Developmental Biology 324 (2008) 322–333

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Developmental Biology

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Genomes & Developmental Control Genetic interaction between the homeobox transcription factors HESX1 and SIX3 is required for normal pituitary development

Carles Gaston-Massuet a, Cynthia L. Andoniadou a, Massimo Signore a, Ezat Sajedi a, Sophie Bird a, James M.A. Turner b, Juan Pedro Martinez-Barbera a,⁎ a Neural Development Unit, Institute of Child Health, University College London, London, UK b Developmental Genetics and Stem Cell Research, National Institute for Medical Research, Mill Hill, London, UK article info abstract

Article history: Hesx1 has been shown to be essential for normal pituitary development. The homeobox gene Six3 is Received for publication 13 May 2008 expressed in the developing pituitary gland during mouse development but its function in this tissue has Revised 6 August 2008 been precluded by the fact that in the Six3-deficient embryos the pituitary gland is not induced. To gain Accepted 8 August 2008 − insights into the function of Six3 during pituitary development we have generated Six3+/ ;Hesx1Cre/+ double Available online 18 August 2008 heterozygous mice. Strikingly, these mice show marked dwarfism, which is first detectable around weaning, and die by the 5th–6th week of age. Thyroid and gonad development is also impaired in these animals. Keywords: +/− Cre/+ HESX1 Analysis of Six3 ;Hesx1 compound embryos indicates that hypopituitarism is the likely cause of these SIX3 defects since pituitary development is severely impaired in these mutants. Similar to the Hesx1-deficient − Ectopic pituitary embryos, Rathke's pouch is initially expanded in Six3+/ ;Hesx1Cre/+ compound embryos due to an increase in Wnt/β-catenin cell proliferation. Subsequently, the anterior pituitary gland appears bifurcated, dysmorphic and occasionally ectopically misplaced in the nasopharyngeal cavity, but cell differentiation is unaffected. Our research has revealed a role for Six3 in normal pituitary development, which has likely been conserved during evolution as SIX3 is also expressed in the pituitary gland of the human embryo. © 2008 Elsevier Inc. All rights reserved.

Introduction essential body functions. On the other hand, the PP is of neural origin and arises from the infundibulum, a ventral evagination of the floor of The pituitary gland is considered a master regulator of homeostasis the diencephalon, which establishes intimate contact with Rathke's in vertebrates as it regulates a multitude of vital physiological pouch that is essential for normal development and function of the AP. functions such as metabolism, growth, fertility and stress response. The PP is devoid of hormone-producing cells, but instead contains the The mature pituitary gland is composed of three lobes; the anterior axonal projections from the hypothalamic nuclei, which produce the and intermediate lobes (anterior pituitary, AP) and the posterior lobe hormones oxytosin (OT) and vasopressin (ADH). Unlike the AP, the PP (posterior pituitary, PP), which arise from different embryonic tissues is highly innervated, but weakly vascularised. Failure to produce one (Cushman and Camper, 2001; Zhu et al., 2007). The anterior pituitary (usually Isolated Growth Hormone Deficiency, IGHD) or several of forms early in mouse development at 8.5 days postcoitum (dpc) these hormones (Combined Pituitary Hormone Deficiency, CPHD) through a thickening of the roof of the oral ectoderm (pituitary causes hypopituitarism in mice and humans (Procter et al., 1998; placode) that contacts the floor of the ventral diencephalon. By Kelberman and Dattani, 2007). 9.5 dpc, this thickened oral ectoderm tissue evaginates and forms HESX1 is a paired-like homeobox transcription factor that is Rathke's pouch, the primordium of the AP. The mature AP houses six essential for normal forebrain and pituitary formation in both mice hormone-producing cell types: corticotropes, thyrotropes, somato- and humans. Hesx1 expression is very dynamic and transcripts can be tropes, lactotropes, gonadotropes and melanotropes, which produce detected transiently within the anterior forebrain from 7.5–8.5 dpc, adrenocorticotrophic hormone (ACTH), thyroid-stimulating hormone and in Rathke's pouch from 8.5–13.5 dpc (Thomas and Beddington, (TSH), growth hormone (GH), prolactin (PRL), luteinising hormone 1996; Hermesz et al., 1996). Hesx1−/− embryos exhibit variable degrees (LH) as well as follicle stimulating hormone (FSH), and melanocyte of anterior forebrain defects, brought about by a cell fate transforma- stimulating hormone (MSH), respectively. Once secreted into the tion of anterior to posterior forebrain, possibly as a consequence of the blood stream, these hormones act upon their target organs (adrenal ectopic activation of the Wnt/β-catenin signalling pathway in the glands, thyroid, bone, mammary glands, gonads and skin) to control anterior forebrain (Dattani et al., 1998; Martinez-Barbera et al., 2000; Andoniadou et al., 2007). Hesx1−/− mutants develop dysmorphic ⁎ Corresponding author. pituitaries, which are enlarged, bifurcated, and often misplaced in E-mail address: [email protected] (J.P. Martinez-Barbera). the nasopharyngeal cavity. In spite of the very dramatic morphological

