Distinct Populations Within Isl1 Lineages Contribute to Appendicular and Facial Skeletogenesis Through the Β-Catenin Pathway

Developmental Biology 387 (2014) 37–48

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Developmental Biology

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Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

Ryutaro Akiyama a,b, Hiroko Kawakami a,b, M. Mark Taketo c, Sylvia M. Evans d, Naoyuki Wada e, Anna Petryk a,f,g, Yasuhiko Kawakami a,b,g,h,n a Department of Genetics, Cell Biology and Development, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455, USA b Stem Cell Institute, University of Minnesota, 2001 Sixth Street SE, Minneapolis, MN 55455, USA c Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8051, Japan d Skaggs School of Pharmacy, and Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA e Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan f Department of Pediatrics, University of Minnesota, 2450 Riverside Avenue, Minneapolis, MN 55455, USA g Developmental Biology Center, University of Minnesota, 321 Church Street SE, Minneapolis, MN 55455, USA h Lillehei Heart Institute, University of Minnesota, 312 Church Street SE, Minneapolis, MN 55455, USA article info abstract

Article history: Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the Received 16 October 2013 contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Received in revised form Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence 27 December 2013 of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; Accepted 3 January 2014 however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of Available online 11 January 2014 nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Keywords: Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand Isl1 chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1- β-catenin expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin Limb accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1/ embryos. Inactivating Branchial arch β Mandible -catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1-β-catenin-Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium. & 2014 Elsevier Inc. All rights reserved.

Introduction budding, and shapes initial limb bud morphology (Gros et al., 2010; Wyngaarden et al., 2010). Simultaneously, the fibroblast growth factor Mechanisms that regulate initiation and early outgrowth of the 10 (Fgf10) gene is activated in limb mesenchyme progenitor cells, vertebrate limb bud have been extensively studied (Duboc and Logan, which induces Fgf8 in the overlying ectoderm to establish an FGF10 2011; Rabinowitz and Vokes, 2012; Zeller et al., 2009). Limb bud (mesenchyme)–FGF8 (ectoderm) positive feedback loop in nascent mesenchymal progenitor cells in lateral plate mesoderm (LPM) limb buds (Min et al., 1998; Ohuchi et al., 1997; Sekine et al., 1999). maintain active proliferation, while proliferation of LPM cells in the Fgf8-expressing ectodermal cells are then confined to form a specia- prospective flank region declines, leading to initial budding (Searls lized limb bud ectodermal tissue, the apical ectodermal ridge, at the and Janners, 1971). Directional movement of LPM cells is coupled with distal edge of the limb bud. FGF8, together with other apical ectodermal ridge-derived FGFs, regulates limb bud mesenchymal cell survival and patterning (Mariani et al., 2008; Sun et al., 2002). n Corresponding author at: Department of Genetics, Cell Biology and Develop- Concomitantly, Gli3 in the anterior region and Hand2 in the posterior ment, University of Minnesota, 321 Church Street SE, 6-160 Jackson Hall, Minnea- region of nascent limb bud pre-pattern the mesenchyme along the polis, MN 55455, USA. – Fax: þ1 612 626 5652. anterior posterior axis (te Welscher et al., 2002a), which leads to E-mail address: [email protected] (Y. Kawakami). Hand2-dependent induction of Shh expression in the posterior

