Fgfr2 is required for the expansion of the early adrenocortical primordium

Molecular and Cellular Endocrinology 413 (2015) 168e177

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Fgfr2 is required for the expansion of the early adrenocortical primordium €fner a, Tobias Bohnenpoll a, Carsten Rudat a, Thomas M. Schultheiss b, Regine Ha Andreas Kispert a, * a b

Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany Department of Genetics and Developmental Biology, Rappaport-Technion Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 May 2015 Received in revised form 21 June 2015 Accepted 22 June 2015 Available online 30 June 2015

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from intermediate mesoderm of the anterior urogenital ridge. Previous work has pinpointed a minor role of the FGFR2IIIb isoform in expansion and differentiation of the fetal adrenal cortex in mice but did not address the complete role of FGFR2 and FGFR1 signaling in adrenocortical development. Here, we show that a Tbx18cre line mediates specific recombination in the coelomic epithelium of the anterior urogenital ridge which gives rise by a delamination process to the adrenocortical primordium. Mice with conditional (Tbx18cre-mediated) deletion of all isoforms of Fgfr2 exhibited severely hypoplastic adrenal glands around birth. Cortical cells were dramatically reduced in number but showed steroidogenic differentiation and zonation. Neuroendocrine chromaffin cells were also reduced and formed a cell cluster adjacent to but not encapsulated by steroidogenic cells. Analysis of earlier time points revealed that the adrenocortical primordium was established in the intermediate mesoderm at E10.5 but that it failed to expand at subsequent stages. Our further experiments show that FGFR2 signaling acts as early as E11.5 to prevent apoptosis and enhance proliferation in adrenocortical progenitor cells. FGFR1 signaling does not contribute to early adrenocortical development. Our work suggests that FGFR2IIIb and IIIc isoforms largely act redundantly to promote expansion of the adrenocortical primordium. © 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Adrenocortical primordium Proliferation Coelomic epithelium Conditional knockout Tbx18 Fgfr2

1. Introduction The adrenal cortex is a critical endocrine tissue that synthesizes and secretes different classes of steroid hormones in the control of body homeostasis and stress response (Walczak and Hammer, 2015). The adrenal cortex develops through a series of complex cellular processes from the intermediate mesoderm of the anterior urogenital ridge. At approximately embryonic day (E) 9.5 in the mouse, cells are supposed to delaminate from the coelomic epithelium covering the urogenital ridge and invade the underlying mesenchyme to form a contiguous adrenogonadal primordium. Starting at E10.5, a small anterior cell cluster separates from this primordium and moves dorso-medially to form the adrenal anlage adjacent to the dorsal aorta, while the remainder of the cells moves ventro-laterally to contribute to somatic cells of the gonads (Hatano

* Corresponding author. Institut für Molekularbiologie, OE5250, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail address: [email protected] (A. Kispert). http://dx.doi.org/10.1016/j.mce.2015.06.022 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.

et al., 1996; Luo et al., 1994; Uotila, 1940). From E11.5 to E12.5, neural crest cells invade the adrenal anlage. While the two progenitor populations proliferate rapidly and sort into a central medulla and surrounding cortex, mesenchymal cells probably of coelomic origin form a fibrous capsule around the composite adrenal anlage by E14.5. Neural crest derived medullary cells differentiate into neuroendocrine chromaffin cells that synthesize catecholamines and secrete them in response to sympathetic inputs (Anderson et al., 1991). The cortical region matures and forms a transient fetal zone (x-zone) and the definitive (adult) cortex. The fetal zone regresses after puberty in mice while the definitive cortex subdivides in a thin outer zona glomerulosa synthesizing mineralocorticoids and a thick inner zona fasciculata producing glucocorticoids. Homeostasis of the adrenal cortex is mediated by capsular and subcapsular progenitors that give rise to steroidogenic cells that move centripetally (for reviews see (Kim et al., 2009; Laufer et al., 2012; Walczak and Hammer, 2015). Analyses of humans with congenital adrenal hypoplasia and of knockout mice have identified a number of molecular regulators of