0012-1606/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2008.08.008 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 323 defects, cell lineage determination and terminal hormone-producing generations, i.e. a proportion of double mutants die before weaning. cell-type differentiation appears normal (Dasen et al., 2001; E. Sajedi Therefore, as in the Hesx1-deficient embryos (Andoniadou et al., and J.P. Martinez-Barbera, unpublished). Hesx1−/− mice are not viable 2007), the genetic background has an effect on the phenotype of the and die perinatally (Dattani et al., 1998; Andoniadou et al., 2007). Six3+/−;Hesx1Cre/+ compound embryos. Genotyping of mice and Mutations in HESX1 have been associated with human phenotypes embryos was carried out by PCR on DNA purified from tail tip biopsies affecting midline telencephalic commissural tracts (corpus callosum from mice and embryos using specific primers (Lagutin et al., 2003; and anterior commissure), the optic nerves and the pituitary gland, Andoniadou et al., 2007). Primers and PCR protocols are available such as septo-optic dysplasia (SOD) and CPHD (Kelberman and upon request. Dattani, 2007). SIX3 is a homeobox transcription factor related to the Drosophila In situ hybridisation, histology, cell death and proliferation analyses sine oculis gene (Oliver et al., 1995). Six3 and Hesx1 expression domains partly overlap during early mouse development, in particular Preparation of mouse tissues and embryos, and in situ hybridisa- within the anterior forebrain and Rathke's pouch. However, Six3 tion on paraffin sections, were performed as described (Dasen et al., expression persists for longer in these tissues compared with Hesx1. 2001; Andoniadou et al., 2007). Cell death in the pituitary gland was Targeted disruption of the Six3 locus has demonstrated the require- analysed by TUNEL staining on wax sections as described (Martinez- ment for Six3 in normal forebrain formation (Lagutin et al., 2003). In Barbera et al., 2002). Proliferation analyses were performed using 6 fact, Six3−/− embryos show forebrain defects, which are comparable to alternate sagittal sections per embryo through the pituitary gland of at those observed in Hesx1−/− mutants, although they are more severe least three embryos per genotype. The mitotic index of the luminal and fully penetrant. As in the Hesx1-deficient embryos, there is a cells represents the percentage of proliferating cells from the total posterior transformation of the anterior forebrain, which is likely to be number of luminal cells (DAPI positive). Anti-phosphorylated-histone due to the activation of Wnt/β-catenin signalling in these cells. H3 (1:1000) (rabbit polyclonal; Upstate) and anti-cyclin D2 (1:250) Mutations in human SIX3 have been linked to holoprosencephaly (Rabbit anti-mouse; Autogen Bioclear) were used for these studies. (Wallis et al., 1999). So far, it has not been possible to determine a role for Six3 during pituitary development as the forebrain defects extend Quantitative real-time PCR to the region of the ventral diencephalon responsible for the initial induction of Rathke's pouch. Total RNA was extracted from 12.5 dpc pituitary glands of wild- The similarities between the Hesx1 and Six3 expression patterns, type, Hesx1Cre/+, Hesx1Cre/Cre, Six3+/− and Six3+/−;Hesx1Cre/+ embryos and the common molecular mechanism underlying the forebrain using the RNeasy Micro kit (Qiagen), including DNase I treatment. defects of targeted mutant mice for these genes, prompted us to Reverse transcription was performed using the Omniscript RT kit investigate the possibility that they might interact during normal (Qiagen) using random hexamers (Promega). Real-time qRT-PCR was anterior forebrain and Rathke's pouch development. Interestingly, we performed using the SYBR GreenER reagent (Invitrogen) on an Applied found that Six3+/−;Hesx1Cre/+ compound heterozygous animals exhibit Biosystems 7500 thermal cycler. The cycling steps were performed as severe growth retardation after weaning, with additional gonadal and follows: one cycle of 50 °C for 2 min, one of 95 °C for 10 min, 40 cycles thyroid gland defects, resulting in a lethal phenotype. We present of 95 °C for 15 s then 57 °C for 1 min. Reactions were run in triplicate evidence that whilst the differentiation programme in the pituitary and repeated for several independent samples for each genotype gland occurs correctly, cell proliferation of AP progenitors is enhanced (n=3–6 for Hesx1, Lef1, Axin2, Pitx2, Cyclin D1, Cyclin D2 and c-Myc). at 12.5 dpc, which leads to hypertrophy and dysmorphogenesis of the Fold changes in gene expression were determined using the ΔΔCT AP in Six3+/−;Hesx1Cre/+ compound embryos. Molecular analysis method, using the 7500 Fast System software (Applied Biosystems), suggests that this enhanced proliferation may be due to an up- by normalizing expression of targets to endogenous Gapdh. Data regulation of Wnt/β-catenin signalling in the pituitary gland in these values were presented as averages for each genotype and the standard embryos. Our results demonstrate a novel role for Six3 in normal error of the mean (SEM) was calculated to determine the error bars pituitary morphogenesis. (±SEM). Forward and reverse primers used are shown in Supplemen- tary Table S1. Materials and methods Analysis of protein–protein interactions Mice Plasmids expressing GAL4-HESX1, SIX3-VP16 or TLE1-VP16 were Hesx1Cre/+ mice carry a null allele in which the entire Hesx1 open generated by cloning the specific coding regions into the pM and reading frame is replaced by the Cre recombinase coding sequence. pVP16 vectors (Clontech). A plasmid containing the entire mouse Six3 Hesx1Cre/Cre embryos show the same forebrain and pituitary defects to coding region was kindly provided by Dr. G. Oliver (Memphis, USA). those observed in Hesx1−/− embryos previously described (Dattani The plasmid expressing untagged HESX1 and the reporter plasmids p- et al., 1998; Martinez-Barbera et al., 2000; Andoniadou et al., 2007). P3-luciferase and p-Gal4BS-luciferase have been previously described Hesx1Cre/+ mice were backcrossed to the C57BL6/J background for (Sajedi et al., 2008). The integrity of the regions encoding the fusion approximately 6–7 generations. Six3+/− mice, originating on a CD1 proteins was confirmed by DNA sequencing. Cell transfection and background, were kindly provided by Dr. G. Oliver (Memphis, USA) immunoprecipitation experiments were performed as described (Lagutin et al., 2003). We crossed Six3+/− founders with Hesx1Cre/+ to (Sajedi et al., 2008). generate Six3+/−;Hesx1Cre/+ mice. The growth study (Fig. 1)used animals from this first filial generation (F1). Animals were weighed Results weekly from weaning (3rd week) for four weeks to generate a growth curve for each genotype. Subsequently, the colony was expanded Growth retardation, hypoplasia of the thyroid gland and gonads and using Six3+/− stud males from F1, which were crossed to C57BL6/J premature lethality in Six3+/−;Hesx1Cre/+ compound mice females. Therefore, although still on a mixed background, these Six3+/− animals incorporated more C57BL6/J genetic background in subse- Intercrosses between Six3+/− and Hesx1Cre/+ animals produced quent generations. For the embryological studies, animals from the offspring that were phenotypically normal until weaning (3rd first 2–4 generations were used. It is worth mentioning that we have postnatal week). Genotyping of embryos revealed the expected observed an increase in the severity of the phenotype in subsequent Mendelian mode of inheritance for all genotypes, whereas there was 324 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333

Fig. 1. Severe growth retardation and hypoplasia of the testis and thyroid gland in Six3+/−;Hesx1Cre/+ mice. (A) Six3+/−;Hesx1Cre/+ compound heterozygote showing growth retardation and smaller body size compared with wild-type, Six3+/− and Hesx1Cre/+ littermates at four weeks of age. (B) Growth graphs of Six3+/−;Hesx1Cre/+ animals (crosses), wild-type (triangles), Six3+/− (squares) or Hesx1Cre/+ (circles). Note the growth retardation and early lethality in Six3+/−;Hesx1Cre/+ animals when compared with other genotypes (pb0.001, one-way ANOVA). (C, D) Transverse sections through the testis stained with hematoxylin–eosin of wild-type (C) and compound heterozygous (D) mice. Note that the Six3+/−;Hesx1Cre/+ testis has small and hypocellular seminiferous tubules compared with the wild-type control (arrows in panels C, D). (E–H) Transverse sections through the thyroid gland stained with hematoxylin– eosin of wild-type (E and G) and Six3+/−;Hesx1Cre/+ (F and H) animals. Note the abnormal histology in the Six3+/−;Hesx1Cre/+ animal, which shows an evident reduction in the number of follicles, when compared with the wild-type (arrows). Photographs of histological analysis are representative of three embryos per genotype.

