Microarray identification of novel genes downstream of Six1, a critical factor in cranial placode, somite, and kidney development

DEVELOPMENTAL DYNAMICS 00:00–00, 2014 DOI: 10.1002/DVDY.24229

PATTERNS & PHENOTYPES

Microarray Identification of Novel Genes Downstream of Six1, a Critical Factor in Cranial Placode, Somite, and Kidney Development a

Bo Yan, Karen M. Neilson, Ramya Ranganathan, Thomas Maynard, Andrea Streit, and Sally A. Moody* 1

Department of Anatomy and Regenerative Biology, The George Washington University, School of Medicine and Health Sciences, Washington, DC Department of Craniofacial Development and Stem Cell Biology, King’s College London, Guy’s, London, UK 3 Department of Pharmacology and Physiology, The George Washington University, School of Medicine and Health Sciences, Washington, DC

DEVELOPMENTAL DYNAMICS

2

Background: Six1 plays an important role in the development of several vertebrate organs, including cranial sensory placodes, somites, and kidney. Although Six1 mutations cause one form of branchio-otic syndrome (BOS), the responsible gene in many patients has not been identified; genes that act downstream of Six1 are potential BOS candidates. Results: We sought to identify novel genes expressed during placode, somite and kidney development by comparing gene expression between control and Six1-expressing ectodermal explants. The expression patterns of 19 of the significantly up-regulated and 11 of the significantly down-regulated genes were assayed from cleavage to larval stages. A total of 28/30 genes are expressed in the otocyst, a structure that is functionally disrupted in BOS, and 26/30 genes are expressed in the nephric mesoderm, a structure that is functionally disrupted in the related branchio-otic-renal (BOR) syndrome. We also identified the chick homologues of five genes and show that they have conserved expression patterns. Conclusions: Of the 30 genes selected for expression analyses, all are expressed at many of the developmental times and appropriate tissues to be regulated by Six1. Many have the potential to play a role in the disruption of hearing and kidney function seen in BOS/BOR patients. Developmental Dynamics C 2014 Wiley Periodicals, Inc. 000:000–000, 2014. V Key words: preplacodal ectoderm; pan-placodal region; otocyst; olfactory; cranial ganglia; BOS syndrome; BOR syndrome Submitted 5 June 2014; First Decision 3 November 2014; Accepted 12 November 2014; Published online 00 Month 2014

Introduction Vertebrate Six genes are highly related to the Drosophila sine oculis gene that is critical for fly visual system development (Cheyette et al., 1994). They play major roles in the development of the eye, but also in other sense organs, cranial sensory ganglia, the central nervous system, muscle, kidney, genitalia, limb buds, and lungs (Oliver et al., 1995; Kawakami et al., 1996; Ohto et al., 1998; Spitz et al., 1998; Pandur and Moody, 2000; Ghanbari et al., 2001; Fougerousse et al., 2002; Laclef et al., 2003; Bessarab et al., 2004; Xu et al., 2003; Zheng et al., 2003; Brodbeck and Englert, 2004; Ozaki et al., 2004; Zou et al., 2004, 2006; Brugmann and Moody, 2005; Grifone et al., 2005; Konishi et al., 2006; Ikeda et al., 2007, 2010; Chen et al., 2009; Sato et al., Grant sponsor: NIH; Grant number: R01 DE022065; Grant number: R03 DE018723; Grant number: R01 NS023158; Grant sponsor: GWU; Grant number: Institute for Neuroscience Biomarker Analysis Core Facility. *Correspondence to: Sally A. Moody, Department of Anatomy and Regenerative Biology, The George Washington University, School of Medicine and Health Sciences, 2300 I Street, N.W., Washington, DC, 20037. E-mail: [email protected] Dr. Yan’s present address is Shandong Provincial Key Laboratory of Cardiac Disease Diagnosis and Treatment, Jining Medical College Affiliated Hospital, 79 Guhuai Road, Jining, Shandong, 272029, China

2010; Guo et al., 2011). Several experiments indicate that Six1 has a central role in controlling development of the cranial sensory placodes, which are patches of thickened embryonic ectoderm that will give rise to the anterior pituitary, olfactory sensory epithelium, lens, auditory and vestibular inner ear structures, and the large, distally located neurons of the cranial sensory ganglia (reviewed in Streit, 2004, 2006; Bailey and Streit, 2006; Schlosser, 2007, 2010; Streit, 2007; Ladher et al., 2010; Park and SaintJeannet, 2010; Graham and Shimeld, 2013; Saint-Jeannet and Moody, 2014). Loss of Six1 function or repression of its downstream targets in Xenopus, chick and zebrafish results in the loss of markers specific to the preplacodal region (PPR) and placodes (Brugmann et al., 2004; Bricaud and Collazo, 2006, 2011; Schlosser et al., 2008; Christophorou et al., 2009). In particular, otic placode defects (Christophorou et al., 2009), and loss of inner ear hair cells (Bricaud and Collazo, 2006, 2011) are observed. Six1-null mice have olfactory, inner ear, and cranial sensory ganglion malformations (Oliver et al., 1995; Laclef et al., 2003; Zheng et al., 2003; Ozaki et al., 2004; Zou et al., 2004; Konishi et al., 2006; Ikeda et al., 2007, 2010; Chen et al., 2009). Conversely, Six1 gain-of-function specifically in the PPR expands Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24229/abstract C 2014 Wiley Periodicals, Inc. V

1

2 YAN ET AL.

TABLE 1. Genes Up-regulated >2-Fold by Six1 in Microarray Assays

DEVELOPMENTAL DYNAMICS

Affymetrix Probe set ID

Fold change

Accession no.

Xl.19919.1.A1_at

4.613

BQ400635

Xl.17484.1.A1_at

4.428

BG812762

Xl.24006.1.S1_at

4.300

AW640051

Xl.3228.1.A1_at Xl.26192.1.A1_at

3.811 3.561

BG022063 CD324856

Xl.20029.2.S1_a_at Xl.21032.1.S1_at Xl.6623.1.S1_at

3.484 3.413 3.200

M80798.1 AF546707.1 BU900288

Xl.23944.1.S1_at

3.057

BG885936

Xl.12902.1.A1_at Xl.19820.1.A1_at

3.046 3.030

BJ092409 BQ399495

Xl.2439.1.A1_at

2.996

BG023545

Xl.23880.1.A1_at

2.989

BJ083747

Xl.19782.1.A1_at

2.958

BQ398913

Xl.12791.1.A1_at Xl.23533.1.S1_at Xl.19504.1.S1_at

2.944 2.918 2.879

BJ090916 BC044278.1 BQ386473

Xl.11568.1.S1_a_at

2.859

AW872117

Xl.21833.1.A1_at Xl.17880.1.A1_at Xl.4077.2.A1_at

2.839 2.826 2.780

BG345845 BG812152 BF024988

Xl.15434.2.A1_at

2.689

BJ079756

Xl.17452.3.A1_at Xl.3765.1.A1_at

2.665 2.641

BG409705 BF428468

Xl.17267.1.S1_at

2.638

BM262144

Xl.6714.1.S1_at

2.620

CD360857

Xl.8697.1.A1_at

2.618

AW764878

Xl.20538.1.S1_at

2.546

CD327089

Xl.21807.1.S1_at

2.526

BC041247.1

Xl.16468.1.A1_at

2.513

BJ048720

Xl.26433.1.A1_at

2.502

BJ077709

Gene title, unigene cluster, or EST name UniGene Xl.940; neuropilin1 (nrp1), aka A5-protein EST: daf30h01.y1; similar to arrestin beta2 (arrb2) UniGene Xl.78601; aldehyde dehydrogenase 7 family, member A1 (aldh7a1) EST: dg21d06.x1 EST: AGENCOURT_14164067; most similar to Xenopus laevis clone CH219-20I13 PDGFa receptor Cryptic tubulin UniGene Xl.22052; heterogeneous nuclear ribonuclear protein R (hnrnpr) UniGene Xl.37859; hypothetical protein LOC100037047 EST: BJ092409 EST: NISC_mp03h05.x1; most similar to Xenopus laevis clone CH219-33D5 UniGene Xl.2439; hypoxanthine phosphoribosyltransferase1 (hrpt1) UniGene Xl.4536; RNA binding motif protein 42 (rbm42-b) UniGene Xl.52003; hypothetical protein LOC 414689 EST: BJ090916 Uncx4.1 homeobox UniGene Xl.19504; xyloside xylosyltransferase 1 (xxylt1) EST: db22e02.y1; matches several genes at 50 end EST: dg42a07.y1 EST: daf66c03.x1 EST: dc82d05.x1; similar to mitogenactivated protein kinase binding protein 1 UniGene Xl.15434; Nacetylglucomsamine-1-phosphate transferase, gamma subunit (gnptg) UniGene Xl.54929; cyclin L1 (ccnl1) UniGene Xl.6193; Polymerase (RNA) II polypeptide I (polr2i) UniGene Xl.17267; Predicted hamartin-like (aka tuberous sclerosis 1; tsc1) UniGene Xl.6714; MACRO domain containing protein 2 (macrod2) UniGene Xl.8697; empty spiracles homeobox 2 (emx2) UniGene Xl.20538; strongly similar to malignant fibrous histiocytoma amplified sequence 1 (mfhas1) Guanine nucleotide binding protein 13 gamma EST: BJ048720; most similar to Xenopus laevis clone CH219-51A4 EST: BJ077709

GO signaling metabolism metabolism

unknown unknown

signaling cytoskeleton nuclear

unknown unknown unknown metabolism RNA binding unknown unknown transcription metabolism unknown unknown unknown signaling

metabolism

transcription transcription protein binding

metabolism transcription unknown

nuclear unknown unknown

NOVEL SIX1 TARGETS 3

TABLE 1. Continued Affymetrix

DEVELOPMENTAL DYNAMICS

Probe set ID

Gene title, unigene Fold change

Accession no.