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adrenocortical development. The steroidogenic factor 1 gene (Sf1, also known as Nr5a1), that encodes an orphan nuclear receptor expressed in the coelomic epithelium, the adrenogonadal primordium and the steroidogenic cells of the adrenal cortex (Ikeda et al., 1994), turned out as a key regulator of progenitor expansion, steroidogenic pathway gene expression and steroidogenic cell identity (Lala et al., 1992; Luo et al., 1994). Sf1 transcription is positively regulated by Cbp/p300-interacting transactivator, with Glu/Asprich carboxy-terminal domain, 2 (CITED2) (Bamforth et al., 2001), and negatively impacted on by Wilms tumor 1 homolog (WT1) (Bandiera et al., 2013; Moore et al., 1999), DAX1 (also known as nuclear receptor subfamily 0, group B, member 1, Nrob1) (Ito et al., 1997) and transcription factor 21 (TCF21) (Tamura et al., 2001). Sonic hedgehog (Shh) is expressed in the adrenocortex underneath the adrenal capsule. It is required for proliferation of capsular and adrenocortical cells but not for differentiation of the adrenocortex (Huang et al., 2010; King et al., 2009). Canonical WNT signaling maintains adrenocortical progenitors between E12.5 and E14.5 (Kim et al., 2008). WNT signaling within the outer adrenal cortex is subsequently involved in recruitment of progenitors, potentially through stimulation of Shh expression, and in differentiation of the zona glomerulosa (reviewed in (Drelon et al., 2015)). Insulin and insulin-like growth factor (IGF)1 receptor signaling have been implicated in induction of cell proliferation and maintenance of Sf1 expression throughout the genital ridge and the adrenogonadal primordium beginning as early as E10.5 (Pitetti et al., 2013). Fibroblast growth factors (FGF)s are a family of 23 secreted proteins that bind with high affinity to at least four members of a family of receptor tyrosine kinases, termed FGFR1-FGFR4. FGFRs occur in different splice variants that signal through different downstream modules to trigger in both a transcriptionally independent and dependent manner changes of cell behavior including migration, proliferation, apoptosis and differentiation in a variety of biological contexts (Laestander and Engstrom, 2014). FGFR2 signaling has been functionally implicated in adrenal development by a number of genetic loss-of-function experiments in vivo. Revest and colleagues showed that mice carrying an Fgfr2 allele in which the IIIb variant is disrupted, exhibit numerous organ defects including hypoplastic adrenals at E16.5 (Revest et al., 2001). A couple of years later Kim and coworkers noted that mice with Sf1cre-mediated deletion of all splice variants of Fgfr2 have a severe adrenal hypoplasia (Kim et al., 2007). More recently, it was found that mice with global deletion of the Fgfr2IIIb variant exhibit smaller adrenals with a thickened mesenchymal capsule and a slightly reduced expression of steroidogenic and zona fasciculata markers (Guasti et al., 2013). Although these studies suggest a (minor) role for the FGFR2IIIb isoform in development of the fetal adrenal cortex, the more severe adrenal hypoplasia noted in mice with conditional deletion of all Fgfr2 splice variants in the adrenogonadal primordium argues for an additional or earlier requirement of other FGFR2 isoforms, and FGFR2 signaling, respectively, in adrenal development. Here, we use a Tbx18cre line (Trowe et al., 2010) to completely delete Fgfr2 in the coelomic epithelium of the anterior urogenital ridge. We show that Fgfr2 (but not Fgfr1) has an early function in the expansion but not the differentiation of the adrenocortical primordium.

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€chsisches Landesamt für Verapproved by the Niedersa braucherschutz und Lebensmittelsicherheit (AZ 33.12-42502-0413/1356). Tbx18GFP (Tbx18tm2Akis) and Tbx18cre (Tbx18tm4(cre)Akis) mice were previously generated in the laboratory at the Medizinische Hochschule Hannover (Christoffels et al., 2006; Trowe et al., 2010). Mice expressing the double fluorescent cre reporter line (Gt(ROSA) 26Sortm4(ACTB-tdTomato-EGFP)Luo, synonym: R26mTmG) (Muzumdar et al., 2007), mice with loxP sites flanking exon 4 of the Fgfr1 locus (Fgfr1tm5.1Sor; synonym: Fgfr1fl) (Hoch and Soriano, 2006), and mice with loxP sites flanking exons 7 to 10 of the Fgfr2 locus (Fgfr2tm1Dor, synonym: Fgfr2fl) (Yu et al., 2003) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained on an NMRI outbred background. Embryos for mutant analysis were derived from matings of Tbx18cre/þ;R26mTmG;Fgfr1fl/þ;Fgfr2fl/þ male and Fgfr1fl/fl;Fgfr2fl/fl female mice. Embryos for Fgfr1/Fgfr2 expression analysis were obtained from NMRI matings. Matings were set up in the evening and vaginal plugs checked in the morning afterward. Noon was taken as E0.5. Urogenital systems and embryos were dissected in PBS, fixed in 4% paraformaldehyde (PFA) in PBS and stored in methanol at 20  C. For genotyping by PCR genomic DNA prepared from yolk sacs or tail biopsies was used. 2.2. Organ cultures Urogenital ridges from E11.5 embryos of Tbx18cre/ ;R26mTmG;Fgfr1fl/þ;Fgfr2fl/þ x Fgfr1fl/fl;Fgfr2fl/fl matings were dissected, explanted on Transwell permeable supports (Corning Inc.) and cultured with DMEM/F12 medium (Gibco) supplemented with 10% fetal calf serum (Lonza) and 1% penicillin/streptomycin solution (Hyclone Laboratories) at the atmosphere-medium interface at 37  C and 5% CO2. Replacement of culture medium and documentation took place every 24 h.