0012-1606/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ydbio.2014.01.001 38 R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 mesenchyme (Galli et al., 2010).Theseprocessesactbothinthe complete lack of hindlimb buds in Hoxb6Cre; β-catenin CKO embryos. forelimb and hindlimb buds; however, recent studies have shown This result indicated that β-catenin functions in a subset of Isl1- striking differences in upstream genetic regulation of limb bud lineages, which contributes to a specificsubdomainwithinthe initiation. More specifically, upstream of limb bud outgrowth and hindlimb bud. Further analysis indicated that β-catenin functions in Fgf10 expression, Tbx5 and Islet1 (Isl1)arespecifically required for Isl1-lineages to maintain survival of a compartment within the initiation of the forelimb and hindlimb buds, respectively (Agarwal posterior mesenchyme of nascent hindlimb bud. In addition, we et al., 2003; Kawakami et al., 2011; Narkis et al., 2012; Rallis et al., found that the lower jaw was completely missing in the mutants. In 2003). Furthermore, retinoic acid signaling is required for initiation of facial tissues, we showed that, in Isl1/ embryos, activation of β- forelimb but not hindlimb buds (Cunningham et al., 2013; Zhao et al., catenin signaling was impaired in epithelium of the mandibular 2009). component of the first branchial arch (BA1), which gives rise to Isl1 encodes a LIM-homeodomain protein whose expression marks Meckel0s cartilage and mandible. Although the Isl1-lineage contributes progenitor populations of various organs in the mouse embryo, broadly to facial epithelium, a requirement for β-catenin in Isl1- including the hindlimb (Yang et al., 2006). Prior to hindlimb bud lineages for facial skeletogenesis was most evident in BA1, where outgrowth, Isl1 is expressed in posterior LPM, and its expression is the epithelial β-catenin–Fgf8 pathway regulates mesenchymal cell confined to the posterior part of the hindlimb-forming region at E9.5 survival, and to a lesser extent in other tissues. Our data identify the (Kawakami et al., 2011; Yang et al., 2006). A genetic lineage tracing contribution of Isl1-expressing cells to hindlimb mesenchyme and BA1 analysis using Isl1Cre and a Rosa26-LacZ reporter (R26R) line demon- epithelium, and describe a requirement for β-catenin within subdo- strated that Isl1-expressing cells contribute to a majority of hindlimb mains of these Isl1 lineages to regulate skeletogenesis by promoting mesenchyme with an anterior (low)–posterior (high) gradient, sug- cell survival of discrete cell populations. gesting heterogeneity within hindlimb mesenchyme progenitors (Yang et al., 2006). Isl1 null embryos arrest development before hindlimb bud formation (Pfaff et al., 1996), thus functional analysis Materials and methods of Isl1 has been performed using conditional knockout (CKO) approaches. Inactivation of Isl1 in early mesoendoderm using Tcre Mouse lines caused a complete failure to initiate hindlimb bud development (Kawakami et al., 2011; Narkis et al., 2012). Furthermore, our previous The mutant mouse alleles used in this study have been previously 3Picc study suggested that Isl1 functions through the β-catenin pathway for reported: BAT-gal (Tg(BAT-lacZ) (Maretto et al., 2003)), conditional β tm2Kem fl26 hindlimb initiation (Kawakami et al., 2011). β-CATENIN is abundantly -catenin knockout allele (Ctnnb1 , Ctnnb1 ),(Brault et al., β tm1Mmt fl3 present at the plasma membrane, and its cytosolic and nuclear levels 2001), conditional -catenin activation allele (Ctnnb1 , Ctnnb1 ), are kept low by constitutive degradation. When stabilized, β-CATENIN (Harada et al., 1999), Isl1 null allele (Itou et al., 2012), Rosa26 LacZ tm1Sor tm1 translocates into the nucleus and forms a complex with transcription reporter (Gt(ROSA)26Sor ,R26R)(Soriano, 1999)andIsl1Cre (Isl1 (cre)Sev Cre þ / factors, such as the members of the Lef1/TCF family. This leads to , Isl1 )(Yang et al., 2006). Ctnnb1 mice were generated by flox (exon26) activation of downstream target genes (Nusse and Varmus, 2012). germline recombination of Ctnnb1 mice using the CMV-Cre β During hindlimb bud initiation, β-catenin signaling is activated in line (Schwenk et al., 1995). To inactivate -catenin in the Isl1-lineage, fl26/fl26 þ /cre þ / LPM. Abrogation of β-catenin broadly in LPM by Hoxb6Cre results in Ctnnb1 mice were crossed with Isl1 ; Ctnnb1 mice, þ /cre /fl24 β the failure to initiate hindlimb formation, similar to Isl1 CKO embryos and Isl1 ; Ctnnb1 (hereafter, referred to as Isl1Cre; -catenin β (Kawakami et al., 2011). However, when the hindlimb bud begins CKO) were obtained. To constitutively activate (CA) -catenin, þ/fl3 þ /cre þ/cre outgrowth, ISL1-positive cells and the active β-catenin signaling Ctnnb1 mice were crossed with Isl1 mice, and Isl1 ; þ/fl3 β domain barely overlap: ISL1-positive cells are located at the ventral- Ctnnb1 (hereafter, referred to as Isl1Cre; CA- -catenin)were proximal domain, while the β-catenin signaling domain is detected in obtained. Mice were maintained on a mixed genetic background. Care the distal area of the hindlimb-forming region. Thus, it remains and experimentation were carried out according to the approval by unknown whether β-catenin signaling functions in Isl1-expressing the Institutional Animal Care and Use Committee of the University of hindlimb progenitor cells or whether Isl1 and β-catenin act in distinct Minnesota. populations of hindlimb progenitor cells. β-catenin is also broadly expressed in craniofacial primordia (in Skeletal preparation and histology analysis both the mesenchyme and the epithelium) and is required for normal craniofacial development, as shown by conditional inactivation of Embryonic day (E) 13.5 and 14.5 embryos were fixed with 50% β-catenin in neural crest cells by Wnt1-Cre (Brault et al., 2001)orby ethanol, and then processed for Alcian Blue cartilage staining as deleting β-catenin in facial epithelium. The latter results in severe previously described (Kawakami et al., 2009; McLeod, 1980). For craniofacial skeletal defects, including deformities of the nasal bone, histological analysis, embryos were fixed in 10% neutral formalin upper jaw, lower jaw and hyoid bone with varying severity and and processed for paraffin sectioning with 6–8 mm thickness as selectivity of affected skeletal elements, depending on Cre lines used previously described (Petryk et al., 2004). Sections were stained (Reid et al., 2011; Sun et al., 2012; Wang et al., 2011). While analyzing with eosin–hematoxylin. β-catenin function in Isl1-lineages during hindlimb development, we found that Isl1-lineages contribute broadly to facial epithelium, where In situ hybridization, LacZ staining and immunofluorescence β-catenin is known to be required for facial development. This suggested a possible relationship between Isl1 and β-catenin, similar Whole mount in situ hybridization and whole mount LacZ staining to the process of hindlimb initiation (Kawakami et al., 2011). However, were performed according to previous publications (Itou et al., 2012; the Isl1 expression pattern in facial tissue, as well as the contribution Kawakami et al., 2011). Section in situ hybridization was performed on of Isl1-lineages to the facial region, has not been studied except in 8 mm thickness paraffin sections according to a standard procedure branchiomeric muscle (Nathan et al., 2008). Furthermore, the relation- (Itou et al., 2012). Sections were counter stained with nuclear fast red. ship between Isl1-lineages and β-catenin in the development of the Immunofluorescence analysis was performed on 14 mm cryosections facial skeleton is unknown. according to a standard procedure (Itou et al., 2012). Mouse anti-ISL1 To test whether β-catenin functions in Isl1-expressing cells, we (39.4D5, Developmental Studies Hybridoma Bank, 4 mg/ml), rabbit inactivated β-catenin in Isl1-lineages. Isl1Cre; β-catenin CKO embryos anti-β-catenin (ab32572, Abcam, 1:100 dilution) and rat anti-E- developed truncated hindlimbs with skeletal defects, in contrast to a cadherin (sc-59778, Santa Cruz Biotechnology, 1:200 dilution) were R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 39 used. Counter staining was done using DAPI. The fluorescent signals Isl1 and β-catenin function in the same cells. To examine the were detected using a Zeiss LSM710 laser scanning confocal micro- requirement of β-catenin in Isl1-lineages, we inactivated scope and analyzed by ZEN2009 software. β-catenin using Isl1Cre. Isl1Cre; β-catenin CKO embryos died at E12.5–E14.5, likely due to cardiovascular defects (Lin et al., 2007). β Cell proliferation and apoptosis analysis Isl1Cre; -catenin CKO embryos exhibited severe hindlimb hypoplasia. Alcian blue staining revealed that mutant embryos developed normal Cell proliferation and apoptosis assays on 14 mm cryosections were forelimb skeletons, consistent with a lack of Isl1 expression in simultaneously performed by using rabbit anti-phospho Histone H3 forelimb progenitor cells and forelimb bud (Kawakami et al., 2011; (Ser 10) (pHis3, Millipore, #06-570. 1:500 dilution) and the In Situ Cell Yang et al., 2006). In contrast, the hindlimb exhibited a short femur, Death Detection Kit (Roche diagnostics) according to the man- truncated zeugopodal cartilage elements, absence of the autopod, and 0 – – ufacturer s instruction. Alexa488 anti-Fluorescein/Oregon green absence of the posterior region of the pelvic girdle (Fig. 1A C, and F ¼ (1:200 dilution) and Alexa594 anti-rabbit IgG (Molecular Probes, H, n 8 at E13.5 or E14.5). These hindlimb defects are distinct from 1:1000 dilution) were used as secondary antibodies. For quantitative the complete lack of the hindlimb bud observed in Hoxb6Cre- β analysis of cell proliferation and cell death in nascent hindlimb bud, mediated inactivation of -catenin in broad regions of LPM pHis3-, TUNEL- and DAPI-positive cells in the LPM were counted from (Kawakami et al., 2011). Formation of the hindlimb with skeletal β two transverse sections from anterior, middle and posterior parts of defects in Isl1Cre; -catenin CKO embryos suggested that Isl1Cre- β each embryo. In the case of the mandibular component of the mediated inactivation of -catenin occurred only in a select subpo- branchial arch, three consecutive transverse sections obtained at the pulation of hindlimb mesenchyme progenitors. same plane of sectioning through the medial region of the arch were examined from each embryo. Statistical significance between control 0 The Isl1-lineage contributes broadly to hindlimb mesenchyme, but and CKO embryo was analyzed by the independent Student s t-test, β-catenin function in Isl1-lineages is required in a discrete posterior 7 and shown as average standard deviation. p Values are indicated region within each panel. The genetic lineage analysis study demonstrated that Isl1- Results lineages contributed to a broad region of hindlimb mesenchyme (Yang et al., 2006). Consistent with this, Isl1-lineages (visualized as Inactivation of β-catenin in the Isl1-lineage causes skeletal dysplasia LacZ signals in Isl1Cre; R26R embryos) occupied the majority of in hindlimb nascent hindlimb bud immediately after initiation of outgrowth, except for a small domain in the anterior part (Fig. S1B, (Yang et al., Isl1 acts upstream of β-catenin during hindlimb bud initiation in 2006)). Previous reports have shown that Isl1 mRNA expression at mice (Kawakami et al., 2011). However, it remains unknown whether E9.0, prior to hindlimb bud development, is broadly detected in

