within the CNS and not clear. We have shown that Notch2 is expressed at 12.5 pituitary. Our data also suggest that Hes1 expression in the and 14.5 dpc in the cells that lie adjacent to the residual pituitary may be responsive to a different Notch receptor, lumen of RP. Notch2 is not expressed in the differentiated such as Notch3, with which it is co-expressed. Hes1 is cells that occupy the ventro-anterior quadrant of the gland. known to repress neuronal differentiation (Bae et al., 2000), Down-regulation of Notch2 expression in differentiated and its expression only in the undifferentiated cells of RP corticotropes and rostral tip thyrotropes suggests that ab- would be consistent with a role in maintaining these cells as sence of Notch signaling may be important to permit precursors. In contrast, Hes6 is thought to promote neural differentiation. Several lines of evidence support the idea differentiation (Bae et al., 2000, Koyano-Nakagawa et al., that Notch2 and Prop1 interact. Notch2 and Prop1 expres- 2000) and repress myogenic differentiation (Cossins et al., sion patterns overlap, Notch2 expression is dependent on 2002; Gao et al., 2001). The presence of Hes6 in the early Prop1, and Notch2 gene contains several putative PROP1 development of the anterior lobe could be necessary either consensus-binding sites. Prop1 alone, however, is insuffi- to promote development of these cells or to prevent prema- cient for ectopic activation of Notch2, because Notch2 ture hormone expression. Expression analysis of specific expression is normal in transgenic embryos that express targets of NOTCH2-mediated signaling should provide Prop1. The fact that Notch2 expression is normal in mice further insight into the consequences of Prop1 mutation. deficient in Hesx1 (a gene expressed just before Prop1 and The mechanism by which Prop1 controls anterior pi- dependent upon Prop1 for repression) suggests that Notch2 tuitary development is of particular interest as this gene is deficiency is specific to Prop1 (data not shown). essential for differentiation of four of the five differenti- In contrast to Notch2, Dll1 expression is elevated in ated cell types and is the most common genetic cause of Prop1-deficient mice. These data raise the possibility that combined pituitary hormone deficiency (CPHD) in Dll1 is normally repressed by Prop1. However, this is humans. The Prop1df/df pituitary is morphologically nor- unlikely as Dll1 and Prop1 are co-expressed in pituitary mal at 12.5 dpc, but by 14.5 dpc has an abnormal progenitor cells. A more likely possibility is that a lateral branched morphology due to an overabundance of the inhibition mechanism similar to that reported in other sys- peri-luminal cells and a deficiency of cells in ventro- tems (de la Pompa et al., 1997; Heitzler et al., 1996) also anterior domain (Raetzman et al., 2002). This defect does operates in the embryonic pituitary. A critical feature of not result from obvious alterations in cellular proliferation lateral inhibition is that progenitor cells within an equiva- or cell death rates (Raetzman et al., 2002). The apparent lence group are sensitive to the level of ligand expressed by excess of peri-luminal progenitor cells in the Prop1 mutant neighboring cells, that is, Notch ligand expression is down- indicates that this gene may be required for progression of regulated in cells that neighbor the Delta positive cell progenitor cells to a differentiated phenotype. As Notch2 through activation of their Notch signaling pathway. In is not expressed in the Prop1df/df pituitary at 14.5 dpc, Prop1 mutants, which have little or no NOTCH2 in the signaling events mediated by Notch2 may therefore be pituitary, Notch ligand expression is not down-regulated, necessary for progenitor cell differentiation. In this respect, resulting in an excess of