þ

2.3. Histological and immunofluorescent analysis For histological analysis, embryos were fixed as stated before, paraffin embedded and sectioned to 5 mm. Sections were stained with hematoxylin and eosin, following standard procedures. Immunofluorescence analysis was done on 5 mm sections with the following antibodies: rabbit anti-SF1 (TransGenic Inc., preparation of antibodies by Dr. Ken-Ichirou Morohashi, 1:200), rabbit anti-TH (ABIN723635, antikoerper-online.de, 1:800), mouse antiGFP (11814460001, Roche, 1:200), rabbit anti-GFP (sc-8334, Santa Cruz, 1:200). Fluorescent staining was performed using Alexa-488/ 555-conjugated secondary antibodies (A11034; A11008; 711-487003; A21202; A21422; A21428, Invitrogen/Dianova; 1:400) or biotin-conjugated secondary antibodies (Dianova; 1:400) and the TSA Tetramethylrhodamine Amplification Kit (PerkineElmer). Tagging with primary antibodies was performed at 4  C overnight after antigen retrieval (Antigen unmasking solution, Vector Laboratories; 10 min, 100  C), blocking of endogenous peroxidases (3% H2O2/ddH2O, 15 min), and incubation in blocking solutions provided with the kits. Sections were mounted with Mowiol (Roth). All sections were counterstained with 40 ,6-diamidino-2phenylindole (DAPI) to visualize the nuclei. 2.4. Proliferation and apoptosis assays

2. Methods 2.1. Mice Mice were housed in rooms with controlled light and temperature. All mouse work was performed according to European and German legislation. The breeding of mutant mouse lines was

To analyze apoptosis with the TUNEL assay, stained sections were treated according to the protocol provided with the ApopTag Fluorescence Apoptosis detection kit (S7111, Millipore) before DAPI-staining and mounting. For cell proliferation analysis the incorporated 5-bromo-20 -deoxyuridine (BrdU) on 5 mm sections was detected fluorescently (1170376, Roche, 1:100) as described

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previously (Ludtke et al., 2013). The BrdU labeling index was defined relative to the number of SF1þ cells. Statistical analyses were performed using the two-tailed Student's t-test. Data are depicted as mean ± s.d. Significances: *: significant P < 0.05, not significant (n.s.): P  0.05. All analyses were performed using at least three specimens of wildtype and mutants. 2.5. Three-dimensional reconstruction To reconstruct early adrenal gland populations serial antibody stainings (5 mm, SF1-GFP counterstaining) of whole embryos were used. Further analysis was performed with the software “Amira” (ATI Technologies Inc., Version 2.1 ATI-1.6.42) as described previously (Soufan et al., 2003). 2.6. In situ hybridization analysis In situ hybridization analysis was performed on 10 mm sections as described previously (Moorman et al., 2001). To detect adrenocortical precursors at E10.5 to E12.5, Sf1 was used as a probe on adjacent sections. 2.7. Image analysis Documentation of whole-mount specimens was performed with a Leica M420 macroscope with Fujix HC-300Z digital camera. Sections were documented with Leica digital camera DFC300 FX. Image processing took place in Adobe Photoshop CS4. 3. Results 3.1. Tbx18 demarcates adrenocortical progenitors We have previously shown that the T-box transcription factor gene Tbx18 is expressed in the urogenital ridge and in the ureteric mesenchyme and that these expression domains contribute to various cell types in the bladder, ureter, kidney, gonad and adrenal gland in the mature urogenital system (Bohnenpoll et al., 2013). To more carefully delineate the expression of Tbx18 in adrenal development, we performed comparative expression and fate analyses with the well-established marker of adrenocortical (progenitor) cells, SF1 (Lala et al., 1992; Rice et al., 1991) (Fig. 1). At E9.5, immunofluorescence analysis did not detect SF1 expression on sections of the urogenital ridge. TBX18 expression (visualized as GFP expression from the Tbx18GFP allele (Christoffels et al., 2006)) was present in the coelomic epithelium and in a cluster of medial mesenchyme close to the dorsal aorta at this stage (Fig. 1A). At E10.5, SF1 was found in the coelomic epithelium and the underlying mesenchyme. TBX18/GFP expression was excluded from the SF1 domain and restricted to a dorsomedial subregion of the mesenchyme in the urogenital ridge (Fig. 1C). To evaluate whether TBX18 expression in the urogenital ridge represents adreno(gonadal) precursors, we irreversibly labeled the descendants of this population using a cre/loxP-based genetic approach with a Tbx18cre line generated in our laboratory and the sensitive Rosa26mTmG reporter (Muzumdar et al., 2007; Trowe et al., 2010). In the Rosa26mTmG reporter line, cells that have undergone recombination express membrane-bound GFP while nonrecombined cells express membrane-bound RFP. At E9.5, immunofluorescence analysis did not detect GFP expression in the urogenital ridge of Tbx18cre/þ;Rosa26mTmG/þ embryos indicating that cre-mediated recombination had not yet occurred (Fig. 1B). At E10.5, GFP reporter expression was found in the coelomic epithelium and the underlying ventromedial mesenchyme completely encompassing SF1 expressing cells at cranial levels, as well as in