Xl.15912.2.A1_at

2.482

BJ055680

Xl.14658.1.A1_at Xl.9485.1.A1_x_at

2.479 2.479

BJ052130 BG264616

Xl.11066.1.A1_at

2.449

AW764707

Xl.9896.1.A1_at

2.432

BG019991

Xl.25648.2.A1_at

2.398

BI940543

Xl.21726.1.S1_at Xl.18330.1.S1_at

2.323 2.311

CA790591 BJ049080

Xl.4441.1.A1_at Xl.11394.1.A1_at

2.305 2.297

BF072093 AW639023

Xl.535.2.S1_a_at

2.290

AF172400

Xl.8063.1. S1_at Xl.22358.1.A1_at

2.255 2.249

AY099224 BG021407

Xl.14141.1.A1_at

2.192

BJ079748

Xl.2873.1.A1_at

2.187

BG018413

Xl.26415.1.S1_at

2.187

CD361045

Xl.18588.1.S1_at

2.184

BI442945

Xl.3096.1.A1_at

2.171

BJ085327

Xl.13487.1.A1_at

2.164

CB564242

Xl.11706.1.S1_at

2.163

AY221506.1

Xl.11463.3.S1_a_at

2.162

BU917037

Xl.1095.1.S1_at

2.162

S71764.1

Xl.13738.1.A1_at

2.149

BJ075690

Xl.16242.1.A1_at

2.145

BJ100481

Xl.16853.1.S1_at

2.137

BG579925

Xl.25532.1.A1_at

2.127

BJ055327

Xl.18739.1.S1_at Xl.12870.1.A1_at

2.119 2.119

BI445731 BJ082868

cluster, or EST name

GO

UniGene Xl.18703; phosphatase, orphan 2 (phospho2) EST: BJ052130 UniGene Xl.10876; strongly similar to Xenopus tropicalis predicted nucleoporin 133kDA (nup133) UniGene Xl.11066; autophagy related 4D, cysteine peptidase (atg4d) EST: dc46e08.x1; most similar to Xenopus tropicalis clone CH216-157J16 UniGene Xl.76517; strongly similar to Xenopus tropicalis predicted tlymphoma invasion and metastasisinducing protein 1 (tiam1) EST: AGENCOURT_10301409 UniGene Xl.64976; moderately similar to Xenopus tropicalis predicted zinc finger protein 335-like EST: db51d10.x1 UniGene Xl.11394; strongly similar to JNK1/MAPK8-associated membrane protein (jkamp) P75 neurotrophin receptor a-2 (p75NTRa) Tumorhead (trhd-a) UniGene Xl.34405; E74-like transcription factor (elf2) EST: BJ079748; most similar to ribosomal protein L14 (rpl14) UniGene Xl.18253; Uncharacterized LOC 100037177 UniGene Xl.26415; testis, prostate and placenta expressed (tepp) UniGene Xl.77328; Uncharacterized LOC496297 UniGene Xl.57373; transmembrane and coiled-coiled domain family 2 (tmcc2) UniGene Xl.13487; Uncharacterized protein MGC154312 Putative polyA-binding protein (PABPN2) UniGene Xl.19374; G protein-coupled receptor kinase-interacting ArfGAP (git2) Sperm-specific basic nuclear protein 5 (sp5) EST: BJ075690; most similar to WNTinhibitory factor 1 (wif1) UniGene Xl.16242; strongly similar to sperm associated antigen 16 protein-like (spag16) UniGene Xl.46098; weakly similar to predicted protein FAM178A UniGene Xl.25532; strongly similar to hypothetical protein LOC100488559 UniGene Xl.18739; KIAA1456 UniGene Xl.12870; moderately similar to zinc finger protein 665-like

metabolism unknown nuclear

metabolism unknown signaling

unknown transcription

unknown metabolism

signaling intracellular transport transcription ribosomal biogenesis unknown unknown unknown membrane

unknown translation intracellular transport

signaling signaling cytoskeleton

unknown unknown metabolism unknown

4 YAN ET AL.

TABLE 1. Continued Affymetrix

DEVELOPMENTAL DYNAMICS

Probe set ID

Gene title, unigene Fold change

Accession no.

Xl.21416.1.S1_at

2.107

AW638131

Xl.12763.1.A1_at Xl.21588.1.A1_at

2.104 2.093

BJ082426 AB054535.1

Xl.13831.3.S1_at

2.088

BJ075928

Xl.9563.1.S1_at Xl.6583.1.S1_at

2.078 2.078

AF480430.1 AW635173

Xl.15729.1.A1_at Xl.401.1.S1_at

2.066 2.058

BJ050119 AF032383

Xl.3198.1.A1_at

2.050

BG021827

Xl.19719.1.A1_at

2.049

BQ398489

Xl.8108.1.A1_at

2.045

BJ047639

Xl.9031.1.A1_x_at

2.019

BG579679

Xl.12209.1.A1_at

2.010

BJ082981

cluster, or EST name

GO

UniGene Xl.28718; Pseudouridylate synthase 7 homolog (pus7) EST: BJ082426 Heparan sulfate 6-O-sulfotransferase (hs6st1) UniGene Xl.72612; moderately similar to predicted coiled-coiled domaincontaining C6orf97 Pbx1b UniGene Xl.6583; weakly similar to wars2 gene product EST: BJ050119 Metalloprotease-disintegrin (MDC11b, aka adam11) UniGene Xl.53283; testis expressed 26 (tex26) EST: NISC_mo08b08.x1; moderately similar to Xenopus laevis canopy FGF signaling regulator 1 (cnpy1) UniGene Xl.47742; Uncharacterized LOC100036828 UniGene Xl.9031; Chromosome 5 open reading frame 22 (c5orf22) EST: BJ082981; most similar to Xenopus laevis hypothetical protein MGC115205

unknown

the domains of placode genes in frog and chick (Brugmann et al., 2004; Christophorou et al., 2009), indicating that it is a key regulator of placode fate. In humans, SIX1 mutations can cause branchio-otic syndrome (BOS, specifically BOS3; Online Mendelian Inheritance in Man [OMIM] #608389; www.omim.org), whose phenotypes include mild craniofacial defects and significant hearing loss (Ruf et al., 2004). Nine mutations in BOS3 patients from 16 unrelated families have been reported to date. Seven are missense mutations in an N-terminal domain, called the Six Domain (SD; Kawakami et al., 2000), which disrupt interactions with co-factors, and two are mutations in the homeodomain, which disrupt interactions with the DNA (Ruf et al., 2004; Ito et al., 2006; Sanggaard et al., 2007; Kochhar et al., 2008; Patrick et al., 2009, 2013; Noguchi et al., 2011). In zebrafish, expression of mutant Six1 mRNA that carries a BOS patient mutation in the SD (R110W) interferes with the Six1/Eya1 interaction that promotes hair cell formation (Bricaud and Collazo, 2011). The Catweasel (Cwe) mouse mutant harbors a missense mutation in the SD (Bosman et al., 2009) that is similar to at least one BOS family (Mosrati et al., 2011). Heterozygous-Cwe mice have an ectopic row of hair cells in the cochlea and homozygous-Cwe mice have a loss of hair cells in the cochlea, semicircular canals, and utricle. Mutations in the Six1 co-factor called EYA1 (Ohto et al., 1999) causes BOS1 (OMIM #602588) and the related branchio-otic-renal (BOR1; OMIM #113650) syndrome, in which kidney development is additionally disrupted (Abdelhak et al., 1997; Kumar et al., 1997; Ozaki et al., 2002; Rodriguez-Soriano, 2003; Spruijt et al., 2006). However, mutations in Six1 and Eya1 only account for

unknown metabolism unknown

transcription translation unknown proteolysis unknown signaling

unknown unknown unknown

approximately half of the BOS and BOR cases, indicating that there are other genes in the Six/Eya genetic pathway that contribute to these syndromes. To identify potential causative genes for BOS and BOR, we generated a more complete list of genes likely to be regulated by the Six1 transcription factor. We expressed Six1 in animal cap ectodermal explants (ACs), which were raised to an early neural plate stage at which time Six1 is highly expressed in the PPR, and compared their expression profile with control, uninjected ACs that will give rise to nonneural epidermis. Herein we describe the expression patterns of 30 genes that were significantly different between these two data sets, most of which have not been previously characterized in Xenopus. We also identified several chick homologues and present their expression patterns. The majority of the genes characterized are expressed at the correct times to be regulated by Six1, particularly in the otic placode and nephric mesoderm. Thus, they may play a role in the disruption of hearing and kidney functions seen in BOS/BOR patients.