Dll1 positive cells. This possibility the role of Notch2 in the pituitary may differ from other is supported be the ability of DLL1 to bind NOTCH2 and tissues such as the cerebellum and blood, where Notch2 activate Notch signaling, as has been shown by others functions as an inhibitor of cell differentiation (Ingles- (Shimizu et al., 2000). While the precise mechanism remains Esteve et al., 2001; Solecki et al., 2001). Alternatively, the to be determined, these data provide the first evidence of absence of NOTCH2 in the Prop1 mutants may cause lateral inhibition in the developing pituitary, and could be pituitary cells to differentiate prematurely, but not become further be explored using gain-of-function and loss-of-func- fully specified. This process may involve other members tion mutations in Notch2 signaling pathway genes. of the Notch signaling family, such as Notch3 and Dll1 Alteration of Notch2 and Dll1 expression in the Prop1 that are expressed in the progenitor cells at 14.5 dpc and mutant pituitary indicates that perturbation of Notch2 sig- down-regulated during differentiation. These studies also naling occurs in this mutant. To investigate the possible indicate that the role of Prop1 may be similar to that of consequences of this signaling defect in more detail, we Mash1 in controlling the timing of cell differentiation by examined the expression of the Hairy and Enhancer of Split modulating the Notch signaling pathway (Casarosa et al., homologs Hes1 and Hes6. We have shown that these genes 1999). L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340 339

Our studies have revealed that Notch signaling genes are Casarosa, S., Fode, C., Guillemot, F., 1999. Mash1 regulates neurogenesis expressed in the developing pituitary and therefore are likely in the ventral telencephalon. Development 126, 525–534. Cossins, J., Vernon, A.E., Zhang, Y., Philpott, A., Jones, P.H., 2002. Hes6 to play a role in differentiation of hormone-producing cells. regulates myogenic differentiation. Development 129, 2195–2207. In addition, we have revealed an interaction between Prop1 Cushman, L.J., Watkins-Chow, D.E., Brinkmeier, M.L., Raetzman, L.T., and Notch2, implicating a role of Notch2 in the differenti- Radak, A.L., Lloyd, R.V., Camper, S.A., 2001. Persistent Prop1 expres- ation of Prop1-dependent lineages. Preliminary examination sion delays gonadotrope differentiation and enhances pituitary tumor of a Notch2 hypomorph (McCright et al., 2001) indicates susceptibility. Hum. Mol. Genet. 10, 1141–1153. Cushman, L.J., Showalter, A.D., Rhodes, S.J., 2002. Genetic defects in the that pituitary morphology and cell specification is normal development and function of the anterior pituitary gland. Ann. Med. 34, just before birth (data not shown). However, as Notch2 null 179–191. mice die too early to examine pituitary cell specification, a Dasen, J.S., O’Connell, S.M., Flynn, S.E., Treier, M., Gleiberman, conditional mutagenesis approach will be necessary to A.S., Szeto, D.P., Hooshmand, F., Aggarwal, A.K., Rosenfeld, explore directly the functional role of Notch2 in pituitary M.G., 1999. Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types. Cell development. Future studies examining a pituitary-specific 97, 587–598. deletion of Notch2 will be necessary to confirm its impor- De Bellard, M.E., Ching, W., Gossler, A., Bronner-Fraser, M., 2002. Dis- tance in the development of the pituitary gland. ruption of segmental neural crest migration and ephrin expression in delta-1 null mice. Dev. Biol. 249, 121–130. de la Pompa, J.L., Wakeham, A., Correia, K.M., Samper, E., Brown, S., Acknowledgments Aguilera, R.J., Nakano, T., Honjo, T., Mak, T.W., Rossant, J., Conlon, R.A., 1997. Conservation of the Notch signalling pathway in mamma- lian neurogenesis. Development 124, 1139–1148. Funding for this