a slight decrease in the proportion of Six3+/−;Hesx1Cre/+ pups at weaning which were hypocellular, and the absence of elongated spermatids (Table 1). Although this decrease was not statistically significant, it is (Figs.1C, D; Supplementary Fig. S1). This phenotype has been observed notable that a small number of Six3+/−;Hesx1Cre/+ mice were found dead in mouse mutants manifesting deficient secretion of the pituitary within the first three weeks after birth (see Materials and methods). hormones FSH and LH (Kendall et al., 1995; Kumar et al., 1997; Soon after weaning, it became evident that a large proportion (∼87%) Nasonkin et al., 2004). Moreover, the thyroid gland showed a dramatic of Six3+/−;Hesx1Cre/+ mice failed to gain weight, resulting in animals reduction in the number of follicles on histological sections (Figs. 1E– with severe growth retardation compared to the wild-type, Six3+/− or H). It has been shown that adequate pituitary input through the Hesx1Cre/+ littermates (Fig. 1A). Both male (n=5) and female (n=6) secretion of TSH is essential for normal thyroid function (Kendall et al., Six3+/−;Hesx1Cre/+ mice showed severe growth deficiency and 1995; Nasonkin et al., 2004). Therefore, abnormal testicular and weighed around four times less than their Six3+/−, Hesx1Cre/+ or thyroid morphology may suggest pituitary dysfunction. Since none of wild-type littermates at five weeks of age. Eventually, these animals these defects were observed in Six3+/−, Hesx1Cre/+ or wild-type died prematurely around the 5th–6th week of age (Fig. 1B). Severe growth retardation resulting in dwarfism has been observed in mouse lines with compromised pituitary development Table 1 and function, such as in the Ames Dwarf (Prop1) and little (Ghrhr) Genotypes of embryos and mice from Six3+/− and Hesx1Cre/+ intercrosses mouse mutants (Lin et al., 1993; Gage et al., 1995; Sornson et al., 1996) Stage Genotypes and the Ghr (growth hormone receptor-deficient embryos) and Cga +/− Cre/+ +/− Cre/+ (αGsu subunit mutant) knockout mice (Kendall et al., 1995; Lupu et al., +/+ Six3 Hesx1 Six3 ;Hesx1 2001). Since Hesx1 has been implicated in hypopituitarism in mice 10.5 dpc 13 10 8 10 and humans (Dattani et al., 1998; Kelberman and Dattani, 2007) and 11.5 dpc 12 9 8 7 12.5 dpc 15 19 17 20 Six3 is reportedly expressed in the pituitary gland (Oliver et al., 13.5 dpc 12 10 12 13 1995), we hypothesised that hypopituitarism could be responsible for 15.5 dpc 6 7 5 8 the growth impairment and perhaps the lethality in the Six3+/−; 17.5 dpc 14 16 14 13 a a a a Hesx1Cre/+ animals. To test this hypothesis we performed morpholo- Total embryos (%) 72 (25.9%) 71 (25.5%) 64 (23%) 71 (25.6%) 3 weeks postnatal 22 21 22 17 gical analyses on the testes and thyroid glands, two pituitary target 8 weeks postnatal 22 21 22 3b tissues (Kendall et al., 1995), in Six3+/−;Hesx1Cre/+ compound mice a fi compared with Six3+/−, Hesx1Cre/+ and wild-type littermates at Chi-square test showed no statistically signi cant deviation from the expected 25% +/− Cre/+ ratios. 5 weeks postnatal. Histological analysis of Six3 ;Hesx1 com- b The reduced survival of Six3+/−;Hesx1Cre/+ at 8 weeks is statistically significant pound mice revealed the presence of small seminiferous tubules, (pb0.01, Z-test). C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 325 littermates, we conclude that there is a genetic interaction between the embryos showed a dysmorphic anterior pituitary with a single lumen two transcription factors during normal pituitary development and (6/9 embryos) (Fig. 2D), whilst in others, the pouch was bifurcated, that hypopituitarism is the most likely condition underlying the had failed to separate from the oral ectoderm and exhibited multiple growth retardation in Six3+/−;Hesx1Cre/+ double heterozygous mice. lumens (3/9 embryos) (Fig. 2E). This latter phenotype may be the consequence of a developmental delay in Rathke's pouch formation, Abnormal morphogenesis, but normal specification and terminal since there was no apparent variability in the morphology of the differentiation of the anterior pituitary in Six3+/−;Hesx1Cre/+ embryos pituitary gland at 15.5 dpc (6/6 embryos) (Fig. 3B). Morphological abnormalities were not generally observed in wild-type, Six3+/− or Hesx1−/− mutants show severe pituitary defects, which are evident Hesx1Cre/+ embryos (Figs. 2A–C), although one Six3+/− embryo (n=17) during embryogenesis (Dattani et al., 1998; Dasen et al., 2001). We showed a slight abnormality in pituitary shape (data not shown). analysed the morphology and gene expression pattern of the Expression of several diagnostic pituitary and/or ventral dience- developing anterior pituitary in Six3+/−;Hesx1Cre/+ compound embryos phalic markers, including Prop1, Six3, Pomc1, Pit1, and Cga (Kendall compared with unaffected littermates. A total of 31 Six3+/−;Hesx1Cre/+ et al., 1995; Cohen et al., 1996; Dasen et al., 2001; Lamonet et al., 2004) compound embryos from 10.5–17.5 dpc were analysed. was essentially normal in Six3+/−;Hesx1Cre/+ compound embryos at In situ hybridisation at 13.5 dpc with Lhx3, a marker of the anterior 13.5–15.5 dpc, although the expression domains were enlarged and pituitary (Sheng et al., 1996), revealed two distinct phenotypes. Some the anterior pituitary was bifurcated in these mutants (Figs. 2F–Y and

Fig. 2. Abnormal morphology, but normal gene expression in Rathke's pouch in Six3+/−;Hesx1Cre/+ embryos. In situ hybridisation on sagittal sections through the pituitary gland of 13.5 dpc embryos. Anterior is to the left. (A–E) Lhx3 is expressed in all genotypes, but note the abnormal morphology of Rathke's pouch in Six3+/−;Hesx1Cre/+ embryos (D, E) compared with single mutants (B, C) or wild-type littermates (A). The phenotype shown in panels D, I, N, S, X was observed in 6/9 Six3+/−;Hesx1Cre/+ embryos, whilst the phenotype displayed in panels E, J, O, T, Y was seen in the remaining three embryos. (F–O) The expression patterns of Prop1 (anterior pituitary) and Six3 (anterior pituitary and ventral diencephalon) are comparable among all genotypes. (P–T) Pomc1 expression in the ventral diencephalon (arrow in panel P) and anterior pituitary (arrowhead in panel P) is reduced (S) or absent (T) in Six3+/−;Hesx1Cre/+ embryos in comparison with control embryos (P–R). (U–Y) Likewise, expression of Pit1 in the ventral region of the anterior pituitary is either down-regulated (X) or not detected (Y) in double mutants compared with Six3+/− (V), Hesx1Cre/+ (W) or wild-type littermates (U). 326 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333