Results and Discussion To identify a more complete list of genes likely to act downstream of Six1 during development, animal cap ectoderm (AC) was explanted at blastula stages from embryos microinjected with Six1 mRNA at the two-cell stage and from control, uninjected sibling embryos. Explants were cultured to neural plate stages when the PPR is specified (Ahrens and Schlosser, 2004; Pieper et al., 2012), and prepared for microarray expression assays as previously described (Yan et al., 2010).

NOVEL SIX1 TARGETS 5

DEVELOPMENTAL DYNAMICS

Fig. 1. Gene Ontology analysis of the genes whose expression levels in animal cap explants were significantly altered >two-fold by Six1 in a microarray analysis. A: The proportions of genes up-regulated by Six1 in each of several GO functional classes. B: The proportions of genes down-regulated by Six1 in each of several GO functional classes.

TABLE 2. Selected Genes Regulated >1.6-Fold by Six1 in Microarray Assays Affymetrix Probe

Fold

Accession

Change

Number

Xl.4323.1.A2_at Xl.418.1.S1_at Xl.647.7.S1_at

1.988 1.876 1.825

BJ078437 NM_001090739.1 AF154558.1

Ism1 Nfya Pax6

signaling transcription transcription

Xl.69.1.S1_at Xl.12075.1.S1_at Xl.7579.1.S1_at

1.757 1.740 1.717

AJ001834.2 AY036894.1 BC077576

transcription transcription Rab GTPase activator activity

Xl.9439.1.S1_at Xl.26468.1.S1_at Xl.4910.1.S1_at

1.674 1.637 1.623

BC041731.1 AF127040.1 BC126014

Xl.1354.2.S1_at

1.615

MGC83633

Irx1 Arnt TBC1 domain family, member 31 (tbc1d31) HoxA3 Mdx4 Cell division cycle associated 7-like (cdca7l) PAP associated domain 4 (papd4-a)

Xl.781.1.S1_at Xl.13309.1.S1_at Xl.20089.1.S1_at Xl.283.1.A1_at

1.852 1.832 1.715 1.642

AY029294.1 X53450 BC042303.1 X62053.1

set ID

Gene Title

otx1 snail1 foxi1 (xema) hoxA1

Six1 Alters the Expression of Numerous Genes in Ectodermal Explants Of the 14,400 genes represented on the Affymetrix GeneChip v1, 72 were expressed at a >two-fold (P < 0.05, analysis of variance [ANOVA]) higher level in the Six1-injected ACs (Table 1). Gene identities were determined by BLAST (http://blast.ncbi.nlm. nih.gov/Blast.cgi) and GOs were determined by searching the Xenopus and mouse gene databases (http://www.xenbase.org; http://www.informatics.jax.org). Based on gene title or most similar match, the largest category of the up-regulated genes is “unknown” function (44.4%). Genes with known function were most commonly involved in metabolic processes (15.2%),

GO

References Pera et al., 2002 Li et al. 1998 Li et al., 1997; Hirsch and Harris, 1997 Gomez-Skarmeta et al., 1998 Bollerot et al., 2001

transcription transcription cell cycle

www.xenbase.org Newman and Krieg, 1999

mRNA processing

Rouhana et al., 2005

transcription transcription transcription transcription

Kablar et al., 1996 Sargent and Bennett, 1990 Suri et al., 2005 Sive and Cheng, 1991

signaling (11.1%), and transcription (9.7%); a small number of other functions also were identified (Fig. 1A). Of these 72 genes, only a few have previously been implicated in cranial placode development: Nrp1, platelet-derived growth factor-alpha (PDGFa) receptor, and p75 neurotrophin receptor. Nrp1-semaphorin signaling regulates neural crest migration, which affects placode contributions to cranial ganglia in Xenopus and mouse (Koestner et al., 2008; Schwarz et al., 2008). PDGF signaling can induce ophthalmic placode formation (McCabe and Bronner-Fraser, 2008) and in combination with fibroblast growth factor (FGF) plays a role in PPR specification (Kwon et al., 2010). The p75 neurotrophin receptor is expressed on both neural crest and placode cells in developing cranial ganglia (Vazquez et al.,

6 YAN ET AL.

1994). In addition, several of the identified genes have functions that are similar to those published for Six1, supporting the idea that they may be in the same genetic pathway. For example, Six1 is known to regulate cell proliferation and is overexpressed in several cancers (Ford et al., 1998; Coletta et al., 2004; Patrick

TABLE 3. qPCR Analysis of Expression Levels in Six1-Injected Animal Cap Explants Relative to Control Explants Fold expression

DEVELOPMENTAL DYNAMICS

over control

P value

Up-regulated BJ02409 BQ399495 AF546707 Pdgfra BG885936 Hrpt1–5047264 Aldh7a1 Hnrnpr Arrb2–4740312 XXylt1–6639045

5.14 3.49 3.37 2.34 1.85 1.83 1.72 1.71 1.35 1.35

0.1

Down-regulated MGC 114680 Image 3399268 Dnaja4.2–4930076 Ralgds-4681426 Cnfn1-a-6316571

0.40 0.55 0.79 1.08 1.25

>0.1 0.1 >0.1 >0.1

et al., 2009; Li et al., 2013; Patrick et al., 2013); genes known to be involved in cell cycle regulation (Uncx4.1; Mitogen-activated protein kinase binging protein 1, Cyclin L1; Trhd1-a) and cell movement/cancer (Tiam1, Elf2, Nrp1, p75, Pbx1; MDC11b) were found in the up-regulated category. Placode development in several vertebrates is enhanced by FGF signaling and reduced by Wnt signaling (Phillips et al., 2001; Leger and Brand, 2002; Maroon et al., 2002; Liu et al., 2003; Brugmann et al., 2004; Ahrens and Schlosser, 2005; Litsiou et al., 2005; Martin and Groves, 2005; Matsuo-Takasaki et al., 2005; Bailey et al., 2006; Hong and SaintJeannet, 2007; Park and Saint-Jeannet, 2008; Esterberg and Fritz, 2009; Patthey et al., 2008, 2009; Kwon et al., 2010; Grocott et al., 2012); putative regulators of FGF (Cnpy1, Elf2, Ism1) and Wnt (Arrb2, Wif1) pathways were in the up-regulated category. We also searched those genes up-regulated >1.6-fold (P < 0.05, ANOVA) to identify previously characterized genes that might be in the Six1 pathway based on published expression patterns (Table 2). Amongst these were genes expressed during cranial sensory placode (Ism1, Pax6, Irx1, Arnt), somite (Arnt, HoxA3) and/or kidney development (Arnt, Mdx4, Irx1). To assess the validity of the microarray results, we performed several experiments. First, we measured the expression of 10 upregulated genes by quantitative polymerase chain reaction (qPCR); 10/10 were expressed at higher levels in Six1-injected ACs compared with uninjected control ACs, half of which reached statistical significance (Table 3). Second, we knocked-down endogenous Six1 in whole embryos by injecting morpholino antisense oligonucleotides; this diminished the expression of 17/ 20 up-regulated candidates (Table 4). Figure 2 shows examples: loss of somite expression of Cdca7l; loss of neural crest expression of HoxA3; loss of neural tube expression of Ism1. Third, we increased Six1 expression: 12/20 up-regulated candidates showed

TABLE 4. Percentage of Embryos Showing Altered Expression Domains of Up-regulated Genes in Response to Changes in Six1 Activity Expanded by Candidate Nrp1/BQ400635 Arrb2/BG812762 BG022063 LOC100037047 BQ399495 Hrpt1/BG023545 Rmb42-b/BJ083747 LOC414689 Uncx4.1/BC044278.1 Xxylt1/BQ386473 Pbx1b/AF480430.1 Ism1/BJ078437 Frizzled10b Nfya/NM_0–01090739.1 Arnt/AY036894.1 Tbc1d31/BC077576 HoxA3/BC041731.1 Mdx4 Cdca7l/BC126014 Papd4/MGC83633