work was provided by the State of De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini, M., Rose- Michigan Life Sciences Corridor Initiative, the National well, I., Dickson, C., 2000. An important role for the IIIb isoform of Institutes of Health (R37HD30428 to SAC; F32DK60306 to fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial LTR; T32-BM0832213; and P60AR20557, P30CA46592 to signalling during mouse organogenesis. Development 127, 483–492. Dunwoodie, S.L., Henrique, D., Harrison, S.M., Beddington, R.S., 1997. the University of Michigan Transgenic Animal Model Core), Mouse Dll3: a novel divergent Delta gene which may complement the and the National Health and Medical Research Council function of other Delta homologues during early pattern formation in (142006 to SLD and 216707 to PQT and SLD). PQT is the mouse embryo. Development 124, 3065–3076. supported by a National Health and Medical Research Dunwoodie, S.L., Clements, M., Sparrow, D.B., Sa, X., Conlon, R.A., Council R.D. Wright Fellowship. We thank Annabelle Scott, Beddington, R.S., 2002. Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with dis- Suntrea Goudeau, and Bayly Wheeler for technical assis- ruption of the segmentation within the presomitic mesoderm. tance. We also thank Tom Gridley and Kelly B. Cha for Development 129, 1795–1806. providing unpublished data and Ryoichiro Kageyama and Ericson, J., Norlin, S., Jessell, T.M., Edlund, T., 1998. Integrated FGF and Francois Guillemot for Hes1 and Hes5 in situ probes. BMP signaling controls the progression of progenitor cell differentia- tion and the emergence of pattern in the embryonic anterior pituitary. Development 125, 1005–1015. Gage, P.J., Roller, M.L., Saunders, T.L., Scarlett, L.M., Camper, S.A., References 1996. Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion. Development Agarwal, G., Bhatia, V., Cook, S., Thomas, P.Q., 2000. Adrenocorticotro- 122, 151–160. pin deficiency in combined pituitary hormone deficiency patients ho- Gaiano, N., Fishell, G., 2002. The role of notch in promoting glial and mozygous for a novel PROP1 deletion. J. Clin. Endocrinol. Metab. 85, neural stem cell fates. Annu. Rev. Neurosci. 25, 471–490. 4556–4561. Gao, X., Chandra, T., Gratton, M.O., Quelo, I., Prud’homme, J., Stifani, S., Akazawa, C., Sasai, Y., Nakanishi, S., Kageyama, R., 1992. Molecular St-Arnaud, R., 2001. HES6 acts as a transcriptional repressor in myo- characterization of a rat negative regulator with a basic helix-loop- blasts and can induce the myogenic differentiation program. J. Cell helix structure predominantly expressed in the developing nervous Biol. 154, 1161–1171. system. J. Biol. Chem. 267, 21879–21885. Hamada, Y., Kadokawa, Y., Okabe, M., Ikawa, M., Coleman, J.R., Tsuji- Apelqvist, A., Li, H., Sommer, L., Beatus, P., Anderson, D.J., Honjo, T., moto, Y., 1999. Mutation in ankyrin repeats of the mouse Notch2 gene Hrabe de Angelis, M., Lendahl, U., Edlund, H., 1999. Notch signalling induces early embryonic lethality. Development 126, 3415–3424. controls pancreatic cell differentiation. Nature 400, 877–881. Heitzler, P., Bourouis, M., Ruel, L., Carteret, C., Simpson, P., 1996. Genes Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: of the Enhancer of split and achaete-scute complexes are required for a cell fate control and signal integration in development. Science 284, regulatory loop between Notch and Delta during lateral signalling in 770–776. Drosophila. Development 122, 161–171. Bae, S., Bessho, Y., Hojo, M., Kageyama, R., 2000. The bHLH gene Hes6, Hrabe de Angelis, M., McIntyre II, J., Gossler, A., 1997. Maintenance of an inhibitor of Hes1, promotes neuronal differentiation. Development somite borders in mice requires the Delta homologue DII1. Nature 386, 127, 2933–2943. 