mesenchyme at a dorsolateral position (Fig. 1D). Immunofluorescence analysis of serial sections in Tbx18cre/þ;Rosa26mTmG/þ embryos revealed that coexpression of SF1 and GFP was restricted to cranial levels of the urogenital ridge. At more posterior levels, the majority of SF1þ cells was negative for GFP (Fig. 1EeH). This suggests that Tbx18cre-mediated recombination occurs in the entire anteriorly located adrenal progenitor pool but only partially in the more extended posterior gonadal subpool of the common adrenogonadal primordium. In fact, in sections of adrenal glands from E11.5 to E18.5 of Tbx18cre/þ;Rosa26mTmG/þ embryos all SF1þ cells coexpressed the lineage marker GFP (Fig. 1IeL) whereas only a subpopulation of SF1þ cells were GFPþ in the gonads (Fig. S1). Together, these analyses confirm that TBX18 expression demarcates (prior to SF1) adrenal progenitor cells in the coelomic epithelium of the urogenital ridge and that the Tbx18cre line is a suitable tool to genetically manipulate this cell population. 3.2. Loss of the Fgfr2 in the urogenital ridge leads to severely hypoplastic adrenal glands To investigate the role of Fgfr2 and the closely related Fgfr1 gene in the urogenital ridge and the ureteric mesenchyme, we used a conditional gene targeting approach with our Tbx18cre line and floxed alleles of Fgfr1 and Fgfr2. For Fgfr1, we used the allele Fgfr1tm5.1Sor in which loxP-sites flank exon 4 that is common to all splice variants (Hoch and Soriano, 2006). For Fgfr2, we used the Fgfr2tm1Dor allele, in which exons 8e10 encoding the ligand binding domains IIIb and IIIc and the transmembrane domain are loxPflanked (Yu et al., 2003). In either case, cre-mediated recombination results in complete null alleles. The urogenital system of Tbx18cre/þ;Fgfr1fl/fl;Fgfr2fl/þ embryos appeared normal at E18.5. In contrast, in Tbx18cre/þ;Fgfr1fl/þ;Fgfr2fl/fl (n ¼ 10) and Tbx18cre/þ;Fgfr1fl/fl;Fgfr2fl/fl embryos (n ¼ 16) adrenal glands were severely hypoplastic or even absent, and weak hydroureter formation was occasionally found (2 out of 10, 5 out of 16) (Figs. 2A, B and S2). Histological analysis by hematoxylin and eosin staining revealed that the tissue architecture was severely disrupted in these mutants and that the fibrous capsular tissue extended strands into the inside of the organ (Fig. 2C, D). Given the similarity of the phenotypic defects, we subsequently interchangeably used Tbx18cre/þ;Fgfr1fl/þ;Fgfr2fl/fl and Tbx18cre/þ;Fgfr1fl/ fl ;Fgfr2fl/fl (Fgfr2cKO) embryos for further analysis. To analyze medullary-cortical subdivision and cortical zonation in more detail, we analyzed expression of specific markers by in situ hybridization (Fig. 2D) and immunofluorescence (Fig. 2E) on sections. In Fgfr2cKO embryos, Sf1/SF1, a marker for steroidogenic cells of the cortex (Lala et al., 1992; Rice et al., 1991), was confined to a lateral cell cluster. In the wildtype, Wnt4 is expressed within the zona glomerulosa (Heikkila et al., 2002), Cyp11b1 marks the zona fasciculata (Yabu et al., 1991). In residual adrenal glands of Fgfr2cKO embryos, Wnt4 was restricted to an outer, Cyp11b1 to the inner region of the SF1þ cell cluster. Expression of heart and neural crest derivatives expressed transcript (Hand)2 and tyrosine hydroxylase (TH) that mark neural crest derived chromaffin cells (Derer et al., 1989; Gestblom et al., 1999), was clustered medially towards the midline of the urogenital system directly abutting the SF1þ domain (Fig. 2D, E). Together this argues that loss of Fgfr2 does not affect cell differentiation in the adrenal gland but strongly impacts on growth and tissue organization of adrenocortical precursors. Fgfr1 may play only a minor role in adrenocortical development. 3.3. Early failure to expand the adrenocortical precursor pool in Fgfr2cKO embryos To detect the onset of the cortico-medullary disorganization in

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Fig. 1. TBX18 demarcates adrenocortical progenitors in the urogenital ridge. Immunofluorescence analysis of SF1 expression (in red), and GFP expression (in green) from the Tbx18GFP allele (A, C) and the Rosa26mTmG reporter allele after recombination with the Tbx18cre line (B, D, EeL), on transverse sections of the urogenital ridge (AeH), and frontal sections of the adrenal gland (primordium) (IeL). Stages and genotypes are as shown. Anterior and posterior designates the relative section level along the body axis. Scale bars are 50 mm (AeJ) and 100 mm (K, L). ce, coelomic epithelium; da, dorsal aorta; dm, dorsal mesentery; ur, urogenital ridge. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fgfr2cKO adrenal glands, we analyzed embryos at E14.5 and E16.5 (Fig. 3). At these stages, the adrenal tissue was encapsulated by a fibrous tissue layer, and a cortico-medullary subdivision with inner THþ chromaffin cells and a surrounding layer of SF1þ steroidogenic cells was established in wildtype embryos. In Fgfr2cKO embryos, the adrenal glands were positioned correctly anterior-medially to the kidney but were dramatically reduced in size (Fig. 3A, B). A fibrous tissue layer surrounded and sometimes intruded into an elongated tissue mass (Fig. 3C) that was subdivided along the medial-lateral axis into a cluster of THþ chromaffin cells and a SF1þ steroidogenic domain (Fig. 3D). This shows that expansion of adrenocortical progenitors and their interaction with neural crest derived chromaffin cells must be strongly affected before encapsulation occurs. To analyze whether the adrenocortical hypoplasia results from a defect in formation, migration and/or expansion of adrenocortical progenitors, we analyzed Fgfr2cKO embryos at E10.5 to E12.5 when these processes occur in wildtype mice. We first visualized the formation of the adrenal primordium by GFP epifluorescence in explant cultures of E11.5 Tbx18cre/þ;Rosa26mTmG/þ (control) urogenital systems. After one day in culture a group of GFPþ cells separated in the anterior region from the rest of the sausageshaped GFPþ-domain within the urogenital ridge and migrated medially towards the dorsal aorta. One day later this cell group had greatly expanded and moved further medially. In urogenital explants of Tbx18cre/þ;Rosa26mTmG/þ;Fgfr2fl/fl embryos a GFPþ cell