Fig. 1. Defects in the hindlimb and lower jaw skeletons in Isl1Cre; β-catenin CKO embryos. Alcian Blue-stained skeletons of wild-type control (A–E) and Isl1Cre; β-catenin CKO (F–J) embryos at E14.5. Lateral views of whole skeletons of control (A) and mutant (F) embryos reveal the absence of the lower jaw cartilage (asterisk) and severe hypoplasia in the hindlimb skeleton (arrow) in mutant embryos. (B and G) Forelimb skeleton developed similarly in control (B) and mutant (G). The scapula (sc), humerus (h), radius (r) and ulna (u) are indicated. The autopod (auto) is indicated with a solid line. (C and H) The hindlimb in mutants contained a hypoplastic pelvic girdle (pg) and femur (fe), and truncated zeugopodal elements. The autopod was completely missing. The tibia (t) and ﬁbula (ﬁ) in the wild type are indicated. (D, E, I, and J) Lateral views (D and I) and bottom views (E and J) of craniofacial skeletons. The image in (E) was taken after removing Meckel0s cartilage. The mutant completely lacks Meckel0s cartilage (asterisk, I). Other cartilaginous elements are formed, although smaller than those in control embryos. The insets in (D) and (I) show the hyoid bone primordia (hy). Cartilage elements are labeled only in control for simplicity: ala temporalis (at), Meckel0s cartilage (mc), nasal cartilage (nc), nasal septum (ns), otic capsle (oc), parachordal plate (pchp), pilapostoptica (ppso), and trabecular basal plate (tbp). 40 R. Akiyama et al. / Developmental Biology 387 (2014) 37–48

LPM (Kawakami et al., 2011). In nascent limb buds, the pattern of sequentially evaluate cell proliferation and death along the anterior– the Isl1Cre; R26R signal was broader than the expression pattern of posterior axis in nascent hindlimb bud (Fig. S2). We found that cell Isl1 mRNA (Fig. S1A). Thus, Isl1Cre-mediated recombination likely proliferation was not affected at any level of the hindlimb bud. occurred in hindlimb progenitor cells in LPM prior to the onset of However, we detected a significant increase in mesenchymal cell hindlimb bud outgrowth (Yang et al., 2006). death, only in the posterior part of Isl1Cre; β-catenin CKO hindlimb To characterize β-catenin function in Isl1-lineages, we moni- buds (n¼3, Fig. 2D, D0,H,H0, and I). Condensed TUNEL-positive signals tored activation of the β-catenin pathway using a BAT-gal trans- in nuclei of apoptotic cells were enriched in sections corresponding to gene that reports activation of Lef1/TCF-β-catenin signaling approximately 1/5 of the hindlimb bud. These results indicated that β- (Maretto et al., 2003). BAT-gal signal was detected in nascent catenin function in Isl1-lineages was required for mesenchymal cell hindlimb bud in E9.75 wild-type embryos, but was downregulated survival in a spatially-restricted domain, which comprises approxi- in the posterior region in Isl1Cre; β-catenin CKO embryos (Fig. S1C mately 1/5 of the posteriormost nascent hindlimb bud. and D). To constitutively activate β-catenin in Isl1 lineages, we excised exon 3 of the Ctnnb1 gene utilizing Isl1Cre, which causes Loss of precursors of Shh-expressing cells in posterior mesenchyme stabilization of β-catenin, and hence, constitutive activation of the in Isl1Cre; β-catenin CKO hindlimbs β-catenin pathway (Harada et al., 1999). BAT-gal signal in Isl1Cre; CA-β-catenin embryos was stronger in the hindlimb bud than BAT- To further investigate the impact of the loss of β-catenin in Isl1- gal signal in controls (Fig. S1E). Thus, β-catenin signaling was lineages, and localized cell death in the posterior region of nascent regulated in nascent hindlimb bud utilizing Isl1Cre-mediated limb bud on outgrowth and patterning processes, we examined recombination to drive loss- or gain-of-function of β-catenin. gene expression in developing hindlimb buds. We first visualized To clarify the role of β-catenin in Isl1-lineages during hindlimb limb buds utilizing antisense probes for Prrx1 (n¼3), a limb development, we examined expression patterns of Msx1 and Fgf10, mesenchyme marker (Cserjesi et al., 1992), and Pitx1 (n¼2), a which are broadly expressed in nascent hindlimb bud at E9.75. gene expressed in the entire hindlimb bud mesenchyme (Lanctot Msx1 expression was significantly downregulated in posteriormost et al., 1997; Shang et al., 1997; Szeto et al., 1996) at E10.5 (Fig. 3A, B, hindlimb bud in Isl1Cre; β-catenin CKO embryos (n¼2, Fig. 2A and F, and G). The anterior–posterior length of the hindlimb bud in E). We also detected a slight reduction in Fgf10 mRNA expression Isl1Cre; β-catenin CKO embryos were reduced by about the length in Isl1Cre; β-catenin CKO embryos (n¼6, Fig. 2B and F). Expression of one somite. Thus, increased cell death at the onset of hindlimb of Fgf8, whose activation in the ectoderm requires FGF10 (Min bud outgrowth likely caused loss of the posterior tissue by E10.5. et al., 1998; Sekine et al., 1999), was significantly downregulated in The posterior mesenchyme of nascent limb bud gives rise to the the posterior part of nascent hindlimb bud (n¼3, Fig. 2C and G). Shh-expressing zone of polarizing activity (Honig and Summerbell, These results suggested that, despite a broad contribution of Isl1- 1985; Riddle et al., 1993). Correlating with the loss of posterior lineages to hindlimb bud mesenchyme, a discrete posterior region mesenchyme, Shh (n¼3), and its transcriptional targets, Gli1 (n¼3) of nascent hindlimb bud was affected in Isl1Cre; β-catenin CKO and Hoxd12 (n¼2) (Hui and Angers, 2011; Litingtung et al., 2002; te embryos. Welscher et al., 2002b), were not detected (Fig. 3C–E, and H–J). Fgf8 expression, whose maintenance requires SHH signaling- β-catenin function in the Isl1-lineage is required for mesenchymal dependent Gremlin1 (Panman et al., 2006; Verheyden and cell survival in a discrete posterior region Sun, 2008), was also downregulated in the posterior apical ectodermal ridge (n¼3, Fig. 3K and O). Contrary to these observa- Genetic experiments have demonstrated that β-catenin functions tions, expression of Alx4, a marker for anterior mesenchyme as a critical factor for cell proliferation and survival in many aspects (Qu et al., 1997; Takahashi et al., 1998), was not altered (n¼2, Fig. 3L (Grigoryan et al., 2008). Thus, we examined pHis3 (proliferation and P). These results suggested that precursors of Shh expressing cells marker) and TUNEL-positive cells (cell death) in serial sections at were lost in nascent hindlimb bud of Isl1Cre; β-catenin embryos, and E9.75. Analysis of alternate transverse sections allowed us to caused selective loss of posterior tissue and gene expression.