717–721. Beatus, P., Lendahl, U., 1998. Notch and neurogenesis. J. Neurosci. Res. Ikeda, H., Yoshimoto, T., 1991. Developmental changes in proliferative 54, 125–136. activity of cells of the murine Rathke’s pouch. Cell Tissue Res. 263, Bulman, M.P., Kusumi, K., Frayling, T.M., McKeown, C., Garrett, C., 41–47. Lander, E.S., Krumlauf, R., Hattersley, A.T., Ellard, S., Turnpenny, Ingles-Esteve, J., Espinosa, L., Milner, L.A., Caelles, C., Bigas, A., 2001. P.D., 2000. Mutations in the human delta homologue, DLL3, cause Phosphorylation of Ser2078 modulates the Notch2 function in 32D cell axial skeletal defects in spondylocostal dysostosis. Nat. Genet. 24, differentiation. J. Biol. Chem. 276, 44873–44880. 438–441. Japon, M.A., Rubinstein, M., Low, M.J., 1994. In situ hybridization anal- 340 L.T. Raetzman et al. / Developmental Biology 265 (2004) 329–340

ysis of anterior pituitary hormone gene expression during fetal mouse quired for cell survival and expansion of the pituitary primordia. Devel- development. J. Histochem. Cytochem. 42, 1117–1125. opment 129, 4229–4239. Kawamura, K., Kouki, T., Kawahara, G., Kikuyama, S., 2002. Hypophy- Scully, K.M., Rosenfeld, M.G., 2002. Pituitary development: regulatory seal development in vertebrates from amphibians to mammals. Gen. codes in mammalian organogenesis. Science 295, 2231–2235. Comp. Endocrinol. 126, 130–135. Shimizu, K., Chiba, S., Hosoya, N., Kumano, K., Saito, T., Kurokawa, M., Kendall, S.K., Gordon, D.F., Birkmeier, T.S., Petrey, D., Sarapura, V.D., Kanda, Y., Hamada, Y., Hirai, H., 2000. Binding of Delta1, Jagged1, O’Shea, K.S., Wood, W.M., Lloyd, R.V., Ridgway, E.C., Camper, and Jagged2 to Notch2 rapidly induces cleavage, nuclear translocation, S.A., 1994. Enhancer-mediated high level expression of mouse pi- and hyperphosphorylation of Notch2. Mol. Cell. Biol. 20, 6913–6922. tuitary glycoprotein hormone alpha-subunit transgene in thyrotropes, Simmons, D.M., Voss, J.W., Ingraham, H.A., Holloway, J.M., Broide, R.S., gonadotropes, and developing pituitary gland. Mol. Endocrinol. 8, Rosenfeld, M.G., Swanson, L.W., 1990. Pituitary cell phenotypes in- 1420–1433. volve cell-specific Pit-1 mRNA translation and synergistic interactions Kiernan, A.E., Ahituv, N., Fuchs, H., Balling, R., Avraham, K.B., Steel, with other classes of transcription factors. Genes Dev. 4, 695–711. K.P., Hrabe de Angelis, M., 2001. The Notch ligand Jagged1 is required Sloop, K.W., McCutchan Schiller, A., Smith, T.P., Blanton Jr., J.R., Rohrer, for inner ear sensory development. Proc. Natl. Acad. Sci. U. S. A. 98, G.A., Meier, B.C., Rhodes, S.J., 2000. Biochemical and genetic char- 3873–3878. acterization of the porcine Prophet of Pit-1 pituitary transcription factor. Kim, S.K., Hebrok, M., 2001. Intercellular signals regulating pancreas Mol. Cell. Endocrinol. 168, 77–87. development and function. Genes Dev. 15, 111–127. Solecki, D.J., Liu, X.L., Tomoda, T., Fang, Y., Hatten, M.E., 2001. Acti- Koyano-Nakagawa, N., Kim, J., Anderson, D., Kintner, C., 2000. Hes6 acts vated Notch2 signaling inhibits differentiation of cerebellar granule in a positive feedback loop with the to promote neuronal neuron precursors by maintaining proliferation. Neuron 31, 557–568. differentiation. Development 127, 4203–4216. Sornson, M.W., Wu, W., Dasen, J.S., Flynn, S.E., Norman, D.J., O’Con- Kusumi, K., Sun, E.S., Kerrebrock, A.W., Bronson, R.T., Chi, D.C., Bu- nell, S.M., Gukovsky, I., Carriere, C., Ryan, A.K., Miller, A.P., Zuo, L., lotsky, M.S., Spencer, J.B., Birren, B.W., Frankel, W.N., Lander, E.S., Gleiberman, A.S., Andersen, B., Beamer, W.G., Rosenfeld, M.G., 1996. 1998. The mouse pudgy mutation disrupts Delta homologue Dll3 and Pituitary lineage determination by the Prophet of Pit-1 homeodomain initiation of early somite boundaries. Nat. Genet. 