cluster also separated anteriorly and moved medially but was dramatically reduced in size at both two points of the culture (Fig. 4A). This suggests that the adrenocortical progenitors form normally but migrate more slowly and/or expand poorly. Initial formation of an adrenal primordium and subsequent lack of expansion in Fgfr2cKO embryos was confirmed by analysis of expression of GFP (from the Rosa26mTmG reporter allele) and SF1 on serial sections at E10.5, E11.5 and E12.5 (Fig. 4B) and subsequent 3dimensional reconstruction of the SF1þ domain (Fig. 4C). Quantification of SF1þ cells in the adrenocortical primordium in serially stained sections revealed a significant two-fold reduction of cell number in the mutant at E11.5. While the cell number in the adrenocortical primordium of the wildtype was more than doubled at E12.5, the number of SF1þ cells in Fgfr2cKO embryos diminished by the factor of two resulting in a more than 10-fold difference of cell number between wildtype and mutant at this stage. We conclude that Fgfr2 is required for the expansion of the early adrenocortical primordium. 3.4. Increased apoptosis and decreased proliferation contribute to adrenocortical hypoplasia To determine the cellular causes of the hypoplasia of the adrenocortical primordium in Fgfr2cKO embryos, we performed the BrdU assay to monitor proliferation and the TUNEL assay to identify changes in apoptosis (Fig. 5A, B). The ratio of BrdUþ to SF1þ cells

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was not significantly changed at E10.5 whereas at E11.5 and E12.5 we found a significant reduction of 58% and 48% in the Fgfr2cKO adrenocortical primordium (Fig. 5C). Furthermore, we found a clear trend to increased apoptosis at E10.5 and E11.5 and a statistically significant increase of 40% in E12.5 Fgfr2cKO adrenocortical cells (Fig. 5D). We conclude that decreased proliferation and increased apoptosis both contribute to hypoplasia of adrenocortical progenitors in Fgfr2cKO embryos. 3.5. Fgfr2 is expressed in the early adrenocortical primordium To better define the spatiotemporal activity of FGFR2 signaling in adrenal development, we analyzed expression of Fgfr2 (and Fgfr1) by in situ hybridization analysis of sections. To detect the adrenocortical primordium in the urogenital ridge from E10.5 to E12.5 we localized Sf1þ cells by in situ hybridization analysis and subsequently used adjacent sections for Fgfr1/Fgfr2 expression analysis. Fgfr1 expression was absent from steroidogenic cells of the urogenital ridge but was weakly detected in subcapsular cortical cells at E14.5 and E16.5. Fgfr2 expression was detected throughout the adrenocortical primordium at E11.5. At E12.5, adrenocortical expression of Fgfr2 became scattered. At E14.5 to E18.5, Fgfr2 expression was confined to an outer cortical subdomain, most likely the zona glomerulosa (Fig. 6). Together, this expression data strongly support that FGFR2 but not FGFR1 acts in the adrenocortical primordium around E11.5 to expand this precursor pool. 3.6. Loss of Fgfr2 does not affect expression of transcription factors and signaling activities in the adrenocortical primordium To investigate whether loss of Fgfr2 in the adrenocortical primordium affects expression of transcription factors and signaling pathways previously implicated in adrenocortical development, we performed in situ hybridization analysis on frontal sections of E10.5 embryos. Expression of the transcription factor genes Cited2, pre B cell leukemia homeobox 1 (Pbx1), Dax1, Tcf21, forkhead box D1 (Foxd1) and Wt1 was indistinguishable in wildtype and mutant Sf1þ adrenocortical cells, as was expression of the targets of canonical Wnt signaling Axin2 and lymphoid enhancer binding factor (Lef)1 (Jho et al., 2002), of Shh and the Shh target gene patched homolog 1 (Ptch1) (Ingham and McMahon, 2001), of insulin receptor 1 (Insr1), Igfr1, Igf1 and Igf2 (Figs. S3 and S4). We conclude that altered expression of these factors does not contribute to the observed phenotypic changes. 4. Discussion 4.1. Tbx18cre is a suitable tool to analyze the molecular control of adrenocortical development Tissue-specific gene targeting approaches using the cre-loxP system circumvent limitations of global gene knockouts such as embryonic lethality or secondary effects in one tissue due to absence of expression at distant site(s). Previous analyses on the genetic control of adrenocortical development largely relied on mouse lines expressing cre under regulatory elements of the Sf1