3C–H). The only relevant difference was a transient delay in the onset Cell death was analysed by TUNEL staining on sections of the of Pomc1 and Pit1 expression in the hypothalamus (Pomc1; Figs. 2S, T) pituitary at 12.5 dpc. The number of apoptotic cells within Rathke's and anterior pituitary (Pomc1 and Pit1; Figs. 2S, T, X, Y) in Six3+/−; pouch and the developing anterior pituitary was almost negligible in Hesx1Cre/+ compound embryos compared with other genotypes. A Six3+/−;Hesx1Cre/+ double heterozygotes, single mutants and wild-type similar delay in Pomc1 expression has been reported in mouse embryos (data not shown) (Ellworth et al., 2008). Therefore, decreased embryos harbouring inactivating Hesx1 mutations (Dasen et al., 2001). cell death is unlikely to be an important contributor to the NeuroD1 expression, which precedes that of Pomc1, was normal in enlargement and bifurcation of the developing pituitary gland in double mutants when compared with wild-type littermates (Figs. 7I, J) double heterozygous embryos. Cell death was not affected in Hesx1- (Lamonet et al., 2004). deficient embryos compared with wild-type littermates (data not Analysis of embryos at 17.5 dpc showed that hormone-producing shown; Dasen et al., 2001). cells were present in the anterior pituitary of the Six3+/−;Hesx1Cre/+ Cellular proliferation was assessed at 11.5 and 12.5 dpc, when the compound embryos, as evidenced by the expression of the terminal pituitary phenotype is first detectable. At 11.5 dpc, more proliferating differentiation markers Gh, Cga, Pomc1, Tshb, Prl, Lhb and Sf1 (Fig. 4). cells were seen in the developing Rathke's pouch of the Six3+/−; The only significant difference was the apparent reduction in Lhb- Hesx1Cre/+ compound embryos compared with other genotypes, but expressing cell numbers, possibly reflecting a developmental delay in differences were not statistically significant (data not shown). In the terminal differentiation of the gonadotrophs. Pre-gonadotroph contrast, at 12.5 dpc, it was evident that the anterior pituitary of the specification was normal in compound embryos when compared Six3+/−;Hesx1Cre/+ mutants was hyperplastic (Fig. 6K). Likewise, with wild-type littermates, evidenced by correct expression of Sf1,a analysis of Hesx1Cre/Cre pituitaries at this stage revealed an increased marker of pre-gonadotrophs. However, we observed two morpholo- mitotic index in three mutants, correlating with mild forebrain gically distinct phenotypes: (i) six Six3+/−;Hesx1Cre/+ compound abnormalities, as previously suggested (n=5; Fig. 6K) (Dasen et al., embryos showed pituitary glands normally located between the 2001). The remaining two mutants did not show an increase in hypothalamus and the basisphenoid bone (Figs. 4B, E, H, K, N, Q, T). proliferation (data not shown). These mutants had significant Morphologically, these were smaller along the medio-lateral axis but forebrain and craniofacial defects, and an apparent developmental expanded along the rostrocaudal axis. In two of the latter, pituitary delay in Rathke's pouch formation, i.e. Rathke's pouch remained tissue could be seen invading the nasopharyngeal cavity (Supple- connected to the oral cavity and resembled that of an 11.5 dpc embryo mentary Fig. S2); (ii) three additional embryos appeared to have an (Supplementary Fig. S4). It is important to emphasise that there is absent pituitary gland, as it could not be morphologically recognised no general developmental delay of Hesx1-deficient embryos, but it in its normal position in paraffin sections. However, in situ is only restricted to anterior forebrain structures, including the hybridisation revealed the presence of a large mass of anterior pituitary tissue ectopically located in the nasopharyngeal cavity (Figs. 4C, F, I, L, O, R). Taken together, these results suggest that there is no major spatial or temporal disruption of the specification or differentiation programmes of the anterior pituitary in the Six3+/−; Hesx1Cre/+ compound embryos, but morphogenesis was severely impaired, occasionally resulting in misplacement of the pituitary into the nasopharyngeal cavity. Ectopic pharyngeal pituitary has also been reported in human embryos in association with craniofacial defects (Kjaer and Hansen, 2000; Osman et al., 2006). Finally, in situ hybridisation analysis of Six3+/−;Hesx1Cre/+ com- pound mice at postnatal week four revealed the presence of all six cell types, although the number of expressing cells appeared lower compared with single heterozygous or wild-type animals (Fig. 5).

Increased cellular proliferation in Six3+/−;Hesx1Cre/+ compound embryos

It has been proposed that increased cellular proliferation and recruitment of additional oral ectoderm into Rathke's pouch may account for the observed enlargement and bifurcation of the anterior pituitary in Hesx1−/− mutants (Dasen et al., 2001). Alternatively or in addition, decreased cell death might also contribute to the observed phenotype. We decided to analyse the contribution of these mechanisms to the pituitary phenotype in Six3+/−;Hesx1Cre/+ compound embryos. The initial induction of Rathke's pouch was assessed by in situ hybridisation with Fgf8 and Lhx3 at 10.5 dpc. Fgf8 was expressed in the ventral diencephalon in Six3+/−;Hesx1Cre/+ double heterozygotes and no major differences were observed in these embryos compared with Six3+/−, Hesx1Cre/+ and wild-type littermates (Supplementary Fig. S3C, D). Likewise, no apparent differences were observed in Lhx3 expression in the developing Rathke's pouch (Supplementary Fig. S3A, B). The expression pattern of Bmp4 was also comparable between wild-type and Six3+/−;Hesx1Cre/+ mutants at 11.5 dpc (Supplementary Fig. S3E, F). From this analysis, we conclude that the initial induction of +/− Cre/+ Rathke's pouch occurs normally in Six3+/−;Hesx1Cre/+ compound Fig. 3. Normal anterior pituitary specification in Six3 ;Hesx1 embryos at 15.5 dpc. In situ hybridisation with Lhx3 (A, B), Pomc1 (C, D), Cga (encoding αGSU) (E, F) and Pit1 embryos and that it is unlikely that the anterior pituitary hyperplasia − (G, H) in wild-type (+/+) and Six3+/ ;Hesx1Cre/+ embryos. The expression domains are in these mutants is primarily caused by the recruitment of additional expanded and the anterior pituitary appears hyperplastic in the double heterozygous oral ectoderm into Rathke's pouch at 10.5 dpc. mutants. C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 327