19, 274–278. factor defective in Ames dwarfism. Nature 384, 327–333. Kusumi, K., Dunwoodie, S.L., Krumlauf, R., 2001. Dynamic expression Suh, H., Gage, P.J., Drouin, J., Camper, S.A., 2002. Pitx2 is required at patterns of the pudgy/spondylocostal dysostosis gene Dll3 in the devel- multiple stages of pituitary organogenesis: pituitary primordium forma- oping nervous system. Mech. Dev. 100, 141–144. tion and cell specification. Development 129, 329–337. Lewis, J., 1998. Notch signalling and the control of cell fate choices in Tang, K., Bartke, A., Gardiner, C.S., Wagner, T.E., Yun, J.S., 1993. Gona- vertebrates. Semin. Cell Dev. Biol. 9, 583–589. dotropin secretion, synthesis, and gene expression in human growth McCright, B., Gao, X., Shen, L., Lozier, J., Lan, Y., Maguire, M., hormone transgenic mice and in Ames dwarf mice. Endocrinology Herzlinger, D., Weinmaster, G., Jiang, R., Gridley, T., 2001. Defects 132, 2518–2524. in development of the kidney, heart and eye vasculature in mice ho- Taniguchi, Y., Kominami, R., Yasutaka, S., Kawarai, Y., 2000. Proliferation mozygous for a hypomorphic Notch2 mutation. Development 128, and differentiation of pituitary corticotrophs during the fetal and post- 491–502. natal period: a quantitative immunocytochemical study. Anat. Embryol. McCright, B., Lozier, J., Gridley, T., 2002. A mouse model of Alagille 201, 229–234. syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Taniguchi, Y., Yasutaka, S., Kominami, R., Shinohara, H., 2001. Prolifer- Development 129, 1075–1082. ation and differentiation of pituitary somatotrophs and mammotrophs Ohuchi, H., Hori, Y., Yamasaki, M., Harada, H., Sekine, K., Kato, S., Itoh, during late fetal and postnatal periods. Anat. Embryol. 204, 469–475. N., 2000. FGF10 acts as a major ligand for FGF receptor 2 IIIb in Tomita, K., Ishibashi, M., Nakahara, K., Ang, S.L., Nakanishi, S., Guille- mouse multi-organ development. Biochem. Biophys. Res. Commun. mot, F., Kageyama, R., 1996. Mammalian hairy and Enhancer of split 277, 643–649. homolog 1 regulates differentiation of retinal neurons and is essential Parks, J.S., Brown, M.R., 1999. Transcription factors regulating pituitary for eye morphogenesis. Neuron 16, 723–734. development. Growth Horm. IGF Res. 9 (Suppl. B), 2–8 (discussion Treier, M., Gleiberman, A.S., O’Connell, S.M., Szeto, D.P., McMahon, J.A., 8–11). McMahon, A.P., Rosenfeld, M.G., 1998. Multistep signaling require- Pernasetti, F., Toledo, S.P., Vasilyev, V.V., Hayashida, C.Y., Cogan, J.D., ments for pituitary organogenesis in vivo. Genes Dev. 12, 1691–1704. Ferrari, C., Lourenco, D.M., Mellon, P.L., 2000. Impaired adrenocorti- Treier, M., O’Connell, S., Gleiberman, A., Price, J., Szeto, D.P., Burgess, cotropin-adrenal axis in combined pituitary hormone deficiency caused R., Chuang, P.T., McMahon, A.P., Rosenfeld, M.G., 2001. Hedgehog by a two- deletion (301–302delAG) in the prophet of Pit-1 signaling is required for pituitary gland development. Development gene. J. Clin. Endocrinol. Metab. 85, 390–397. 128, 377–386. Przemeck, G.K., Heinzmann, U., Beckers, J., Hrabe de Angelis, M., 2003. Vasiliauskas, D., Stern, C.D., 2000. Expression of mouse HES-6, a new Node and midline defects are associated with left–right development in member of the Hairy/Enhancer of split family of bHLH transcription Delta1 mutant embryos. Development 130, 3–13. factors. Mech. Dev. 98, 133–137. Pulichino, A., Vallette-Kasic, S., Peih-Ying Tsai, J., Couture, C., Gauthier, Wu, W., Cogan, J.D., Pfaffle, R.W., Dasen, J.S., Frisch, H., O’Connell, Y., Drouin, J., 2003. Tpit determines alternate fates during pituitary cell S.M., Flynn, S.E., Brown, M.R., Mullis, P.E., Parks, J.S., Phillips III, differentiation. Genes Dev. 17, 738–747. J.A., Rosenfeld, M.G., 1998. Mutations in PROP1 cause familial com- Raetzman, L.T., Ward, R., Camper, S.A., 2002. Lhx4 and Prop1 are re- bined pituitary hormone deficiency. Nat. Genet. 18, 147–149.