Expanded by

Diminished by Six1 MO

Expanded by

Activating

Repressive

knock-down (n)

wild-type Six1 (n)

Six1-VP16 (n)

EnR-Six1 (n)

41.2 0.0 0.0 43.8 0.0 8.3 45.0 31.6 28.6 28.6 64.3 50.0 57.1 75.0 54.5 26.7 71.4 38.5 66.7 66.7

(17) (20) (20) (16) (16) (24) (20) (19) (14) (14) (14) (12) (14) (16) (11) (15) (14) (13) (12) (12)

4.8 0.0 5.9 15.6 7.1 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

(42) (52) (34) (45) (28) (49) (47) (45) (24) (40) (41) (31) (23) (39) (41) (53) (33) (30) (46) (21)

27.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 11.1 85.0 0.0 72.2 47.1 0.0 10.0 53.8 7.7 0.0 0.0

(18) (13) (8) (10) (7) (12) (14) (12) (11) (18) (20) (15) (18) (17) (14) (10) (13) (13) (13) (14)

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

(28) (24) (10) (20) (20) (16) (16) (12) (10) (14) (14) (15) (10) (10) (9) (11) (7) (9) (11) (8)

DEVELOPMENTAL DYNAMICS Fig. 2. Examples of changes in gene expression of putative Six1 targets after knock-down of endogenous Six1 levels by injection of antisense morpholino oligonucleotides (Six1 MOs), increased levels of wild-type Six1 by mRNA injection (Six1 WT), activation of Six1 targets by mRNA injection of an activating construct (Six1-VP16), or repression of Six1 targets by mRNA injection of a repressive construct (EnR-Six1) (see Brugmann et al., 2004 for validation of these constructs). Six1 MOs: Expression of Cdca7l in the segmenting somites (arrows on control [ctrl] side) is lost on the injected (inj) side (side views, anterior to left). Expression of HoxA3 in the neural crest (nc) is greatly reduced on the injected side, whereas hindbrain (hb) expression is maintained (side views, anterior to left). Expression of Ism1 is lost in the midbrain (arrow) (frontal view, dorsal to the top). Expression of IMAGE 3399268 is expanded in the neural plate (line depicts width from midline to lateral border) on the injected side (dorsal view, anterior to the bottom). Expression patterns of Cnfn1-a in the hatching gland (arrows) and of Snail1 in the migrating neural crest are reduced and disrupted on the injected sides (frontal views, dorsal to the top). Six1 WT: Expression of BG022063 is broader in the neural plate (line) and neural crest (bracket) on the injected side (frontal view, dorsal to the top). Expression of Nfya is broader in the neural tube (line) on the injected side (dorsal view, anterior to the top). Expression of IMAGE 3399268 in the segmented somites (arrows, ctrl side) is lost on the injected side (side views, anterior to left). Expression of IMAGE 4057931 in the neural crest (bracket), otocyst (oto) and trigeminal ganglion (arrow) is greatly diminished on the injected side (side views, anterior to left). Six1-VP16: Expression of Frizzled10b, Pbx1, and Zic2 in the neural plate is expanded (line) on the injected side (frontal views, dorsal to the top). EnR-Six1: Expression of Frizzled10b in the neural plate is reduced (line) on the injected side (frontal view, dorsal to the top). Expression of IMAGE 4057931 is reduced in the PPR (arrows on the control side; frontal view, dorsal to the top). Expression of Zic2 and Otx1 in the neural tube is reduced (line) on the injected sides (arrows; frontal views, dorsal to the top). In MO-injected embryos, injected sides were confirmed by the use of lysamine-labeled MOs (not shown). In mRNA-injected embryos, injected sides were confirmed by the presence of nuclear b-galactosidase lineage labeling (pink dots visible in most images).

8 YAN ET AL.

DEVELOPMENTAL DYNAMICS

TABLE 5. Genes Down-regulated >2-Fold by Six1 in Microarray Assays Affymetrix probe set ID

Fold change

Accession no.

Xl.22313.1.S1_at Xl.26171.1.A1_s_at Xl.14963.1.A1_x_at

3.876 3.610 3.378

BC080322 BC079829 BG264797

Xl.9924.1.A1_at Xl.2213.1.A1_at

3.322 3.311

BG020210 BG022871

Xl.4337.1.A1_at

3.215

BC097548

Xl.15435.1.A1_at

3.185

BJ081509

Xl.16561.1.A1_at

3.135

CF521493

Xl.16590.1.S1_at

3.115

BJ049628

Xl.23897.1.S1_at Xl.7794.1.A1_at

2.994 2.833

BC068675 BQ897357

Xl.2209.1.S1_at

2.695

CB562491

Xl.1064.1.S1_at Xl.13604.1.A1_at

2.695 2.646

U63818.1 BJ088530

Xl.23980.1.A1_at Xl.2443.1.S1_at

2.618 2.604

AW764542 BC046660.1

Xl.9975.1.A1_at

2.597

BJ088036

Xl.23110.1.A1_at

2.584

BJ076210

Xl.22434.1.A1_at

2.525

CB560725

Xl.2361.1.A1_at

2.475

BQ399921

Xl.16796.1.S1_at

2.463

BJ085230

Xl.166.1.S1_at Xl.15263.1.A1_at

2.439 2.421

U66003.1 BQ400687

Xl.16177.1.A1_at

2.421

BJ055605

Xl.16447.1.A1_at

2.387

BE505669

Xl.15778.1.A1_at Xl.2755.1.S1_at Xl.541.1.S1_s_at Xl.11153.1.A1_at

2.370 2.336 2.315 2.288

BJ056356 AY062263.1 BC041183.1 BJ057396

Xl.23887.1.A1_at Xl.738.1.S1_at Xl.15014.1.A1_at Xl.15701.1.S1_at Xl.25379.1.A1_at

2.283 2.268 2.262 2.247 2.242

BJ090458 AF317656.1 BJ048100 AY150813.1 BM262157

Xl.15834.1.S1_at

2.222

BJ050466

Gene title, unigene cluster, or EST name Xenopus laevis cDNA clone IMAGE 4174145 Xenopus laevis cDNA clone IMAGE 3399268 EST: daa32g06.x1; IMAGE 4057931; most similar to 50 biphosphate nucleotidase 1 (bpnt1) EST: dc48g12.y1; IMAGE 3400511 UniGene Xl.2213; suppressor of cytokine signaling 3 (Socs3) Xenopus laevis hypothetical protein MGC114680 UniGene Xl.79199; Fin and gill-specific type1 keratin (fgk) EST: AGENCOURT_15529711; IMAGE 7010138 UniGene Xl.63625; Glucocorticoid induced transcript 1 (glcci1) Cornifelin gene1 (Cnfn1-a) EST: AGENCOURT_8670989; most similar to Xenopus tropicalis ral guanine nucleotide dissociation stimulator (ralgds) UniGene Xl.55075, complement component 3 (c3) Ring finger protein EST: BJ088530; most similar to Xenopus laevis IMAGE clone 4965486 EST: da88e01.x1 DnaJ (Hsp40) homolog, subfamily A, member 1 (Dnaja4.2) EST: BJ088036; most similar to ras homolog family member T1 (rhot1) UniGene Xl.83926; Regulator of calcineurin 1 (rcan1) UniGene Xl.22434; Nacetylgalactosaminidase alpha (naga-a) UniGene Xl.2361; phosphoinositide-3-kinase, regulatory subunit 5 (pik3r5) EST: BJ085230; most similar to Xenopus tropicalis Rab interacting lysosomal proteinlike 1 (rilpl1) Adam13 UniGene Xl.63771; predicted sickle tail protein like (etl4) UniGene Xl.16177; Axin 2 (Axis inhibition protein 2) UniGene Xl.16447; strongly similar to mouse CDC42 small effector protein 2 (cdc42ep2) EST: BJ056356 Sp1-like zinc-finger protein (XSPR-2) jun oncogene EST: BJ057396; most similar to Xenopus tropicalis clone ISB1–168-L6 UniGene Xl.23887; Rab40B Akt EST: BJ048100 Kremen2 (krm2) UniGene Xl.25379; weakly similar to meteorin-like protein-like (metrn) UniGene Xl.63885; Xenopus laevis MGC84728 protein, most similar to similar to mouse/human prohibitin 2 (phb2)

GO unknown unknown metabolism

unknown signaling unknown cytoskeleton unknown unknown unknown signaling

signaling transcription unknown unknown protein folding signaling signaling metabolism metabolism protein transport

proteolysis unknown signaling cytoskeleton unknown transcription transcription unknown signaling signaling unknown signaling unknown transcription

NOVEL SIX1 TARGETS 9

TABLE 5. Continued

DEVELOPMENTAL DYNAMICS

Affymetrix probe

Fold

Accession

set ID

change

no.