Fig. 4. Normal terminal differentiation of hormone-producing cells in Six3+/−;Hesx1Cre/+ anterior pituitary glands. In situ hybridisation on transverse sections through the pituitary glands of wild-type (A, D, G, J, M, P, S) and Six3+/−;Hesx1Cre/+ compound embryos showing either mild (B, E, H, K, N, Q, T) or severe (C, F, I, L, O, R, U) defects. In the mild phenotype, the pituitary gland is positioned normally and all cell types are detectable. However, in severe cases, the pituitary gland is ectopically misplaced in the nasopharyngeal cavity (np in panel C). In general, all cell types are identifiable in Six3+/−;Hesx1Cre/+ mutants, although Lhb expression was barely detectable in panel O. This is likely to be as a consequence of delayed differentiation of the gonadotrophs, as Sf1 expression was normal in the anterior pituitary (S–U). The apparent increase of signal in the hypothalamic area in panel U is due to the axial level of the section. telencephalon, eyes, frontonasal mass and pituitary gland. From these (Six3) and with the normal pattern of expression of these two genes data, we conclude that the Six3+/−;Hesx1Cre/+ mutants phenocopy the during pituitary development (Dasen et al., 2001; Dyer, 2003). Second, mild end of the spectrum of forebrain and pituitary defects observed the increased proliferation at early stages leads to an abnormal in Hesx1 null embryos. morphogenesis of the pituitary gland in compound embryos without Cell death and proliferation analyses were also performed at affecting lineage specification or final differentiation at 17.5 dpc. 17.5 dpc and at 30–33 days of age (postnatal week 4.5). Cell death, as However, there is a knock-on effect in compound embryos that causes measured by TUNEL staining, was comparable between Six3+/−; a reduction in the proliferation rate of differentiated progenitors at Hesx1Cre/+ mutants and wild-type littermates (data not shown). 17.5 dpc and postnatally, possibly leading to hypopituitarism. However, cellular proliferation in the pituitary gland was significantly reduced in compound mutants (Supplementary Fig. S4). At 17.5 dpc, Increased proliferation might be mediated by up-regulation of the Wnt/ the pituitary glands of Six3+/−;Hesx1Cre/+ mutants and wild-type β-catenin pathway through LEF1–PITX2 littermates were similar in size (although the mutant pituitary was generally smaller). However, by 4.5 weeks postnatally, the wild-type Having established that increased proliferation is the primary pituitary was much larger than that of compound mutants (Supple- cellular defect responsible for the pituitary phenotype of the Six3+/−; mentary Fig. S4). In fact, the latter appears similar in size to that of Hesx1Cre/+ compound embryos, we next sought a possible molecular 17.5 dpc embryos. mechanism. Hesx1 and Six3 have been proposed to antagonise the Taken together, two main conclusions can be drawn from the ectopic activation of the Wnt/β-catenin signalling pathway within the marker and proliferation analyses. First, the Hesx1/Six3 interaction anterior forebrain (Lagutin et al., 2003; Andoniadou et al., 2007). appears to be relevant for the normal control of proliferation in Compelling evidence indicates that the Wnt/β-catenin signalling periluminal progenitors of Rathke's pouch at 12.5 dpc. This is pathway controls proliferation during normal development in a consistent with previous results showing the involvement of these variety of cell types, including the early pituitary (van Noort and two genes in the growth of the pituitary gland (Hesx1) and retina Clevers, 2002; Zhu and Rosenfeld, 2004; Arce et al., 2006; Hoppler and 328 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333

(ventral diencephalon), was unaffected in Six3+/−;Hesx1Cre/+ compound embryos compared with single heterozygous and wild-type embryos (Figs. 7C–F; n=3)(Treier et al.,1998; Cha et al., 2004). We also analysed the expression of known Wnt/β-catenin targets such as Lef1, Pitx2 and Axin2. Axin2 is a known Wnt/β-catenin signalling target, which is ectopically expressed within the anterior forebrain in both Hesx1−/− (Jho et al., 2002; Andoniadou et al., 2007) and Six3−/− embryos (G. Oliver, personal communication). Axin2 is normally expressed in a high-ventral to low-dorsal gradient in the anterior pituitary at 12.5 dpc (Olson et al., 2006)(Fig. 7G). In Six3+/−;Hesx1Cre/+ compound embryos this characteristic gradient of expression was less evident and Axin2 transcripts were detected in more dorsal regions of the developing anterior pituitary (Fig. 7H; n=4). Moreover, the hybridisation signal appeared stronger in the anterior pituitary of the compound embryos when compared with wild-type littermates. An apparent up-regula- tion of Pitx2 expression in the developing pituitary of Six3+/−;Hesx1Cre/+ compound embryos compared with wild-type littermates was also observed (Supplementary Fig. S3; n=3). However, in situ hybridisation with Lef1 antisense riboprobes detected comparable low levels of expression in the developing pituitary of compound embryos and wild-type littermates at 12.5 dpc (data not shown). In conclusion, the up-regulation of Axin2 and Pitx2 expression in the mutant Rathke's pouch and developing anterior pituitary suggests that over-activation of Wnt/β-catenin signalling might contribute to the pituitary hyper- plasia observed in the double heterozygous mutants. Next, quantitative real-time PCR was used to analyse the expression levels of some components of the Wnt/β-catenin–LEF1–PITX2 path- way. Lef1, Pitx2 and Cyclin D2 expression levels were significantly higher in the pituitary gland of Six3+/;Hesx1Cre/+ compound embryos compared with control pituitaries from other genotypes (Fig. 7M). Immunostaining with a specific antibody against cyclin D2 revealed the presence of higher numbers of cycling cells in the pituitary gland of the double heterozygotes compared with wild-type littermates (Figs. 7K,L). Axin2 expression was elevated in Six3+/−;Hesx1Cre/+ and surpris- ingly, in Six3+/− embryos, suggesting that Six3 haploinsufficiency alone may cause up-regulation of Axin2, which may contribute to the mild pituitary phenotype observed sporadically in Six3+/− embryos. This analysis also revealed that Lef1 and Cyclin D2 expression was higher in Hesx1Cre/Cre embryos compared with wild-type littermates, but Pitx2 and Axin2 expression was unaffected in these mutants (Supplementary Fig. S5). However, Cyclin D1 and c-Myc expression levels remained unchanged in compound heterozygotes compared with single mutants and wild-type embryos (data not shown). Two conclusions can be drawn from these results: (i) Although the cellular consequences of Fig. 5. Hormone-producing cells are present in the postnatal pituitary gland of Six3+/−; lacking either the two Hesx1 alleles in Hesx1Cre/Cre embryos, or only one Cre/+ Hesx1 animals. (A, B) Photographs of pituitary glands isolated from wild-type (A) or of each in Six3+/−;Hesx1Cre/+ mutants are comparable (cellular hyper- Six3+/−;Hesx1Cre/+ (B) mice at four weeks of age. (C–N) In situ hybridisation on transverse proliferation), there are molecular differences in the transcription sections through the pituitary glands shown in panels A, B. Note the specific signal in Six3+/−;Hesx1Cre/+ sections with all the probes analysed, suggesting that hormone- levels of particular Wnt/β-catenin components associated with producing cells are still present at this stage. specific genotypes; (ii) The increased proliferation observed in Six3+/−;Hesx1Cre/+ and Hesx1Cre/Cre embryos may be partly due to the Kavanagh, 2007). For instance, the β-catenin/LEF1/PITX2 pathway has up-regulation of the Wnt/β-catenin/LEF1 pathway. been shown to stimulate the expression of growth-regulating genes, including Cyclin D1 (Ccnd1), Cyclin D2 (Ccnd2) and c-Myc (Kioussi Molecular nature of the Hesx1/Six3 interaction et al., 2002; Baek et al., 2003; Briata et al., 2003). We hypothesised that over-activation of the Wnt/β-catenin signalling pathway might The possibility that Hesx1 is regulating Six3 expression appears to be − contribute, at least in part, to the pituitary phenotype in the Six3+/ ; unlikely. Firstly, Six3 expression is normal in the pituitary of Hesx1- Hesx1Cre/+ compound embryos. deficient embryos (Supplementary Fig. S3). Unfortunately, Six3−/− Firstly, we performed in situ hybridisation with several markers embryos fail to form any pituitary tissue, which precludes the analysis that have been shown to act upon the Wnt/β-catenin signalling of Hesx1 expression. However, levels of Hesx1 mRNA in Six3+/− and pathway. SOX2 and SOX3, two inhibitory β-catenin partners that are Six3+/+ (wild-type) pituitaries are comparable by qRT-PCR (Fig. 7M). This required for normal pituitary development (Figs. 7A–B; data not does not support a regulatory role for Six3 on the Hesx1 locus. Moreover, shown) (Zorn et al., 1999; Rizzoti et al., 2004; Mansukhani et al., 2005; within the anterior forebrain, Hesx1 is expressed in Six3−/− mutants and Kelberman et al., 2006), were expressed normally in the ventral Six3 is expressed in Hesx1−/− embryos. Taken together, the data suggest diencephalon (Sox2 and Sox3) and developing Rathke's pouch (Sox2)in that it is unlikely that Six3 and Hesx1 regulate each other. − Six3+/ ;Hesx1Cre/+ compound embryos at 12.5 dpc (n=3). Likewise, We next explored whether the mechanism underlying the genetic expression of Wnt4 (ventral diencephalon and pituitary) and Wnt5a interaction between Hesx1 and Six3 might be a direct protein–protein C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 329