Fig. 2. Loss of Fgfr2 in the urogenital ridge leads to severely hypoplastic adrenal glands with a disturbed tissue organization at E18.5. (A, B) Morphological analysis of whole urogenital systems (A) and adrenal glands (B); and (C, D) histological analysis by hematoxylin and eosin staining of frontal sections of embryos with Tbx18cre mediated conditional loss of Fgfr1 and Fgfr2. Arrows point to hypoplastic mutant adrenal glands. Images in (D) represent magnifications of the boxed areas in (C). (E) In situ hybridization analysis of steroidogenic cells (Sf1) of the cortex, of the zona glomerulosa (Wnt4), of the zona fasciculata (Cyp11b1), and chromaffin cells of the medulla (Hand2)

of the adrenal gland. (F) Immunofluorescence analysis of SF1 and TH expression in the adrenal gland (both in red). Nuclei are counterstained with DAPI (in blue). GFP expression (in green) marks recombined cells in the adrenal cortex of Tbx18cre/ þ ;Rosa26mTmG/þ;Fgfr1fl/þ;Fgfr2fl/þ control and Tbx18cre/þ;Rosa26mTmG/þ;Fgfr1fl/fl;Fgfr2fl/fl embryos. Markers and genotypes are as shown. Scale bars are 2 mm (A), 0.3 mm (B), 100 mm (C), 25 mm (D), 100 mm (E, F). a, adrenal gland; b, bladder; c, fibrous capsular tissue; hu, hydroureter; ki, kidney; t, testis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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(Bohnenpoll et al., 2013; Greulich et al., 2012; Norden et al., 2011; Trowe et al., 2010), domains, the manipulation of which are unlikely to impact on adrenocortical development. 4.2. The adrenocortical primordium arises by a localized mesenchymal transition from the coelomic epithelium of the urogenital ridge

Fig. 3. Radial organization of cortex and medulla is not established at E14.5 and E16.5 in mice with loss of Fgfr2 in the urogenital ridge. (A) Morphological analysis of adrenal glands. (B, C) Histological analysis by hematoxylin and eosin staining of frontal sections of embryos. Images in (C) represent magnifications of the boxed areas in (B). Arrows point to the fibrous capsular tissue. (D) Immunofluorescence analysis of SF1 and TH expression in the adrenal gland (both in red). Nuclei are counterstained with DAPI (in blue). Markers and genotypes are as shown. Scale bars are 0.3 mm (A), 100 mm (B), 25 mm (C), 100 mm (D). a, adrenal gland; ki, kidney. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

gene. Sf1 is expressed in the steroidogenic cells of the gonads and adrenal gland but also in the spleen, the ventromedial hypothalamic nucleus and the anterior pituitary, the latter of which might cause secondary effects. Using BAC transgenes two lines were developed that differ in copy number (five copies for Sf1/Crehigh versus one copy for Sf1/Crelow) but recapitulate endogenous expression of Sf1 including that in the adrenogonadal primordium of the urogenital ridge from E10.5 onwards (Bingham et al., 2006). Our analysis shows that the Tbx18cre line previously generated in our laboratory provides a second independent tool for loxP-mediated gene analysis in the adrenocortical lineage as it continuously labels SF1þ steroidogenic cells from E10.5 in the adrenocortical primordium until at least E18.5 in the cortex of the fetal adrenal gland. Tbx18cre additionally mediates recombination in the anterior gonadal primordium, the ureteric and (partially) the bladder mesenchyme, the epicardium, the venous pole of the heart, the otic mesenchyme, the limb buds and the anterior somite halves