Gene title, unigene cluster, or EST name

Xl.13799.1.A1_at Xl.16517.1.A1_at Xl.8910.1.S1_at Xl.13511.1.A1_at Xl.2612.1.A1_at

2.217 2.217 2.212 2.203 2.188

BJ085895 AW199946 BC042269.1 BJ085865 BJ097651

Xl.13965.1.A1_at

2.179

BJ081606

Xl.11640.1.A1_at

2.169

BJ075244

Xl.2784.1.S1_at Xl.17176.1.A1_at Xl.1085.1.S1_at Xl.11033.1.A1_at

2.159 2.141 2.132 2.114

M96729.1 BM261509 U57453.1 BM191782

Xl.4175.2.A1_at Xl.15853.1.A1_at

2.101 2.062

BJ081456 BJ089315

Xl.15479.1.A1_at

2.062

CD328587

Xl.25273.1.A1_x_at

2.058

AW782822

Xl.3057.2.S1_at

2.053

BU901475

Xl.9994.1.S1_s_at

2.028

BC043990.1

Xl.24413.1.S1_at

2.020

BQ397907

Xl.428.1.S1_at Xl.13762.1.A1_at Xl.15997.1.A1_at

2.012 2.012 2.008

X64762.1 BJ085361 BJ043804

Xl.22640.1.A1_at

2.008

BJ086587

Xl.19274.1.S1_at

2.004

BI442114

EST: BJ085895 UniGene Xl.16517; zinc finger protein 830 Keratin type I cytoskeletal 18-A (krt18-a) EST: BJ085865 UniGene Xl.2612; A-kinase anchor protein 2 (akap2) UniGene Xl.4228; Adaptor-related protein complex 4, mu 1 subunit (ap4m1) UniGene Xl.78983; strongly similar to related RAS viral (rras2) oncogene homolog Metallothionein (mti) EST: dai45d06.x1 Zic2 UniGene Xl.75442; phosphatidylinositol glycan anchor biosynthesis class C (pigc) EST: BJ081456 UniGene Xl.15853; strongly similar to Xenopus tropicalis von Willebrand factor C domain-containing protein 2-like, gene 2 precursor (vwc2l) UniGene Xl.15479; Xenopus laevis uncharacterized protein MGC116516; weakly similar to mouse/human mitogen-activated protein kinase 12 (mpk12) UniGene Xl.25885; weakly similar to Xenopus laevis zinc finger protein 33A UniGene Xl.3057; Xenopus laevis uncharacterized protein LOC100158313; weakly similar to human ZYX gene product Hypothetical protein MGC64520; strongly similar to Xenopus laevis growth associated protein 43 (gap43-b) UniGene Xl.24413; moderately similar to Xenopus tropicalis acid sphingomyelinaselike phosphodiesterase 3a precursor HNF1 homeobox A (hnf1a-a) EST: BJ085361 UniGene Xl.15997; synaptotagmin-like 1 (sytl1) UniGene Xl.22640; Vps20-associated 1 homolog (vta1) UniGene Xl.19274; Tumor suppressor candidate 3 (tusc3)

expanded expression domains by injection of mRNAs encoding either wild-type Six1 or the activating Six1-VP16 construct, and 0/20 was expanded by the repressive EnR-Six1 construct (Table 4). As examples, Figure 2 shows: broader neural plate and/or neural crest expression by wild-type Six1 (BG022063, Nfya) or Six1-VP16 (Frizzled 10b, Pbx1); reduced Frizzled 10b by EnRSix1. Although nearly all of the tested candidates require Six1, it was surprising that only approximately half of them were increased/expanded by wild-type or activating Six1. However, these results are consistent with SO activity in the fly eye field: overexpression rarely induces ectopic eye tissue (Jusiak et al., 2014) and it promotes eye fate mainly by repressing alternate

GO unknown cell cycle cytoskeleton unknown cytoskeleton protein transport signaling

metabolism unknown transcription metabolism unknown signaling

cell cycle

unknown unknown

cell adhesion

metabolism

transcription unknown protein transport protein transport ion transport

fates (Anderson et al., 2012). Thus, many of the up-regulated genes identified by the microarray assay are likely downstream of Six1 based on published function in Xenopus, chick, or mouse, published expression patterns and/or changes in expression after Six1 loss- or gain-of-function. However, Six1 ChIP analyses are required to confirm if any of these candidates are direct transcriptional targets. Of the genes represented on the GeneChip, 58 were expressed at a >two-fold (P < 0.05, ANOVA) lower level in the Six1expressing ACs (Table 5). The largest category of the downregulated genes is “unknown” function (36.2%). Genes with known function were most commonly involved in metabolic

10 YAN ET AL.

TABLE 6. Percentage of Embryos Showing Altered Expression Domains of Down-regulated Genes in Response to Changes in Six1 Activity

DEVELOPMENTAL DYNAMICS

Candidate IMAGE 4174145 IMAGE 3399268 IMAGE 4057931 IMAGE 3400511 Socs3/BG022871 MGC114680 IMAGE 7010138 Cnfn.1a/BC068675 Ralgds/BQ897357 Dnaja4.2/BC046660.1 Otx1 Snail1 Foxi1 (Xema) HoxA1 Zic2

Diminished (#) or expanded (") by

Diminished by

Six1 MO knock-down (n)

wild-type Six1 (n)

0.0 (12) 70.0 (10) " 20.0 (10) # 70.0 (10) # 0.0 (15) 0.0 (14) 0.0 (11) 75.6 (14) # 42.9 (14) # 50.0 (14) # 11.8 (17) # 78.6 (14)# 71.4 (14) # 0.0 (14) 42.9% (14) "

66.7 (21) 82.6 (46) 73.0 (37) 59.4 (32) 58.5 (41) 37.8 (37) 40.0 (45) 50.0 (26) 50.0 (16) 45.0 (20) 73.3 (15) 84.2 (19) 61.1 (18) nd nd

Diminished by repres- Diminished by activatsive EnR-Six1 (n) 92.9 91.7 100.0 88.2 100.0 100.0 87.5 83.3 88.9 86.7 77.8 83.3 90.9 80.0 80.0

(14) (12) (13) (17) (15) (12) (16) (12) (9) (15) (9) (15) (11) (15) (5)

ing Six1- VP16 (n) 0.0 0.0 0.0 0.0 0.0 17.6 0.0 0.0 14.3 5.9 5.9 0.0 0.0 0.0 0.0

(16) (14) (16) (10) (5) (17) (21) (17) (14) (17) (17) (10) (16) (16) (17)

nd, not done

Fig. 3. Semiquantitative RT-PCR assays illustrate the temporal expression patterns of previously uncharacterized up-regulated (A) and downregulated (B) genes in normal, unmanipulated Xenopus embryos. RT, minus RT step; numbers across the bottom indicate the developmental stages. H4 is an internal control. Im, IMAGE

processes (10.3%), signaling (19.0%), and transcription (10.3%); a small number of other functions also were identified (Fig. 1B). The expression of most of the 58 genes have not been characterized across Xenopus developmental stages, but by searching a mouse expression database (www.informatics.jax.org), we found

that many of those for which there is an identified homologue are expressed in developing tissues regulated by Six1. These include: cranial placodes (Glcci1, Rcan1, Naga, Rilpl1, Etl4, Xspr2, Jun, Phb2, Mt1, Pigc, Gap43, Tusc3), neural crest (Zic2), muscle (Rcan1, Jun, Krm2, Phb2, Rras2, Pigc, Sytl1), and nephric

AC

AC

AC, eq

animal

animal

animal, eq

Xxylt1 BQ386473 6639045 Trhd-a AY099224

AC, eq

animal

Hprt1 BG023545 5047264 Rbm42-b BJ083747 6635289 LOC414689 BQ398913 6636835 Uncx4.1 BC044278.1 5543082

AC, eq

AC, eq

animal

2. present*

AC

animal

BQ399495 4965752

AC

animal

AC

animal

LOC100037047 BG885936 3437507

AC

animal

Arrb2 BG812762 4740312 BG022063 3749434

AC

animal

st 8–9

Nrp1 BQ400635 4969039

Blastula

Cleavage

OB Clone #

Gene title

accession #

ecto, weak meso

ecto, weak meso

animal ecto

ecto

ecto

ecto, weak meso

ecto

animal ecto

ecto

animal ecto

animal ecto, dorsal ecto

st 10–11.5

Gastrula

E: dorsal ecto, NP, BZ, epi, meso E: dorsal ecto, NP, BZ, epi, meso

E: dorsal ecto, NP, BZ, epi, meso E: dorsal ecto, NP, BZ, epi, meso

epi L: dorsal

epi L: dorsal

epi L: dorsal

epi L: dorsal

E: dorsal ecto, epi L: NP, BZ, epi

E: dorsal ecto L: NP, BZ, epi, dorsal meso

E: dorsal ectoL: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, cem, dorsal meso E: dorsal ecto L: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, stripe in midbrain, noto