Fig. 6. Increased cellular proliferation in the anterior pituitary of Six3+/−;Hesx1Cre/+ and Hesx1Cre/Cre embryos. (A–J) Fluorescent images of sections of 12.5 dpc embryos at the level of the pituitary gland (anterior to the left) stained with either anti-phospho-histone H3 antibody (A–E) or DAPI (F–J). (K) Mitotic index of luminal cells in the anterior pituitary of different genotypes. Six3+/−;Hesx1Cre/+ and Hesx1Cre/Cre have a significantly higher mitotic index (pb0.001 one-way ANOVA) compared with wild-type, Six3+/− or Hesx1Cre/+ embryos. Note the slight increase in the mitotic index in Six3+/− embryos, which failed to reach statistical significance compared with wild-type.

interaction. Firstly, this possibility was analysed in a mammalian two- coronal paraffin sections through the developing anterior pituitary hybrid system. Co-transfection of CHO and 293T cells with plasmids and prospective hypothalamus of human embryos at Carnegie Stage expressing either full-length HESX1 protein or a fusion of SIX3 to the (CS) 17 and 20, and Fetal Stage (F) 1 (Fig. 8A). SIX3 expression was activation domain of the herpes simplex virus protein VP-16 (SIX3- detected in Rathke's pouch and overlying ventral diencephalon at CS VP16) failed to reveal any significant activation over the basal levels of 17 (Fig. 8A) and in the anterior and posterior lobes of the pituitary at a luciferase reporter containing five copies of a consensus paired-like CS 20 (Fig. 8B). At F1, SIX3 expression was observed in the anterior binding site (P3) (Supplementary Fig. 6A). In contrast, co-expression of pituitary, and the ventricular zones of the hypothalamus and HESX1 and TLE1-VP16 led to a significant increase in luciferase telencephalic vesicles (Fig. 8C). This analysis indicates that SIX3 activity. TLE1 has been previously shown to interact with HESX1 expression occurs during normal human pituitary gland embryogen- (Dasen et al., 2001). Similar results were obtained when cells were esis, suggesting that its function in this organ may be conserved transfected with p-Gal4BS-luciferase reporter plasmid, which har- between mice and humans. bours GAL4 binding sites, and plasmids expressing GAL-4-HESX1 and SIX3-VP16 fusion proteins (data not shown). Secondly, four attempts Discussion to co-immunoprecipitate tagged HESX1 and SIX3 in mammalian cells were unsuccessful, even though both proteins were expressed at high In this manuscript we show that haploinsufficiency of both Six3 levels and could be immunoprecipitated with specific anti-tag and Hesx1 leads to severe pituitary dysmorphogenesis during antibodies (Supplementary Fig. S6B). Taken together with the fact development, which results in hypopituitarism with dramatic post- that a HESX1/SIX3 interaction has not been reported in several natal growth retardation of Six3+/−;Hesx1Cre/+ compound mice. This screening studies aiming to identify HESX1 and SIX3 protein partners research provides the first evidence indicating an essential role for the (Zhu et al., 2002; Lopez-Rios et al., 2003; Sajedi et al., 2008), we homeobox gene Six3 during normal pituitary formation. conclude that it is unlikely that HESX1 and SIX3 interact at the protein level. Hesx1 and Six3 control cell proliferation of anterior pituitary progenitors during development SIX3 is expressed in the pituitary gland of the human embryo Our data indicate that the primary cellular defect underlying the It has been previously reported that SIX3 is expressed in the eye of anterior pituitary phenotype in Six3+/−;Hesx1Cre/+ compound embryos the developing human embryo (Granadino et al., 1999), but it is is an increased proliferation of anterior pituitary progenitors between unknown whether SIX3 may also be expressed in the human pituitary 11.5 and 12.5 dpc. This defect is also observed in a proportion of gland. To investigate this, we performed in situ hybridisation on Hesx1-deficient (Hesx1Cre/Cre) embryos, suggesting that a similar 330 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333