Fig. 2. Cell death in the posterior margin of the mesenchyme in nascent hindlimb buds in Isl1Cre; β-catenin CKO embryos. (A–C, and E–G) Expression patterns of Msx1 (A and E), Fgf10 (B and F) and Fgf8 (C and G) in control (A–C) and mutant (E–G) embryos. Msx1 and Fgf8 expressions were missing in the posterior margin of nascent hindlimb bud in mutants. Fgf10 expression was slightly downregulated in the mutant hindlimb bud. Black and blue arrowheads point to normal and reduced expression, respectively, and asterisks indicate absence of signals. (D, D', H, H' and I) Cell proliferation and cell death analysis in control (D and D0) and mutant (H and H0) embryos. Shown are representative transverse section images of the posterior part of nascent hindlimb bud. Colors represent pHis3 (red), TUNEL (green) and DAPI (blue) signals. (D0) and (H0) show close up of the boxed areas in (D) and (H), respectively. Dotted lines indicate boundary between the limb bud and the trunk. (I) Quantitative analysis of cell death (TUNEL) and cell proliferation (pHis3) in nascent limb buds along the anterior–posterior axis. The y axis represents percentage of TUNEL or pHis3 positive cells per DAPI positive nuclei. Ant, Mid and Post represent anterior, middle and posterior regions of the limb bud, respectively. R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 41

Fig. 3. Selective loss of tissue and gene expression in the posterior of hindlimb buds and reduced Sox9 expression in Isl1Cre; β-catenin CKO embryos. Expression of Prrx1þUncx4.1 (A and F), Pitx1 (B and G), Shh (C and H), Gli1 (D and I), Hoxd12 (E and J), Fgf8 (K and O), Alx4 (L and P), and Sox9 (M, N, Q and R) in control (A–E, and K–N) and Isl1Cre; β-catenin CKO (F–J, and O–R) hindlimb bud. Dorsal views are shown. The stages are E10.5, except for Sox9 (N and R) at E11.5. (A, B, F, and G) Visualizing limb bud mesenchyme by Prrx1 and Pitx1 indicates a lack of posterior tissues (asterisks in F and G), corresponding to approximately one somite level. Somite borders are indicated by dotted lines. Expression of Prrx1 and Uncx4.1 labels limb bud mesenchyme and the posterior border of somites, respectively. Somite numbers are indicated in each panel. (C– E, and H–J) Expression of Shh and its targets, Gli1 and Hoxd12, are missing in the posterior mesenchyme in the mutant hindlimb bud. (K and O) Fgf8 expression in the apical ectodermal ridge is downregulated in the posterior region (blue arrow in O), but not in the anterior region in the mutant hindlimb bud. (L and P) Alx4 expression in the anterior mesenchyme appears to not to be affected. (M, N, Q, and R) Sox9 expression was reduced at E10.5 (M and Q) and detected in small domain at E11.5 (N and R) in the mutant hindlimb bud. fe, ﬁ, p, and ti indicate primordia for the femur, ﬁbula, pelvic girdle and tibia, respectively. Arrowheads and arrows point to expression in the anterior and posterior regions, respectively. Asterisks indicate lack of expression or tissues. Black color and blue color indicate normal and reduced expressions, respectively.

The loss of posterior mesenchymal cells, as well as the lack of of the β-catenin pathway. Isl1Cre; CA-β-catenin embryos died SHH signaling that is required for expansion of chondrogenic around E10.5–E11.0, likely due to cardiovascular defects (Kwon progenitors (Zhu et al., 2008), would cause reduction of Sox9- et al., 2007). We detected comparable expression of Fgf10 (n¼3) expressing chondrogenic progenitor cells in the hindlimb bud and Hand2 (n¼3) in nascent hindlimb bud at E9.75 (Fig. 4A, B, G, (Fig. 3M, N, Q, and R). Sox9 expression was also missing in the and H), suggesting that hindlimb progenitor cells in LPM were posterior-proximal region at E10.5 (n¼3, Fig. 3M and Q), which not affected by Isl1Cre-mediated activation of β-catenin signal- was correlated with absence of the posterior region of the pelvic ing. However, at E10.0 (30–31 somite stages), we detected girdle (Fig. 1H). At E11.5, the Sox9 expression domain in mutant posterior expansion of Gli3, normally excluded from the posterior hindlimb bud looked more condensed, and did not extend along region of nascent limb bud in wild-type embryos (n¼3, Fig. 4C the proximal–distal axis as observed in control hindlimb bud and I) (te Welscher et al., 2002a). Consistent with the mutual (n¼2, Fig. 3N and R). This Sox9 expression pattern correlated with antagonism between anterior Gli3 and posterior Hand2,we the truncated, shorter cartilage elements at E14.5 (Fig. 1). Collec- observed increased downregulation of Hand2 in posterior tively, these results indicated that β-catenin deletion in the Isl1- mesenchyme at E10.0 in Isl1Cre; CA-β-catenin mutants lineage resulted in a speciﬁc loss of the posterior mesenchyme of (n¼2, Fig. 4DandJ,32–33 somite stages). In agreement with the hindlimb bud, which resulted in failure to maintain the the known role of Hand2 in inducing Shh in the limb bud posterior gene expression program. Although the loss of mesench- (Galli et al., 2010), expression of Shh (n¼3) and Gli1 (n¼2) was yme was restricted to the posterior region, the absence of the signiﬁcantly downregulated in Isl1Cre; CA-β-catenin hindlimb posterior gene expression program and failure to expand chon- buds at E10.5 (Fig. 4E, F, K, and L). These results suggested that drogenic progenitor cells would cause the truncated short skeletal proper levels of β-catenin signaling were critical for normal elements in the Isl1Cre; β-catenin CKO hindlimb. activation of the Hand2–Shh pathway in posterior mesenchyme. Our results have indicated that loss- and gain- of β-catenin Constitutive activation of β-catenin signaling in the Isl1-lineage function in Isl1-lineages caused loss or downregulation of impairs the Hand2–Shh pathway in the hindlimb through Shh in hindlimb buds by distinct mechanisms, namely loss of upregulation of Gli3 precursor cells (Isl1Cre; Ctnnb1 CKO) and dysregulation of Hand2–Gli3 antagonism (Isl1Cre; CA-β-catenin). Thus, maintain- To further examine β-catenin function in Isl1-lineages, we ing proper levels of β-catenin function in Isl1-lineagesiscrucial examined developmental consequences of constitutive activation for Shh expression in limb buds. 42 R. Akiyama et al. / Developmental Biology 387 (2014) 37–48