Our expression analysis has shown that TBX18 is not coexpressed with SF1 in adrenocortical development. At E9.5, TBX18 is expressed in the coelomic epithelium while SF1 is not yet expressed in the ridge; at E10.5 TBX18 expression is restricted to dorsomedial mesenchymal cells whereas SF1 is found in the coelomic epithelium and underlying mesenchymal cells in a ventrolateral position. As our Tbx18cre-mediated lineage tracing showed that TBX18-derived cells localize to the ventrolateral mesenchyme and express SF1, this argues that the adrenocortical primordium arises by a localized delamination process from TBX18þ cells in the coelomic epithelium. Histological analyses indicated that a common adrenogonadal primordium arises within the intermediate mesoderm of the urogenital ridge. As sources of this primordium cells from mesonephric tubules of the urogenital ridge, the lateral plate mesoderm and/or the overlying coelomic epithelium were discussed (Uotila, 1940; Wrobel and Suss, 1999). An origin of adrenocortical cells from the coelomic epithelium gained support from the expression of SF1 both in the coelomic epithelium and the underlying mesenchymal cells in the urogenital ridge at E10.5 (Hatano et al., 1996; Ikeda et al., 1994). However, expression of a gene both in the cells of origin and the cells possibly derived from them does not permit the conclusion that such a lineage relation exists. Furthermore, cre-loxP based lineage tracing cannot be used in such a case to prove that one cell derives from another cell. While the origin of adrenocortical cells from the coelomic lining of the urogenital ridge had not been unambiguously shown to date, DiI labeling in the mouse has clearly proven that coelomic epithelial cells of both sexes migrate into the gonad, and give rise to Sertoli cells, as well as interstitial cells in the male (Karl and Capel, 1998). This together with our Tbx18cre-based genetic lineage tracing argues that the adrenocortical and the gonadal primordia arise as a continuum from the overlying coelomic epithelium. 4.3. FGFR2 signaling is required for expansion of the early adrenocortical primordium in the urogenital ridge Mice with a targeted disruption of Fgfr2 die shortly after implantation with a reduced inner cell mass and a lack of the visceral endoderm indicating a contribution of FGFR2 to outgrowth, differentiation, and maintenance of the inner cell mass possibly as a receptor for FGF4 (Arman et al., 1998). Conditional gene targeting approaches subsequently revealed a variety of cellular requirements for this signaling pathway in diverse developmental contexts including proliferation of osteoprogenitors (Yu et al., 2003), collecting duct morphogenesis (Zhao et al., 2004) and proliferation and Sertoli differentiation during male sex determination (Kim et al., 2007) just to name a few. Our conditional deletion experiment revealed that FGFR2 signaling acts in the early adrenocortical primordium as a proproliferative and anti-apoptotic factor. We have shown that this primordium is formed within the mesonephric region of the intermediate mesoderm but fails to expand starting from around E11.5 onwards. Our analysis does not exclude that FGFR2 signaling plays a minor role in the initial delamination process from the coelomic epithelium. Such a requirement has recently been suggested for FGF10/FGFR signaling in limb bud formation (Gros and

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Fig. 4. Early failure to expand the adrenocortical precursor pool in Fgfr2cKO embryos. (A) Morphology and GFP epifluorescence of explants of E11.5 urogenital ridges at 0, 1 and 2 days of culture. Arrowheads mark the adrenocortical primordium. GFP epifluorescence derives from Tbx18cre-mediated recombination of the Rosa26mTmG reporter allele. Red fluorescence marks unrecombined cells. (B) Immunofluorescence analysis of expression of the steroidogenic marker SF1 (in red) and the lineage marker GFP (in green, from the Rosa26mTmG reporter allele) on frontal section through the anterior urogenital ridge to visualize the adrenocortical primordium (a) and the gonadal primordium (g), in proximity to the dorsal aorta (da) at E10.5, E11.5 and E12.5. Scale bar is 400 mm (A) and 50 mm (B). (C) Three-dimensional reconstruction of the SF1þ adrenocortical primordium (a, in green) in association with the dorsal aorta (da, in grey). (D) Quantification of SF1þ cells at E11.5 and E12.5 in control (black bars) and in the Fgfr2cKO adrenocortical primordium (grey bars). E11.5: control: 1586 ± 172, mutant: 861 ± 31, p ¼ 0.002; E12.5: control: 3235 ± 1122, mutant: 361 ± 102, p ¼ 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Tabin, 2014). Continued expression of Fgfr2 in the zona glomerulosa of the adrenal cortex is compatible with a continued requirement as a mitogen for these cells; a hypothesis that can only be addressed by inducible cre lines not available at this point for this lineage. It is interesting to note that a mitogenic role of FGF signaling in adrenal cells was suggested a long time ago by in vitro studies. First, it was shown that administration of FGF to cultures of human fetal adrenocortical cells strongly increases the proliferation of these cells (Crickard et al., 1981; Feige and Baird, 1991), and that acidic and basic FGF are also mitogenic for adrenal chromaffin cells (Claude et al., 1988; Stemple et al., 1988). Later expression studies supported the idea that FGF2 might act as an autocrine mitogen for adrenocortical cells (Meisinger et al., 1996). Work in the chick identified FGF9 as a potent mitogen in the mesonephric region acting on the gonadal, thus possibly also on the adrenal primordium (Yoshioka et al., 2005). A RT-PCR analysis of Fgf and Fgfr

expression in laser capture microdissected E15.5 adrenal capsule and cortex confirmed expression of Fgf1 in the adrenal cortex, and of Fgf2 and Fgf9 in the capsule, identifying the encoded proteins as possible ligands of cortical FGFR2 but also FGFR1 and FGFR3 at least at this stage (Guasti et al., 2013). Our analysis excludes a role of FGFR2 signaling in differentiation of steroidogenic cells from uncommitted precursors. Fgfr2-deficient adrenocortical precursors express SF1 from E10.5 onwards and undergo further differentiation into specific sublineages of the fasciculata and glomerulosa zones. Furthermore, we have failed to detect changes of signaling factors and transcription factors implicated in regulation of steroidogenic differentiation and/or Sf1 expression. Fgfr2cKO mice display a failure of encapsulation of chromaffin cells by cortical steroidogenic cells. It is possible that FGFR2 signaling has an independent function in this morphogenetic