E: st 12–13L: st 14–17

Neural plate

NT, NC, PPR, epi, somites

NT-lat stripes in Mb, Hb and spcd, NC, olf, head epi, somites, nephric meso NT, NC, PPR, somites

NT, NC, PPR, head epi, cem, somites

NT, NC, PPR, dorsal epi

NT, NC, PPR, dorsal epi, noto, somites

NT, NC, epi, somites

NT, NC, head epi, cem, somites

NT, NC, olf, lens, oto, somites

NT, NC, dorsal epi, cem, somites

1. spcd, NC, PPR

st 18–22

Neural tube closure

Tail bud st 23–31

brain, ret, spcd, lens, oto, epi, BA, somites brain, ret, spcd, oto, epi, BA, somites

brain, ret, stripe in Mb, lens, oto, head epi, BA brain, ret, spcd, olf, lens, oto, head epi, BA, somites brain, ret, spcd, olf, lens, oto, head epi, cem, BA, somites brain, ret, spcd, olf, oto, head epi, BA, somites, nephric meso

brain, ret, spcd, olf, lens, oto, BA, somites brain, ret, spcd, olf, lens, oto, cem, BA, somites brain, ret, spcd, oto, BA, somites

1. spcd, NC, PPR, dorsal gut; Hb, ret, olf, lens, oto, dorsal epi, epibranchial placodes, cem, BA, nephric meso Hb, ret, oto, BA, somites

TABLE 7. Developmental Expression Patterns of Up-regulated Genes

DEVELOPMENTAL DYNAMICS

Larva

same, plus nephric meso, pharyngeal pouches

same, plus nephric meso, heart

same, plus lens

same, plus cranial ganglia, nephric meso same, plus nephric meso, heart

same, plus lens, nephric meso, heart same, plus nephric meso

same, plus nephric meso

same, plus dorsal spcd, lens, head epi, nephric meso same, plus nephric meso

same, plus pineal, spcd, IX/Xg, BAdistal patches, heart

st 32–38

NOVEL SIX1 TARGETS 11

AC

AC

AC

5. present* animal

6. animal

animal

animal, weak

animal

8. present* animal

Nfya/ NM001090739.1 6631251

Arnt AY036894.1 4203590

Tbc1d31 BC077576 5079674

HoxA3 BC041731.1 4683538 Cdca7l BC126014 6864704 Papd4 MGC83633 5084876

Gastrula

ecto, meso

ecto, weak meso

ecto, weak meso

ecto, weak meso

6. animal ecto, meso

ecto, weak meso

4. ventro-lateral blastopore lip

ecto, meso

st 10–11.5

Neural plate

E: dorsal ecto, NP, BZ, epi, meso E: dorsal ecto, NP, BZ, epi, meso E: dorsal ecto, NP, BZ, epi, meso epi L: dorsal

epi L: dorsal

epi L: dorsal

E: dorsal ecto, epi L: NP, BZ, epi dorsal meso

6. NP, BZ, epi, dorsal meso

4. E: same, plus noto 4. L: ant NP, BZ, pharyngeal endoderm E: dorsal ecto, epi L: NP, BZ, epi dorsal meso

3. NP, BZ, dorsal meso

E: st 12–13L: st 14–17

NT, NC, PPR, epi, somites

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, somites

NT, NC, PPR, epi, somites

NT, NC, olf, lens, oto, somites

4. Mb, post spcd, NC, Dlp, paraxial meso

3. brain, ret, ant spcd, BA, somites

st 18–22

Neural tube closure

Tail bud

brain, ret, spcd, lens, oto, epi, BA, somites

brain, ret, ant spcd, olf, lens, oto, BA, somites, nephric meso 6. brain, ret, spcd, lens, oto, BA, somite, nephric meso, olf, cranial ganglia brain, ret, spcd, olf, lens, oto, diffuse epi, BA, noto, somites 7. spcd, caudal BAs, somites, Strong stripe in Hb brain, ret, epi, somites

3. same, plus nephric meso, lateral plate meso, olf, lens, oto, somites 4: Di, Mb, oto, BA, tailbud, ventral blood islands

st 23–31

Larva

same, plus scattered epi cells, BA, nephric meso same, plus nephric meso

same

same, plus nephric meso

9. same

same

same, plus somites

st 32–38

ISH expression patterns at different developmental stages for 14 genes for which there are no ISH expression data. ISH patterns also were completed for 5 genes for which there are available descriptions. For 3 genes (Trhd-a, Nfya, Papd4) presence of expression (*) was detected only by Northern blot, RNase protection, RT-PCR or RNA-Seq. AC, animal cap; BA, branchial arches; BZ, border zone; cem, cement gland; dlp, dorso-lateral placode; E, early; ecto, ectoderm; epi, epidermis; eq, equatorial; Hb, hindbrain; IX/Xg, glossopharyngeal/vagal ganglion; L, late; Mb, midbrain; meso, mesoderm; NC, neural crest; NP, neural plate; NT, neural tube; OB, Open Biosystems; olf, olfactory placode; oto, otocyst; PPR, pre-placodal region; ret, retina; spcd, spinal cord; st, stage; Vg, trigeminal ganglion; VIIg, facial ganglion. Abbreviations in bold refer to previous published expression patterns; bold numbers indicate references for previously described expression data:1. Koestner et al., (2008); 2. Wu et al. (2001); 3. Maeda et al. (2002); 4. Pera et al. (2002); 5. Li et al. (1998); 6. Bollerot et al. (2001); 7. Xenbase: http://www.xenbase.org/gene/ expression.do?method¼displayGenePageExpression&geneId¼482266&tabId¼1; 8. Rouhana et al. (2005).

AC

6. AC

AC

AC

4. animal

Ism1 BJ078437 4888159

AC, eq

animal

st 8–9

Pbx1b

Blastula

Cleavage

OB Clone #

Gene title

accession #

TABLE 7. Continued

DEVELOPMENTAL DYNAMICS 12 YAN ET AL.

DEVELOPMENTAL DYNAMICS

NOVEL SIX1 TARGETS 13

Fig. 4. Whole-mount in situ hybridization assays at early developmental stages illustrating the common expression patterns of 14 upregulated genes: Nrp1, Arrb2, BG022063, LOC100037047, BQ399495, Hrpt1, Rbm42-6, LOC414689, Uncx4.1, Xxylt1, Nfya, Tbc1d31, Cdca7l, Papd4-a. For all genes, expression is enhanced in the animal cells (an) of cleavage and blastula embryos (side views). At gastrula stages, expression is throughout the ectoderm, occasionally enhanced in the animal cells (e.g., Arrb2) or the dorsal cells (e.g., Nrp1) (side views). At early neural plate stages, all genes are expressed in the epidermis (epi), often more intensely on the dorsal (d) side, including neural plate (np) and border zone (bz) (all except Nrp1 are side views with anterior [a] to the left; Nrp1 is an anterior view with dorsal to the top). At late neural plate and neural tube stages, expression is strongest in the neural crest (nc), pre-placodal region (ppr), neural tube (nt) and occasionally cement gland (cem) (all are anterior views). For Nrp1, embryos were sectioned in the horizontal plane to demonstrate that the ppr stripe in the whole embryo is due to expression in the deep (d) layer of the ectoderm, not the superficial (s) layer of the ectoderm or the pharyngeal endoderm (phe); ph, lumen of pharynx.

mesoderm (Naga, Pik3r5, Cdc42ep2, Xspr2, Rab40B, Akt, Rras2, Pigc, Vwc2, Gap43, HNF1a, Sytl1, Tusc3). Several also are transcribed in tissues in which Six1 is not normally expressed. These

Fig. 5. Whole-mount in situ hybridization assays at tail bud to larval stages illustrating more divergent expression patterns of 14 up-regulated genes: Nrp1, Arrb2, BG022063, LOC100037047, BQ399495, Hrpt1, Rbm42-6, LOC414689, Uncx4.1, Xxylt1, Nfya, Tbc1d31, Cdca7l, Papd4a. Side views at tail bud (left column) and larval (middle column) stages, with anterior to the left and dorsal to the top, and transverse sections at larval stages (right side) with dorsal to the top. ba, branchial arches; cem, cement gland; ep, epibranchial placodes; epi, epidermis; fb, forebrain; h, heart; hb, hindbrain; IX/Xg, glossopharyngeal/vagal ganglion; l, lens; mb, midbrain; ne, nephric mesoderm; olf, olfactory placode; oto, otocyst; p, pineal; r, retina; spcd, spinal cord; so, somites; Vg, trigeminal ganglion; VIIg, facial ganglion.