Fig. 4. Constitutive activation of the β-catenin pathway negatively impacts on the Hand2–Shh pathway in the posterior mesenchyme. Expression pattern of Fgf10 (A and G), Hand2 (B and H), Gli3 (C and I), Hand2 (D and J), Shh (E and K) and Gli1 (F and L) in control (A–F) and Isl1Cre; β-catenin CKO (G–L) hindlimb bud. Dorsal views at indicated stages are shown. (A, B, G, and H) Expression pattern of Fgf10 (A and G) and Hand2 (B and H) was comparable (arrows) in nascent hindlimb buds in control and Isl1Cre; CA-β-catenin embryos. (C and I) Gli3 expression was not detected in the posterior margin of the control hindlimb bud (asterisk, C), but was extended to the posterior region in Isl1Cre; CA-β-catenin hindlimb buds (red arrow, I) at E10.0. Dotted lines indicate the posterior margin of the nascent hindlimb bud. (D and J) Hand2 was expressed in the posterior mesenchyme at E10.0 in control embryos (arrow, D), but was downregulated in Isl1Cre; CA-β-catenin hindlimb buds (blue arrow, J). (E, F, K, and L) Shh was expressed in the posterior mesenchyme and its target Gli1 was broadly detected in the posterior mesenchyme in control embryos at E10.5 (arrows, E and F). Expression of both Shh and Gli1 was downregulated in Isl1Cre; CA-β-catenin hindlimb buds (blue arrows, K and L).

The Isl1-lineage through β-catenin contributes to craniofacial The recombination in Isl1Cre; R26R embryos were consistent development with the expression pattern of Isl1, and LacZ staining was detected in BA1 at E8.5 and E9.0 (Fig. S4A and B), indicating early and In addition to hindlimb defects, Isl1Cre; β-catenin CKO embryos efficient recombination in this tissue. At E9.5, Isl1-lineages were exhibited defects in craniofacial development (Figs. 1A and F, and detected broadly in the maxillary and mandibular components of S3). Mutant embryos exhibited agnathia, a complete lack of the BA1, as well as BA2 (hyoid arch) (Fig. S4C and D). Transverse and lower jaw, a loss of tongue, and hypoplasia of nasal and maxillary sagittal sections indicate that Isl1-lineages were present in epithe- processes (Fig. S3). Alcian blue staining demonstrated that lium of ectoderm and endoderm, consistent with the ISL1 signal mutants lacked Meckel0s cartilage, while other cartilaginous ele- (Fig. S4E–G). Isl1-lineages were also detected in medial and lateral ments, such as hyoid bone primordia, were slightly reduced in size nasal processes at E10.5 (Fig. S4H and I). At E13.5, Isl1-lineages (Fig. 1D, E, I, and J, n¼8). Previous studies have shown that were specifically detected in epithelia of the nasal process, lower deletion of β-catenin causes severe skeletal defects in the cranio- jaw and the distal tip of the tongue (Fig. S4J and K). These results facial region (Huh and Ornitz, 2010; Joeng et al., 2011; Reid et al., demonstrated highly localized Isl1 expression in facial epithelium 2011; Sun et al., 2012; Wang et al., 2011). The complete loss of the and efficient recombination by Isl1Cre in a broad region of facial lower jaw, which is derived from the mandibular prominence of epithelium. BA1 (Depew and Simpson, 2006; Minoux and Rijli, 2010)inIsl1Cre; β-catenin CKO embryos indicated that β-catenin function in Isl1- Isl1 is necessary for nuclear accumulation of β-CATENIN in BA1 lineages contributed to a substantial degree to BA1-derived epithelium craniofacial structures. The absence of Meckel0s cartilage in Isl1Cre; β-catenin CKO embryos, as well as expression of ISL1 in facial epithelium where Expression of Isl1 in BA1 epithelium and broad contribution β-catenin is required for facial development, raised the possibility of Isl1-lineages to facial epithelium that Isl1 regulates Meckel0s cartilage development through the β-catenin pathway, similar to the pathway required for initiation The Isl1 lineage has been shown to contribute to subpopula- of hindlimb buds (Kawakami et al., 2011). Isl1 null embryos arrest tions of head muscle (Nathan et al., 2008); however, Isl1 expres- at E9.5 (Pfaff et al., 1996), excluding the possibility of direct sion in other craniofacial tissue has not been characterized. Thus, examination of Isl1 function in the development of Meckel0s we examined Isl1 mRNA and protein expression, as well as Isl1- cartilage. However, visualizing BA1 by Prrx1 expression at E9.0 lineages during development of BA1. Isl1 expression was detected showed hypoplasia of the mandibular component of BA1 in Isl1/ as early as E8.5 in the BA1 prominence (Fig. 5A). Immunoreactive mutants (n¼2, Fig. 6A and G), demonstrating a requirement ISL1 signals were predominantly detected in the epithelium, in for Isl1 in BA1 development. Fgf8 in BA1 epithelium is crucial addition to some scattered mesenchymal signals (Fig. 5B and C). At for the development of Meckel0s cartilage (Macatee et al., 2003; E9.0, ISL1 signals in BA1 (as well as BA2) were broadly detected in Trumpp et al., 1999). Indeed, we found that Fgf8 expression in BA1 the epithelium, and the scattered mesenchymal signals, which was lost in Isl1/ embryos, while Fgf8 expression in the mid- likely represent branchiomeric muscle precursors, became more brain–hindbrain boundary and forelimb bud ectoderm was main- prominent (Fig. 5D–F). Transverse sections at E9.5 demonstrated tained (n¼2, Fig. 6B, C, H, and I). These results suggested that Isl1 that ISL1 signals were in both ectodermal and endodermal regulated BA1 development through Fgf8 expression in components of the mandibular epithelium, in addition to the epithelium. branchiomeric muscle primordia in the core of the mesenchyme It has been recently demonstrated that β-catenin signaling (Fig. 5G–J). The epithelial ISL1 signal continued to be detected, but regulates Fgf8 expression in facial epithelium (Reid et al., 2011; became weaker at E10.5 and 11.5 (Fig. 5K–P). Sun et al., 2012; Wang et al., 2011), suggesting that Isl1 regulates R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 43

Fig. 5. Expression of Isl1 in the epithelium of the branchial arches. (A, D, E, G, H, K, and L) Whole mount Isl1 mRNA expression pattern at indicated stages. (B, C, F, I, J, and M–P) ISL1 immunoﬂuorescence on 14 mm cryosections. Approximate sectioning planes are indicated in the whole mount images. In (B) and (C), an epithelial marker, E-cadherin (red) was co-stained. Arrows in (A), (D), (E), (G), and (K) point to Isl1 expression in BA1. (C), (J), (N) and (P) are close up of the boxed areas in (B), (I), (M) and (O), respectively. end, ect and md indicate endoderm, ectoderm and the mandibular component of BA1, respectively. ba1 and ba2 indicate BA1 and BA2, respectively.