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Fig. 5. Changes of proliferation and apoptosis in the adrenocortical primordium of Fgfr2cKO embryos. (A, B) Immunofluorescence analysis of expression of the steroidogenic marker SF1 (in green) and BrdUþ cells (in red) (A) and TUNELþ cells (in green) (B) on frontal sections through the adrenocortical primordium at E10.5, E11.5 and E12.5. Scale bar is 50 mm (A, B). (C) Quantification of cell proliferation as ratio of BrdUþ to SF1þ cells. E10.5: wt: 13.4 ± 6.1, mutant: 15.9 ± 1.4, p ¼ 0.51; E11.5: wt: 28.4 ± 5.6, mutant: 16.4 ± 3.1, p ¼ 0.03; E12.5, wt: 40.9 ± 8.9, mutant: 19.6 ± 8.2, p ¼ 0.02. (D) Quantification of cell death as ratio of TUNELþ cells to SF1þ cells. E10.5: wt: 4.7 ± 4.1, mutant: 11.6 ± 3.0, p ¼ 0.19; E11.5: wt: 9.8 ± 1.4, mutant: 14.8 ± 6.5; p ¼ 0.26; E12.5: wt: 5.2 ± 1.9, mutant: 13.1 ± 2.1, p ¼ 0.03. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

process. However, this disintegration may simply be secondary to the massive reduction of cortical cells that may physically prevent the engulfment of chromaffin cells or lead to the reduction of signals from cortical cells required in this process. A failure of encapsulation of chromaffin cells was also noted in mice with Sf1cre-mediated deletion of Shh (Ching and Vilain, 2009). In this case, however, normal cortico-medullary zonation is initially established but is subsequently disturbed by hyperproliferation of zona fasciculata cells making a causal relation between FGFR2 and SHH signaling unlikely. Our analysis did not detect a requirement for Fgfr1 in adrenocortical development. Fgfr1 is not expressed in the early primordium but is weakly found in subcapsular mesenchyme adjacent to the Fgfr2 expression domain. This leaves the possibility that FGFR1 has a (minor) role in proliferation of these cells.

4.4. Fgfr2 isoforms may act redundantly in adrenocortical development FGFR1-3 are alternatively spliced in the third Ig domain such that an invariant exon 7 is spliced to either exon 8 or 9 to produce IIIb or IIIc isoforms. Different isoforms can bind to different ligands, and exhibit differential expression (Zhang et al., 2006). For FGFR2 it was shown that the isoform IIIb is predominantly expressed in epithelial cells whereas the IIIc isoform preferentially localizes to mesenchymal cells (Orr-Urtreger et al., 1993; Peters et al., 1992). While this suggests that the two isoforms of FGFR2 exhibit unique functions at unique sites, we favor the idea that in adrenocortical development the two isoforms act redundantly to control expansion of the adrenocortical primordium. First, coexpression of Fgfr2IIIb and Fgfr2IIIc was detected in subcapsular mesenchymal

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Fig. 6. Fgfr2 is expressed in the adrenocortical primordium. In situ hybridization analysis of Fgfr1 and Fgfr2 expression in the adrenocortical primordium in the urogenital ridge at E10.5 to E12.5 (highlighted by a black line defined as Sf1þ area on adjacent sections) and in the adrenal gland at E14.5 to E18.5 on frontal sections of wildtype embryos. Markers and genotypes are as shown. Scale bars are 50 mm for E10.5 to E12.5 and 100 mm for E14.5 to E18.5. a, adrenal gland; ki, kidney.

cells of the adrenal cortex, the zona glomerulosa at E15.5 (Guasti et al., 2013). Our in situ hybridization analysis which did not discriminate between the two isoforms, detected Fgfr2 in the same domain from E14.5 onwards. Moreover, we found Fgfr2 expression in the adrenocortical primordium at E11.5 clearly arguing that FGFR2 signaling acts throughout adrenocortical development possibly through both isoforms. Second, analysis of mice with a global deletion of the Fgfr2IIIb isoform detected (mildly) hypoplastic adrenal glands with normal cortico-medullary subdivision and cortical zonation. Proliferation was slightly reduced in the inner cortical region. Cortical markers appeared mildly reduced indicating a weak reduction in steroidogenic differentiation (Guasti et al., 2013). Third, hemizygous mice with global deletion of the Fgfr2IIIc splice variant have no discernible phenotypic differences between wildtype and mutant adrenal glands at postnatal day 2 (Hajihosseini et al., 2001). Our analysis has shown that conditional Tbx18cre-mediated deletion of all Fgfr2 splice variants results in a very severe adrenal hypoplasia. Our analysis is in line with the observation of Kim and coworkers who used a Sf1cre line to delete Fgfr2 in the adrenogonadal primordium. They noted a severe reduction or absence of adrenal glands at E15.5 in their mutant mice (Kim et al., 2007). Although it cannot be excluded that the global deletion of Fgfr2IIIb might have ameliorated the phenotypic consequences of loss of this isoform in the adrenal gland, we deem it likely that FGFR2IIIb and IIIc isoforms act redundantly in adrenocortical development to promote the proliferative expansion of this tissue. Acknowledgments This work was financially supported by the state of LowerSaxony, Hannover, Germany as a joint research project within the Niedersachsen-Israel Research Cooperation program. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mce.2015.06.022. References Anderson, D.J., Carnahan, J.F., Michelsohn, A., Patterson, P.H., 1991. Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in vivo and reveal the timing of commitment to neuronal differentiation in the sympathoadrenal lineage. J. Neurosci. 11, 3507e3519.

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