Fig. 7. Up-regulation of the β-catenin/LEF1–PITX2 pathway in Six3+/−;Hesx1Cre/+ pituitary glands. (A–J) In situ hybridisation on sagittal sections at the level of the pituitary gland of 12.5 dpc (A–F) and 13.5 dpc (G–J) wild-type and Six3+/−;Hesx1Cre/+ embryos. (A, B) Sox2 expression in the ventral diencephalon and infundibulum (arrows in panel A), and pituitary gland (arrowhead, A) is similar between genotypes. (C, D) Wnt4 is expressed in the anterior pituitary gland of Six3+/−;Hesx1Cre/+ (D) and wild-type (C) embryos. (E, F) Wnt5a is expressed in the ventral diencephalon and infundibulum of the double mutant (F) and wild-type (E) embryos. (G, H) Axin2 is expressed in a high-ventral to low-dorsal gradient within the wild-type anterior pituitary (G). (H) Axin2 expression is increased in the ventral anterior pituitary of the Six3+/−;Hesx1Cre/+ embryo and appears to reach more dorsal levels than in the wild-type littermate. (I, J) NeuroD expression is detected in the ventral side of the anterior pituitary in both genotypes. (K, L) Pituitary glands of 13.5 dpc wild-type (K) and Six3+/−;Hesx1Cre/+ (L) embryos stained with an antibody against Cyclin D2. Note the increase of Cyclin D2-positive cells in the mutant pituitary gland (L). (M) Quantitative real-time PCR analysis of Hesx1, Lef1, Pitx2, Cyclin D2 and Axin2 expression in pituitary glands dissected from wild-type (black bars), Hesx1Cre/+ (white bars), Six3+/− (grey bars) and Six3+/−;Hesx1Cre/+ (striped bars) embryos at 12.5 dpc. Note an up-regulation of Lef1 (pb0.005, one-way ANOVA) and Pitx2 (pb0.05, one-way ANOVA followed by Dunn's method) in Six3+/−;Hesx1Cre/+ compound embryos when compared with other genotypes. CyclinD2 is increased in double mutants (pb0.05, one-way ANOVA) in comparison with wild-type and Six3+/−, but not compared with Hesx1Cre/+ embryos. Axin2 is up-regulated in Six3+/−;Hesx1Cre/+ double heterozygotes compared with wild-type and Hesx1Cre/+ embryos (pb0.05, one-way ANOVA). Axin2 expression appears elevated in Six3+/− embryos compared with wild-type and Hesx1Cre/+, but the differences do not reach statistical significance.

molecular mechanism might underlie the pituitary phenotype in both β-catenin is not detected in the developing pituitary gland from 10.5 mutants. to 13.5 dpc (Brinkmeier et al., 2007). Thirdly, activation of Wnt/β- Normal pituitary growth is dependent on several signalling catenin signalling through LEF1 has been proposed to induce Pitx2 pathways, including the FGF, TGFβ and Wnt/β-catenin pathways expression, which in turn, controls the transcription of critical cell (Treier et al., 1998; Ericson et al. 1998). Different lines of evidence cycle regulators that promote cell-type-specific proliferation (Kioussi suggest that down-regulation of the Wnt/β-catenin signalling path- et al., 2002; Baek et al., 2003; Briata et al., 2003). Finally, whilst way in anterior pituitary precursors during early stages of pituitary mutations in Wnt4 are associated with pituitary hypoplasia (Treier development is required for normal pituitary formation. Firstly, et al., 1998), embryos carrying a mutation in Tcf4, which usually acts conditional removal of normal β-catenin function with a Pitx1-Cre as a Wnt/β-catenin repressor (van Noort and Clevers, 2002; Arce et driver has no phenotypic consequences from 9.5 to 11.5 dpc, al., 2006; Hoppler and Kavanagh, 2007), exhibit elevated cell suggesting that β-catenin does not play an essential role during proliferation (Brinkmeier et al., 2007). In the forebrain, both Hesx1 early pituitary development (Olson et al., 2006). Secondly, nuclear and Six3 function to antagonise the activation of Wnt/β-catenin C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 331 signalling within the anterior forebrain precursors to prevent a posterior cell fate transformation. Our data indicate that expression of Lef1, Pitx2 and Cyclin D2 is elevated in both Six3+/−;Hesx1Cre/+ and Hesx1Cre/Cre embryos and Axin2 (another Wnt/β-catenin target) is expressed ectopically and at high levels in Six3+/−;Hesx1Cre/+ compound heterozygotes compared with wild-type littermates. Therefore, it is feasible that enhanced or ectopic Wnt/β-catenin signalling might be a major contributor to the pituitary phenotype observed in the Six3+/−; Hesx1Cre/+ compound and Hesx1Cre/Cre embryos. Although we cannot exclude a role for the FGF and TGFβ signalling pathways, we have shown that Fgf8 and Bmp4 expression is not grossly affected in either Six3+/−;Hesx1Cre/+ or Hesx1-deficient embryos compared with single mutants or wild-type littermates at 10.5–11.5 dpc. It is worth noting that the phenotypic consequences of enhanced Wnt/β-catenin signal- ling within the anterior forebrain (cell fate transformation) and anterior pituitary progenitors (increased proliferation) are different, supporting the idea that the specific response to a de-regulation of Wnt/β-catenin signalling is highly context dependent (van Noort and Clevers, 2002; Arce et al., 2006; Hoppler and Kavanagh, 2007). The molecular basis of the genetic interaction between Hesx1 and Six3 is not fully understood. We present data suggesting that a HESX1/ SIX3 protein–protein interaction is unlikely. Whether these two transcription factors act in parallel pathways during pituitary development or alternatively, they directly regulate transcription of a subset of common genes, acting synergistically to regulate Wnt/β- catenin signalling in the pituitary, is an important question that merits further molecular studies.