Fig. 6. Isl1 is essential for nuclear β-catenin accumulation and Fgf8 expression in BA1 epithelium. (A–C, and G–I) Expression pattern of Prrx1 (A and G) and Fgf8 (B, C, H, and I) in wild-type (A–C) and Isl1/ (G–I) embryos at E9.0. Lateral views of the craniofacial region (A, B, G, and H), and distal views of the forelimb bud (C and I) are shown. Arrowheads and arrows in (A) and (G) point to the maxillary and mandibular components of BA1, respectively. The blue arrow in (G) indicates hypoplastic mandibular component of BA1 in the Isl1/ embryo. Arrowheads in (B) and (H) point to the expression in the midbrain–hindbrain boundary. Arrow and asterisk in (B) and (H) indicate normal and absence of Fgf8 expression in BA1 in the control and Isl1/ embryo, respectively. (D–F, and J–L) Immunoﬂuorescence of β-CATENIN in the epithelium of BA1 in wild-type and Isl1/ embryos at E9.0. Cells denoted by arrows in (D) and (J) are shown in (E and F) and (K and L), respectively. In (D), (F), (J) and (L), β-CATENIN signals are only shown, and in (E) and (K) DAPI signals are only shown. (M) Optic sectioning and line scanning of DAPI (blue) and β-CATENIN (green) signals. Upper and lower panels show cells in (E and F) and (K and L), respectively. The x and y axes represents pixel number along the cell and ﬂuorescent intensity, respectively.

Fgf8 via β-catenin signaling. To address this possibility, we exam- embryos, defects were more severe in Meckel0s cartilage than ined nuclear accumulation of β-CATENIN, a hallmark of activation other skeletal elements (Fig. 1). Thus, we next investigated the of β-catenin signaling, in BA1 epithelium. In addition to strong activation status of β-catenin signaling by examination of BAT-gal membrane signals, we detected β-CATENIN in the nuclei of reporter signals in facial tissue. We observed BAT-gal signals in epithelial cells in wild-type embryos (Fig. 6D–F). By contrast, maxillary and mandibular components of BA1 and BA2 (Fig. S6A nuclear β-CATENIN levels were low in the Isl1/ epithelium and B), consistent with the previous report of active β-catenin (Fig. 6J–L). The different levels of nuclear β-CATENIN were further signaling in these tissues (Brugmann et al., 2007). In Isl1Cre; confirmed by optical sectioning (cells indicated by arrows are β-catenin CKO embryos, severe downregulation of BAT-gal signals shown in Fig. 6M; cells indicated by arrows and arrowheads are was observed in the mandibular component of BA1, while effects shown in Fig. S5). These results supported the idea that Isl1 on the maxillary process of BA1 and BA2 seemed to be milder regulated β-catenin signaling in BA1 epithelium, and β-catenin, (Fig. S6C and D). Contrary to this, activation of β-catenin signaling in turn, regulated Fgf8 expression important for lower jaw in Isl1Cre; CA-β-catenin embryos resulted in stronger BAT-gal development. signal, which appeared in a punctate pattern and was broadly detected in BA1 and BA2 (Fig. S6E and F). These results confirmed β-catenin function in Isl1-lineages is required for mesenchymal cell efficient loss- and gain-of function of β-catenin by Isl1Cre in facial survival in BA1 through epithelial Fgf8 tissues, and further demonstrated that the requirement for β-catenin in Isl1-lineages was more significant in the mandibular LacZ signals in Isl1Cre; R26R embryos demonstrated efficient component of BA1 than other craniofacial regions. recombination by Isl1Cre and a broad contribution of Isl1-lineages Consistent with this notion, in situ hybridization of Prrx1 at E9.5 to facial epithelium (Fig. S4). However, in Isl1Cre; β-catenin CKO demonstrated selective defects in the mandibular component of BA1, 44 R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 while the maxillary process was comparable in control and Isl1Cre; recombine in the myogenic core of the mesenchyme (Fig. S4) β-catenin CKO embryos (Fig. 7AandD).Meckel0s cartilage develops (Nathan et al., 2008). Thus, β-catenin regulation of Fgf8 in the Isl1- from cranial neural crest cell-derived mesenchyme in BA1 (Gross and lineage was specific to the epithelium. Hanken, 2008; Ito et al., 2002), while ISL1 expression and Isl1-lineage Barx1 expression seems to be unchanged in the mandibular contribution are specific to the epithelium (Figs. 5 and S4). Thus, to component of BA1, suggesting that FGF8 signaling was above a investigate how β-catenin function in Isl1-lineages affected Meckel0s threshold for Barx1 expression in the Isl1Cre; CA-β-catenin cartilage development, we examined cell proliferation and survival in (Fig. 8M, n¼2). However, Barx1 signals in the maxillary process the mandibular component of BA1. Surprisingly, cell proliferation and were stronger than control embryos (Fig. 8M, arrowhead), likely cell survival were not affected in BA1 epithelium of Isl1Cre; β-catenin due to upregulated Fgf8 expression in this domain. Dusp6 expres- CKO embryos compared to wild-type embryos (Fig. 7B, D, and E). sion was expanded towards the medial domain, and the signals However, we detected increased cell death without changes in cell became stronger compared to control wild-type embryos (Fig. 8N, proliferation in BA1 mesenchyme in Isl1Cre; β-catenin CKO embryos n¼2). These data further supported observed alterations of Fgf8 (Fig. 7B, D, and F). TUNEL signals condensed in the nuclei of apoptotic expression in the facial region in Isl1Cre; β-catenin CKO and Isl1Cre; cells were clustered close to the epithelium. Thus, deletion of CA-β-catenin embryos. β-catenin in the Isl1-lineage caused cell death specifically in the In addition to Barx1 and Dusp6, which are lateral markers of the mesenchyme. mandibular component of BA1, a medial mandibular marker, Given downregulation of β-catenin signaling and loss of Fgf8 Hand2 (Thomas et al., 1998), was also downregulated in Isl1Cre; expression in epithelium of the mandibular component of BA1 in β-catenin CKO embryos at E9.75 (Fig. 8E and J, n¼3). In Isl1Cre; Isl1/ embryos (Fig. 6), we examined how Fgf8 expression was CA-β-catenin mutants Hand2 expression in the mandibular com- affected in Isl1Cre; β-catenin CKO embryos. Fgf8 expression was ponent of BA1 appeared to be slightly expanded to the lateral severely downregulated in the mandibular component of BA1, region (Fig. 8O, n¼4). while weak expression was detectable in the maxillary component and in the frontonasal process at E9.75 in Isl1Cre; β-catenin CKO embryos (Fig. 8A, B, F, and G, n¼3). We also examined expression Discussion of Barx1 and Dusp6, targets of FGF8 signaling (Kawakami et al., 2003; Trumpp et al., 1999). In Isl1Cre; β-catenin CKO embryos, both Isl1 lineages and heterogeneity in nascent hindlimb bud mesenchyme genes were downregulated to different degrees (Dusp6 to a greater and facial epithelium degree than Barx1), which could reflect different threshold responses to FGF8. The residual Fgf8 expression in the maxillary In this study, we demonstrated that Isl1-lineages contributed to process at this stage (Fig. 8F and G) appeared sufficient to maintain skeletogenesis of the hindlimb and lower jaw through β-catenin a low level of Barx1 expression in the lateral region (Fig. 8C and H, signaling. While abrogating β-catenin has been shown to cause n¼2). Contrary to this, Dusp6 expression was significantly down- severe defects in the development of the hindlimb and facial tissue regulated in the entire BA1 (Fig. 6D and I, n¼2), likely because the (Kawakami et al., 2011; Reid et al., 2011; Sun et al., 2012; Wang residual Fgf8 expression was not sufficient to maintain Dusp6 et al., 2011), deletion of β-catenin in Isl1-lineages caused severe expression. defects in more restricted tissues. In Isl1Cre; CA-β-catenin mutants, Fgf8 expression was detected Our previous study showed that Isl1 acts upstream of the broadly in BA1 and BA2 (n¼3, Fig. 8K and L). Fgf8 in situ mRNA β-catenin pathway during hindlimb initiation (Kawakami et al., detection on transverse and sagittal sections at E9.75 demon- 2011). However, ISL1-positive cells and nuclear β-catenin-positive strated ectopic Fgf8 expression in epithelium as well as epithelial cells barely overlap just prior to hindlimb initiation. Sensitivity of thickening in BA1 (Fig. S7, n¼4). In contrast, no ectopic Fgf8 was antibodies in our previous study hampered further examination of induced in the mesenchyme of BA1 (Fig. S7), although Isl1Cre can the possibility of β-catenin signaling in Isl1-lineages at earlier