DEVELOPMENTAL DYNAMICS

14 YAN ET AL.

Fig. 6. Whole-mount in situ hybridization assays illustrating the expression patterns of previously characterized up-regulated (Trhd-a, Pbx1b, Ism1, Arnt, HoxA3) or down-regulated (Foxi1-Xema) genes at stages previously unpublished. Abbreviations are as in Figures 4 and 5.

Fig. 7. Four examples of cleavage stage gene expression in embryos that were bisected before the in situ hybridization protocol to facilitate probe access to all of the cells. In each case the probe was detected in the animal pole blastomeres (a) and in marginal zone animal blastomeres (arrows), but not in vegetal blastomeres (v).

include: neural plate/tube (Socs3, Glcci1, Dnaj4.2, Rhot1, Pik3r5, Axin2, Xspr2, Jun, Rab40b, Akt, Krm2, Metrn, Phb2, Akap2, Ap4m1, Rras2, Mt1, Zic2; Pigc, Vwc2, Gap43, HNF1a, Sytl1, Vta1, Tusc3), epidermis (Fgk, Cnfc1, Axin2, Krm2, Phb2, Krt18-a, Pigc, Tusc3) and mesoderm (Socs3, Pik3r5, Adam13, Xspr2; Jun, Krm2, Rras2, Mt1, Pigc, HNF1a). We also searched genes down-regulated >1.6-fold (P < 0.05, ANOVA) to identify previously characterized genes expected to be repressed by Six1,

based on studies in chick and Xenopus (Brugmann et al., 2004; Christophorou et al., 2009). Of note are genes expressed in neural plate (Otx1), neural crest (Snail1, HoxA1) and epidermis (Foxi1/ Xema) (Table 2). To assess the validity of the microarray results, we performed several experiments. First, we measured the expression of five down-regulated genes by qPCR; 3/5 were expressed at lower levels in Six1-injected ACs compared with uninjected control ACs, one of which reached statistical significance (Table 3). Second, we knocked-down endogenous Six1 in whole embryos by injecting morpholino antisense oligonucleotides; this altered the expression patterns of 10/15 down-regulated candidates (Table 6). The expression domains of two were expanded (e.g., IMAGE 3399268), whereas the remainders were reduced (e.g., Cnfn1-a, Snail1; Fig. 2). Third, we increased Six1 expression: 15/15 downregulated candidates had diminished expression domains by injection of mRNAs encoding either wild-type Six1 or the repressive EnR-Six1 construct, and only 4/15 by the activating Six1VP16 construct (Table 6). Figure 2 shows examples: reduced somite (IMAGE 3399268) and neural crest/placode (IMAGE 4057931) expression by wild-type Six1; reduced PPR (IMAGE 4057931) and neural tube (Zic2, Otx1) expression by EnR-Six1. Of interest, Zic2 neural tube expression was expanded by Six1VP16 (87.5%, n ¼ 17; Fig. 2). Thus, many of the down-regulated genes identified by the microarray assay are likely downstream of Six1 based on published function in Xenopus, chick or mouse, published expression patterns and/or changes in expression after Six1 loss- and gain-of-function. However, Six1 ChIP analyses are required to confirm if any of these candidates are direct transcriptional targets.

Expression Patterns of Uncharacterized Genes The data above suggest that many of the genes whose expression patterns are altered by Six1 are likely to be involved in Six1-related developmental processes. Because a large number of

NOVEL SIX1 TARGETS 15

these genes are of unknown function (Fig. 1) and/or their developmental expression patterns have not been reported in Xenopus, we randomly picked 10 genes of unknown function up-regulated >two-fold (shaded in Table 1) and 10 genes of unknown function down-regulated >two-fold (shaded in Table 5) and performed expression analyses. We also chose a few uncharacterized >1.6fold altered genes to analyze, and provide more detail for a few characterized genes (shaded in Tables 1, 2, and 5).

DEVELOPMENTAL DYNAMICS

RT-PCR To demonstrate expression levels over time, RT-PCR was performed for 12 of the up-regulated genes (Fig. 3A). Nrp1 is strongly expressed maternally, is reduced at blastula stages, strongly expressed during neural ectoderm/neural plate stages, and is reduced at tail bud stages. Arrb2 is expressed at very low levels until late tail bud stages. BG022063 and LOC10003704 are expressed at low levels before gastrulation, are highly expressed from neural plate through tail bud stages, after which expression decreases. BQ399495 is strongly expressed from cleavage through gastrulation stages, followed by peaks at early and late tail bud stages. Hrpt1 is highly expressed at cleavage and blastula stags, followed by reduced expression through the remaining stages. LOC414689 is weakly detected at all stages, but more highly at neural tube through tail bud stages. Uncx4.1 is strongly expressed nearly uniformly across all stages. Xxylt1 is highly expressed at cleavage and blastula stages, decreasing at neural plate stages, and peaking again at late tail bud stages. Ism1 is expressed nearly uniformly across all developmental stages, Arnt is strongly expressed from early neural plate to late tail bud stages and Hox3A is highly expressed maternally, then uniformly at lower levels from gastrula through early tail bud, then at increased levels at later tail bud stages. RT-PCR was performed for 10 of the down-regulated genes (Fig. 3B). IMAGE 4174145 is weakly expressed at all stages. IMAGE 3399268 is moderately expressed at all stages. IMAGE 4057931 is detected at early neural plate and early tail bud stages. IMAGE 3400511 is weakly expressed until early neural plate and late tail bud stages. Socs3 is weakly expressed from gastrulation through tail bud stages. MGC114680 and Ralgds are expressed at uniform levels from cleavage through tail bud stages, with less expression at later stages. IMAGE 7010138 is highly expressed at gastrulation and expressed at low levels after that (but see tissue expression patterns below). Cnfn1-a is highly expressed at cleavage to gastrulation stages, and again at late neural plate through early tail bud stages. Dnaja4.2 is strongly expressed at cleavage stages and then moderately expressed from gastrula though tail bud stages. These temporal expression profiles partially overlap with that of Xenopus Six1. Six1 expression is not detected by RTPCR or in situ hybridization at cleavage or blastula stages (Pandur and Moody, 2000). This indicates that during these early stages Six1 does not regulate the expression of the upregulated or the down-regulated genes. However, Six1 expression is detected by RT-PCR at gastrula (st 10.5), neural plate (st 12), neural tube (st 19), tail bud (st 26/27) and larval (st 37/38; st 40) stages when placodes, somites, and kidney anlage form (Pandur and Moody, 2000). This partial overlap with the RT-PCR data for each of the tested genes (Fig. 3) is consistent with the possibility that Six1 may regulate these genes after the onset of gastrulation.

Fig. 8. Examples of embryos bisected after the in situ hybridization protocol to reveal expression patterns in internal cells. BQ399495: At blastula (left image, sagittal section), expression is primarily in the animal cap (AC) cells, and barely detected in the equatorial (eq) zone. At neural plate stages (right image, transverse section), expression is observed in the neural plate (np), somites (so), and epidermis (epi). Hprt1: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At gastrula stages (right image, sagittal section, anterior to the left), expression is observed in the surface ectoderm (ecto) and involuting mesoderm (meso); bl, dorsal blastoporal lip. Xxylt1: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At neural plate stages (right image, sagittal section, anterior to the left), expression is observed in the neural plate and underlying dorsal mesoderm (meso). IMAGE 3399268: At blastula (left image, sagittal section), expression is in both animal cap and equatorial cells. At gastrula stages (right image, sagittal section, anterior to the left), expression is observed in the animal ectoderm, but barely detected in the involuting mesoderm. Cnfn1-a: At gastrula stages (sagittal section, anterior to the left), expression is observed in the animal ectoderm, but not in the involuting mesoderm. IMAGE 7010138: At blastula (left image, sagittal section), expression is primarily in the animal cap cells, and barely detected in the equatorial zone. At gastrula stages (middle image, sagittal section, anterior to the left), there is strong expression in both the ectoderm and involuting mesoderm. At neural plate stages (right image, sagittal section, anterior to the left), weak expression is observed in both the neural plate and underlying dorsal mesoderm.