Haploinsufficiency of Hesx1 and Six3 leads to hypopituitarism in mice

A puzzling aspect of the phenotype in the Six3+/−;Hesx1Cre/+ Fig. 8. SIX3 is expressed in the developing pituitary of human embryos. In situ compound heterozygotes is that the pituitary gland is hyperplastic hybridisation on coronal sections of human embryos at Carnegie Stages (CS) 17 and 20, during development but it becomes hypoplastic postnatally. More- and at Fetal Stage 1 specified on the left side of the panel. (A) SIX3 is expressed in the over, normal pituitary function is impaired as judged by the arrested ventral diencephalon (arrows) and developing anterior pituitary (arrowhead) at CS17. growth of the compound mice and the histological defects observed in (B) Note the expression in the infundibulum (arrows) and anterior pituitary (arrow- pituitary target organs such as the thyroid and the testes. However, all heads) at CS20. (C) At Fetal Stage 1 (F1), SIX3 transcripts are detected in the pituitary gland (arrowhead) and in the ventricular zone of the hypothalamus (hy, inset) as well as hormone-producing cell types are present in the pituitary gland of in the telencephalic vesicles (tv, inset). bs: basisphenoid cartilage. Six3+/−;Hesx1Cre/+ compound mice. This observation can be partly explained when considering that a proportion of anterior pituitary tissue escapes from its normal location (between the hypothalamus This is exemplified by the fact that although developmentally, the − and basisphenoid bone) into the nasopharyngeal space. Although this pituitary defects in Six3+/ ;Hesx1Cre/+ embryos are very similar to those tissue contains hormone-producing cells, it is likely that its function described in embryos carrying these inactivating Hesx1 mutations may be impaired as there is no contact with the hypothalamus, which (Dasen et al., 2001; E.Sajedi and J.P. Martinez-Barbera, unpublished), controls expression and secretion of anterior pituitary hormones. the phenotypic consequences in newborn mice are rather different. − − Moreover, this ectopic tissue might be lost once the affected pups are The great majority of Hesx1 / mice, which carry a null allele, die soon born, either by degeneration or by physical damage, as we have found after birth (Andoniadou et al., 2007). In contrast, newborns harbour- that it is often absent when the embryos are processed for histological ing a Hesx1 hypomorphic allele, in which there is a substitution of the analysis. This is consistent with our observations that there is a conserved isoleucine 26 by threonine, are generally viable and show significant reduction of pituitary tissue in Six3+/−;Hesx1Cre/+ compound no growth defects (E. Sajedi and J.P. Martinez-Barbera, unpublished). − embryos at 17.5 dpc. Finally, Six3+/ ;Hesx1Cre/+ mice exhibit dwarfism and die by the 5th– However, a significant amount of anterior tissue remains in its 6th week of age. It is important to emphasise that the main pheno- normal location in the compound mutants at 17.5 dpc. Our data typic difference between these genotypes is the presence of severe − − indicate that this remaining tissue fails to proliferate postnatally and forebrain defects in Hesx1 / embryos, whereas both Hesx1I26T/I26T and − the pituitary gland of Six3+/−;Hesx1Cre/+ compound mice is very small Six3+/ ;Hesx1Cre/+ mutants exhibit only mild forebrain defects (data not when compared with normal littermates. This suggests that the shown). Since the pituitary defects are very similar between the three hypothalamic–pituitary axis may be disrupted in these mutants, as it genotypes, it is conceivable that there may be a neural contribution to − has been shown that hypothalamic input through the hypophysio- the growth retardation in Six3+/ ;Hesx1Cre/+ compound mice. In fact, trophic factors is essential for normal postnatal expansion of Hesx1 is expressed in the prospective hypothalamic region between differentiated anterior hormone-producing cells (Billestrup et al., 8.5 and 9.5 dpc, and Six3 expression remains high in this area during 1986; Korbonits etal., 2004; Ward et al., 2005, 2006; Zhu et al., 2007). development until 16.5 dpc, making it feasible that a gene dosage Analysis of the Six3+/−;Hesx1Cre/+ compound mice (this study) and reduction of these two transcription factors could affect normal other mouse models carrying inactivating Hesx1 mutations initially diencephalic development, including the hypothalamus. Alternatively, identified in humans suffering from hypopituitarism and septo-optic or in addition, defects in the portal blood system or impaired dysplasia (E.Sajedi and J.P. Martinez-Barbera, unpublished), suggests a connection between hypothalamus and anterior pituitary might hypothalamic contribution to the pituitary phenotype in these mutant contribute to the postnatal hypopituitarism (Ward et al., 2006). mice. Although further research is needed to clarify the reasons underlying 332 C. Gaston-Massuet et al. / Developmental Biology 324 (2008) 322–333 the postnatal phenotype of the Six3+/−;Hesx1Cre/+ compound mice, it is Dattani, M.T., Martinez-Barbera, J.P., Thomas, P.Q., Brickman, J.M., Gupta, R., Martensson, I.L., Toresson, H., Fox, M., Wales, J.K., Hindmarsh, P.C., Krauss, S., Beddington, R.S.P., interesting that conditional inactivation of Six3 either within the brain Robinson, I.C.A.F., 1998. Mutations in the homeobox gene HESX1/Hesx1 associated or the pituitary gland leads to postnatal growth retardation and with septo-optic dysplasia in human and mouse. Nat. Genet. 19, 125–133. premature death (A. Lavado and G. Oliver, personal communication), Dyer, M.A., 2003. Regulation of proliferation, cell fate specification and differentiation by the homeodomain proteins Prox1, Six3 and Chx10 in the developing retina. Cell implying that Six3 is required not only within the pituitary gland but Cycle 2, 350–357. also in the forebrain for normal pituitary function. Ellworth, B.S., Butts, D.L., Camper, S.A., 2008. Mechanisms underlying pituitary It has been shown that dominant mutations in HESX1 associated hypoplasia and failed cell specification in Lhx3-deficient mice. Dev. Biol. 313, – with human cases of hypopituitarism show a marked variability in 118 129. Ericson, J., Norlin, S., Jessell, T.M., Edlund, T., 1998. Integrated FGF and BMP signaling both the severity and penetrance of the phenotype (Dattani et al., controls the progression of progenitor cell differentiation and the emergence of 1998; Kelberman and Dattani, 2007). Often, this variability is observed pattern in the embryonic anterior pituitary. Development 125, 1005–1015. between individuals carrying the same heterozygous mutation within Gage, P.J., Lossie, A.C., Scarlett, L.M., Lloyd, R.V., Camper, S.A., 1995. Ames dwarf mice exhibit somatotrope commitment but lack growth hormone-releasing factor the same family, suggesting that factors other than the HESX1 response. Endocrinology 136, 1161–1167. genotype might be involved. Our research has revealed that whilst Granadino, B., Gallardo, M.E., López-Ríos, J., Sanz, R., Ramos, C., Ayuso, C., Bovolenta, P., single heterozygous individuals are normal, double heterozygous Rodríguez de Córdoba, S., 1999. Genomic cloning, structure, expression pattern, and – +/− Cre/+ chromosomal location of the human SIX3 gene. Genomics 55, 100 105. Six3 ;Hesx1 animals exhibit hypopituitarism. This raises the Hermesz, E., Mackem, S., Mahon, K.A., 1996. Rpx: a novel anterior-restricted homeobox possibility that some of the HESX1 heterozygous human patients gene progressively activated in the prechordal plate, anterior neural plate and might also be heterozygous for SIX3. The fact that SIX3 expression in Rathke's pouch of the mouse embryo. Development 122, 41–52. Hoppler, S., Kavanagh, C.L., 2007. Wnt signalling: variety at the core. J. Cell Sci. 120, the developing pituitary gland is conserved in human embryos 385–393. supports this idea. Future research will evaluate the role of SIX3 in Jho, E.H., Zhang, T., Domon, C., Joo, C.K., Freund, J.N., Costantini, F., 2002. Wnt/β-catenin/ the aetiology of these human conditions. 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Martin, Kendall, S.K., Samuelson, L.C., Saunders, T.L., Scarlett, L.M., Camper, S.A., 1995. Targeted disruption of the pituitary glycoprotein hormone alpha-subunit produces hypogo- M. Rosenfeld, R. Lovell-Badge, Sally Camper and the MRC Geneservice nadal and hypothyroid mice. Genes Dev. 15, 2007–2019. for probes, and the MRC/Wellcome-funded Human Developmental Kioussi, C., Briata, P., Baek, S., Rose, D., Hamblet, N., Herman, T., Ohgi, K., Lin, C., Biology Resource for human embryo sections. We are grateful to Gleiberman, A., Wang, J., Brault, V., Ruiz-Lozano, P., Nguyen, H.D., Kemler, R., Glass, C.K., Wynshaw-Boris, A., Rosenfeld, M.G., 2002. Identification of a Wnt/Dvl/β- E. Pauws for help with thyroid gland dissections. This work was catenin-Pitx2 pathway mediating cell-type-specific proliferation during develop- supported by grants 068630 and 078432 from The Wellcome Trust. ment. Cell 111, 673–685. 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