Fig. 7. Inactivation of β-catenin in the Isl1-lineage causes cell death in the mesenchyme not in the epithelium of BA1. (A, C) Expression pattern of Prrx1 in wild-type (A) and Isl1Cre; β-catenin CKO (C) embryos at E9.5. Arrowheads and arrows point to the maxillary and mandibular components of BA1, respectively. The blue arrow in (C) indicates hypoplastic mandibular component of BA1. (B, D) Confocal images of pHis3 (red) and TUNEL (green) staining on transverse sections of the mandibular component of BA1 at E9.75. Medial–lateral and proximal–distal axes are indicated with arrows. Massive cell death is visualized by condensed TUNEL signals (arrows, D). (E and F) Quantitative analysis of cell death and cell proliferation in the epithelium (E) and mesenchyme (F) of BA1. Blue bars and red bars represent control and Isl1Cre; β-catenin CKO embryos, respectively. The y axis represents percent positive cells, compared to DAPI-positive nuclei. Asterisk indicates statistical signiﬁcance. Increased cell death in the BA1 mesenchyme showed signiﬁcant difference. R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 45

Fig. 8. Altered Fgf8 expression and FGF8 signaling in loss and gain of β-catenin signaling in Isl1-lineages in the facial region. Expression pattern of Fgf8 (A, B, F, G, K, and L), Barx1 (C, H, and M), Dusp6 (D, I, and N) and Hand2 (E, J, and O) in control (A–E), Isl1Cre; β-catenin CKO (F–J) and Isl1Cre; CA-β-catenin (K–O) embryos at E9.75. Arrowheads and arrows point to expression in the maxillary and mandibular components of BA1, respectively. In (F–J), asterisks and blue arrows/arrowheads indicate absence of and reduced expression, respectively. In (K–O), red arrows and arrowheads indicate upregulated expression. Small arrows in (K) and (L) point to ectopic Fgf8 expression. For simplicity, not all ectopic signals are labeled. Shown are frontal images, except for (A), (F), and (K), which are lateral images. For (B–E), (G–J) and (L–O), close up of the mandibular component of BA1 is shown under each panel.

stages. A genetic approach in this study using Isl1Cre to inactivate formed, but was slightly smaller. Similarly, the hyoid bone pri- β-catenin provided evidence that β-catenin was required in Isl1- mordium that is derived from BA2 was present, but hypoplastic. lineages, but this requirement was limited to a portion of the Thus, the functional significance of β-catenin also appeared to hindlimb bud mesenchyme progenitors, which contributes to the differ within Isl1-lineages in facial tissue. posterior region of nascent hindlimb buds. This is evident by the observations that localized cell death in nascent hindlimb buds Relationship between Isl1 and β-catenin in limb development was restricted to posterior one somite level, and the anterior– posterior length of hindlimb buds was reduced by approximately The relationship between Isl1 and β-catenin function during one somite length in mutants (Figs. 2 and 3). The contribution of embryonic development has been extensively studied in the heart, Isl1-lineages to a large portion, but not the entire hindlimb where β-catenin positively regulates Isl1 expression in cardiac mesenchyme, as well as the requirement of β-catenin in Isl1- progenitor cells in the second heart field (Ai et al., 2007; Cohen lineages, indicated that the seemingly homogenous nascent limb et al., 2012; Klaus et al., 2012; Klaus et al., 2007; Kwon et al., 2007; bud mesenchyme is in fact heterogeneous from the onset of Lin et al., 2007; Qyang et al., 2007). These studies indicate that hindlimb development. β-catenin acts upstream of Isl1 expression and/or Isl1-lineage In facial tissue, Isl1-lineages broadly contributed to facial development. In contrast, our current findings and previous study epithelium, including the epithelium of BA1 and BA2 (Fig. S4). (Kawakami et al., 2011) suggest that Isl1 functions upstream of Similar to hindlimbs, inactivating β-catenin in Isl1-lineages exhib- β-catenin in hindlimb and BA1. Contrary to the heart where ited severe skeletal defects in a localized manner. More specifi- β-catenin regulates proliferative expansion of cardiac progenitors, cally, the mandibular component of BA1 was most severely our analysis in nascent hindlimb buds indicated that a loss of affected, leading to the absence of Meckel0s cartilage and lower β-catenin did not cause defects in proliferation in Isl1-lineages jaw (Figs. 1 and S3). By contrast, the upper jaw, which is largely (Fig. 2). Instead, our analysis highlighted the function of β-catenin derived from the maxillary process and the frontonasal process, in the survival of a portion of Isl1-lineages. Cell survival seems to 46 R. Akiyama et al. / Developmental Biology 387 (2014) 37–48 be a common target of mesenchymal β-catenin signaling during disrupting an epithelial Isl1-β-catenin-Fgf8 pathway. Therefore, different steps of limb development. For instance, early inactiva- our study identified a novel role of Isl1 as a regulator of tion of β-catenin in LPM prior to initiation of hindlimb bud β-catenin-Fgf8 pathway during craniofacial skeletogenesis. outgrowth by Hoxb6Cre caused cell death broadly in hindlimb Analysis of Lef1/TCF-β-catenin reporters has shown that progenitor cells as well as the complete failure to activate the β-catenin signaling is broadly activated in the craniofacial region Fgf10–Fgf8 feedback loop (Kawakami et al., 2011). In the case of ((Brugmann et al., 2007) and Fig. S4). In addition, a functional inactivating β-catenin with Prx1Cre in the developing limb bud analysis of epithelial β-catenin suggested differential requirements mesenchyme, a failure to maintain the apical ectodermal ridge and for β-catenin in the upper and lower jaws, implying that high apoptosis of the proximal mesenchyme was detected during limb levels of epithelial β-catenin signaling support lower jaw devel- bud elongation (Hill et al., 2006). Cell death in proximal mesench- opment (Sun et al., 2012). Given that ISL1 is necessary for nuclear yme is likely to be secondary to reduced secretion of FGFs from the accumulation of β-catenin (Fig. 6), Isl1 might function in creating apical ectodermal ridge, whose loss is known to cause proximal higher β-catenin levels in the epithelium of BA1 to promote cell death in developing limb buds (Mariani et al., 2008; Sun et al., normal development of the lower jaw. 2002). The present study also found a requirement for β-catenin in cell survival in Isl1-lineages. However, unlike previous reports, An evolutionarily conserved β-catenin–Fgf8 pathway in branchial only a part of Isl1-lineages located in posteriormost nascent arch and limb bud, and implications for evolutionary origins of a hindlimb buds was affected. Morphological and gene expression genetic module analyses in Isl1Cre; β-catenin CKO hindlimb buds suggested that apoptotic cells in posteriormost hindlimb included precursors of The present study and previous studies highlight a common Shh-expressing cells (Fig. 3), which are located at the posterior role for the β-catenin–Fgf8 pathway in the epithelium of the limb margin of the developing limb bud (Riddle et al., 1993). This idea is bud and BA1. In the limb bud, high levels of β-catenin signaling are in agreement with our recent study, which demonstrated Isl1 necessary for Fgf8 expression in the apical ectodermal ridge regulation of the Hand2–Shh morpho-regulatory pathway in the (Barrow et al., 2003; Kawakami et al., 2001; Kengaku et al., posterior mesenchyme, specifically in hindlimb buds (Itou et al., 1998; Soshnikova et al., 2003). Moreover, ectopic activation of 2012). β-catenin signaling in limb ectoderm can induce ectopic Fgf8 By contrast, constitutive activation of β-catenin in Isl1-lineages expression in a punctate manner, which was associated with caused expansion of Gli3 expression into the posterior margin of ectoderm thickening that resembles the pseudostratified apical nascent hindlimb buds (Fig. 4). It has been demonstrated that Gli3 ectodermal ridge (Barrow et al., 2003; Kawakami et al., 2001, in the anterior part and Hand2 in the posterior part of nascent limb 2004; Kengaku et al., 1998; Soshnikova et al., 2003). The buds are mutually antagonistic (te Welscher et al., 2002a), and β-catenin–Fgf8 pathway is activated during early limb develop- Hand2, in combination with Hox genes, induces Shh expression in ment both in forelimb and hindlimb bud. However, upstream posterior limb bud mesenchyme (Galli et al., 2010; Kmita et al., genetic regulation differs in forelimbs and hindlimbs. Specifically, 2005; Tarchini et al., 2006). The expansion of Gli3 expression mesenchymal Isl1 is genetically upstream of the epithelial toward the posterior margin and increased downregulation of β-catenin–Fgf8 pathway in the hindlimb bud (Kawakami et al., Hand2 in the posterior mesenchyme of nascent hindlimb buds 2011), while forelimb buds use another pathway, likely through correlate with downregulation of Shh in Isl1Cre; CA-β-catenin Tbx5 (Agarwal et al., 2003; Rallis et al., 2003). mutant hindlimb buds (Fig. 4). Thus, constitutive activation of Similar to the limb bud epithelium, the present study and β-catenin signaling likely impacts the balance between Gli3 and recent studies demonstrated β-catenin regulation of Fgf8 in the Hand2. Phenotypes resulting from either up- or down-regulating epithelium of BA1 (Reid et al., 2011; Sun et al., 2012; Wang et al., ß-catenin functions in Isl1-lineages suggested that levels of 2011). Furthermore, ectopic activation of the β-catenin pathway in β-catenin signaling need to be properly regulated to establish the facial epithelium was associated with surface thickening posterior gene expression and cell survival for proper develop- (Fig. S7). The common epithelial β-catenin–Fgf8 pathway in limb ment of the hindlimb. buds and BA1 supports the idea of deep homology between the pharyngeal arch and limb bud (Schneider et al., 1999; Shubin et al., Isl1 and function of β-catenin in the craniofacial region 1997, 2009). Preservation of the molecular machinery of the epithelial In this study, we found that Isl1 is expressed in the epithelium β-catenin–Fgf8 pathway in vertebrate limb and jaw development of BA1 and that Isl1 is necessary for nuclear accumulation of is also important from an evolutionary standpoint. More specifi- β-CATENIN (Figs. 5 and 6), similar to hindlimb bud progenitors cally, analysis of gene expression and patterning in the chon- / (Kawakami et al., 2011). Absence of Fgf8 expression in BA1 in Isl1 drichthyan gill arch and fin, as well as chick limb buds, suggest embryos (Fig. 6) as well as the requirement of β-catenin for Fgf8 that developmental genetic modules controlling limb develop- expression (Reid et al., 2011; Sun et al., 2012; Wang et al., 2011), ment may have been co-opted from modules functioning in gill strongly suggested a pathway (Isl1-β-catenin-Fgf8) to regulate arch development (Gillis et al., 2009). The epithelial β-catenin–Fgf8 BA1 development. This idea was supported by selective cell death pathway might be an example of such a shared genetic module in mesenchyme of BA1 in Isl1Cre; β-catenin CKO embryos (Fig. 7), between limbs and gill arches. even though ISL1 is detected in the epithelium. The Isl1-lineage also contributes to branchiomeric muscle (Nathan et al., 2008). However, lack of ectopic Fgf8 expression in Acknowledgments mesenchyme of Isl1Cre; CA-β-catenin embryos indicated that the Isl1-β-catenin-Fgf8 pathway is specific to epithelium of the We are grateful to Dr. Juan Carlos Izpisúa Belmonte for in situ branchial arch (Fig. S7). Given that epithelial Fgf8 is essential for probes, and Dr. Yasushi Nakagawa and Dr. Michael O0Connor for survival of BA1 mesenchyme that will give rise to Meckel0s the use of their equipment. We thank Thu Quach, Elizabeth cartilage (Macatee et al., 2003; Trumpp et al., 1999), it is unlikely West, Jenna Matson, Julia Wong and Brian Schmidt for their that β-catenin function in branchiomeric muscle contributes to excellent technical support, and Austin Johnson for editorial Meckel0s cartilage development. Our data support the idea that assistance. This work was supported by the National Institute of loss of Meckel0s cartilage in Isl1Cre; β-catenin CKO is caused by Dental and Craniofacial Research of NIH to A.P. 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