In situ expression of up-regulated genes To demonstrate spatial expression patterns, whole embryo in situ hybridization (ISH) analyses were performed for 14 up-regulated genes for which no ISH data are available, and for 5 up-regulated genes for which the data are incomplete (Nrp1, Pbx1b, Ism1, Arnt, HoxA3). At the early developmental stages, all 19 genes share very similar expression patterns (Figs. 4–6; Table 7). All are expressed maternally, with enhanced localization to the animal hemisphere at cleavage stages (Fig. 4). To be assured that this was not due to lack of probe penetration into the yolky vegetal cells,

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16 YAN ET AL.

Fig. 9. Examples of Xenopus embryos hybridized with sense RNA probes to evaluate the extent of nonspecific staining at the early stages when expression patterns are diffuse (cf. Figs. 4, 5, 6, 10).

we also bisected embryos before ISH; enhanced animal hemisphere expression and lack of vegetal expression was consistently observed (Fig. 7). At blastula stages, expression remained in the animal half of the embryo (Fig. 4), in some cases extending into the equatorial region (Table 7; Fig. 8). At gastrula stages, all genes but Ism1 are diffusely expressed throughout the ectoderm germ layer; as previously reported (Pera et al., 2002), Ism1 instead is expressed in the ventrolateral portion of the blastopore lip of the gastrula (Table 7). Some genes had enhanced expression in the animal pole ectoderm (Nrp1, Arrb2, LOC100037047, Uncx4.1); sectioning the embryos showed that others also had weak expression in the involuting mesoderm (Hprt1, Xxylt1, Trhd, Pbx1b, Nfya, Arnt, Tbc1d31, HoxA3, Cdca7l, Papd4) (Figs. 4, 8; Table 7). To be assured that the diffuse staining at these early stages was not the result of nonspecific background, embryos also were processed with sense directed RNA probes; very little background was detected (Fig. 9). At neural plate stages, all 19 genes are expressed in the ectoderm/epidermis, with enhanced expression on the dorsal side of the embryo, including the early neural plate and neural border zone (BZ); for nearly all of these genes sectioning the embryos also showed expression in the dorsal mesoderm (Table 7; Fig. 8). At early neural plate stages, Arrb2 is additionally expressed in the cement gland, and Ism1 in the pharyngeal endoderm. At neural tube closure stages, all 19 genes are expressed in the neural tube and neural crest; 15/19 genes are additionally expressed in the PPR or a subset of cranial placodes. At later tail bud and larval stages, expression patterns diverge (Fig. 5; Table 7). How-

ever, each of the 19 genes is expressed in at least two tissues whose development is known to be regulated by Six1: at least one placode-derived structure (17/19), somite (18/19), or nephric mesoderm (17/19); the latter is consistent with a potential role in the BOR kidney deficit. Of those with placode expression, 17/17 are expressed in the otocyst, consistent with a potential role in the BOS3 hearing deficit. Although Six1 is thought to be primarily important in placode development, recent work also shows it is required for some neural crest derivatives (Yajima et al., 2014; Garcez et al., 2014); consistent with these reports we find that all 19 of the up-regulated genes are detected in the cranial neural crest and the branchial arch mesoderm. However, it should be noted that many of the 19 genes also have prominent expression in tissues that do not have detectable levels of Six1 at these developmental stages: neural tube/retina (19/19), heart (4/19), cement gland (3/19), pharyngeal endoderm/pouches (2/19), and blood islands (1/19). Therefore, additional transcriptional pathways must regulate these genes in these additional tissues. Because the expression patterns of five of the genes identified in the microarray analysis were previously published, but not for all stages of development, we completed their developmental expression patterns (Figs. 4–6; Table 7). Of note, Nrp1, a receptor for Sema3a, is expressed in the deep layer of the PPR (Fig. 4) and in several placodes including otic (Fig. 5). Trhda is a maternally expressed nuclear protein that inhibits neural differentiation (Wu et al., 2001); because no ISH data are available we present the entire developmental series (Fig. 6). Pbx1, a homeoboxcontaining transcription factor that interacts with Meis and Hox

DEVELOPMENTAL DYNAMICS

NOVEL SIX1 TARGETS 17

Fig. 10. Whole-mount in situ hybridization assays at early stages illustrating common expression patterns of 10 down-regulated genes: IMAGE 4174145, IMAGE 3399268, IMAGE 4057931, IMAGE 3400511, Socs3, MGC114680, IMAGE 7010138, Cnfn1-a, Ralgds, Dnaja4.2. For all genes, expression is enhanced in the animal cells (an) of cleavage and blastula embryos, and is throughout the ectoderm at gastrula stages (side views). At early neural plate stages, all genes are expressed in the epidermis (epi), often more intensely on the dorsal (d) side, including neural plate (np) and border zone (bz) (all except IMAGE 3400511 are side views with anterior [a] to the left; IMAGE 3400511 is an anterior view). At later neural plate and neural tube stages, expression is strongest in the neural crest (nc), pre-placodal region (ppr), neural tube (nt) and epidermis (epi) (anterior views). Cnfn1-a is uniquely expressed in the hatching gland (hg).

proteins to influence hindbrain and neural crest gene expression (Maeda et al., 2002), is additionally expressed in the animal ectoderm, some placodes and somites (Fig. 6). Ism1 is a novel secreted factor co-expressed in FGF8-expressing domains (Pera et al.,

2002); we found that, in addition to the published expression pattern, it is expressed in the pharyngeal endoderm and somites (Fig. 6). Arnt is a bHLH/PAS factor that regulates many aspects of vertebrate development and tumorigenesis (Crews and Fan, 1999;

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18 YAN ET AL.

Fig. 11. Whole-mount in situ hybridization assays at tail bud to larval stages illustrating more divergent expression patterns of 10 down-regulated genes: IMAGE 4174145, IMAGE 3399268, IMAGE 4057931, IMAGE 3400511, Socs3, MGC 114680, IMAGE 7010138, Cnfn1-a, Ralgds, Dnaja4.2. Side views at tail bud (left column) and larval (middle column) stages, with anterior to the left and dorsal to the top, and transverse sections at larval stages (right side) with dorsal to the top. Abbreviations are as in Figure 10. For Cnfn1-a: hg, hatching gland; clo, cloaca.

Ralgds BQ897357 4681426

MGC114680 BC097548 6316942 IMAGE 7010138 CF521493 7010138 Cnfn1-a BC068675 6316571

IMAGE 4057931 BG264797 4057931 IMAGE 3400511 BG020210 3400511 Socs3 BG022871 4173655

IMAGE 4174145 BC080322 4174145 IMAGE 3399268 BC079829 3399268

animal

AC

AC 2. present*

AC, eq

animal

animal 2. present*

AC

animal

AC 1. not detected*

AC

animal

animal 1. not detected*

AC

AC, eq

animal

animal

AC

animal

st 8–9

OB Clone #

Cleavage

Blastula

Gene title accession #

ecto, meso

ecto 2. present*

ecto, weak meso

ecto

ecto 1. not detected*

ecto

ecto, weak meso

ecto

ecto

st 10–11.5

Gastrula

E: dorsal ecto L: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, dorsal meso E: dorsal ecto L: NP, BZ, epi, dorsal meso 1. strong expression* E: dorsal ecto L: NP, BZ, epi, dorsal meso E: dorsal ecto L: NP, BZ, epi, dorsal meso E: dorsal ecto L: scattered cells in NP and BZ 2. present*

E: ecto L: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, dorsal meso

E: dorsal ecto L: NP, BZ, epi, dorsal meso

14–17

Neural plate E: st 12–13 L: st

NT, NC, PPR, dorsal epi, somites

hatching gland, dorsal epi 2. present*

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, somites

NT, NC, PPR, dorsal epi, somites 1. strong expression*

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, dorsal epi, somites

NT, NC, PPR, dorsal epi, somites

st 18–22

Neural tube closure

Tail bud st 23–31

brain, ret, spcd, olf, lens, oto, cranial ganglia, head epi, BA, somites

brain, ret, spcd, olf, lens, oto, cranial ganglia, epi, BA, somites brain, pineal, ret, spcd, lens, oto, BA, somites brain, ret, spcd, lens, head epi, BA, somites brain, ret, spcd, lens, oto, BA, somites 1. strong expression* brain, ret, spcd, lens, oto, epi, BA, somites brain, ret, spcd, lens, oto, epi, somites hatching gland, cem, scattered cells in epi 2. present*

brain, ret, spcd, lens, oto, head epi, BA, somites

TABLE 8. Developmental Expression Patterns of Down-regulated Genes

DEVELOPMENTAL DYNAMICS

Larva

same, plus brain, ret, spcd, oto, Vg, BA, somites, nephric meso, ventral pharynx, caudal noto 2. present* same, plus nephric meso

same, plus BA, nephric meso

same, plus cranial ganglia, nephric meso

same, plus cranial ganglia, nephric meso 1. strong expression*

same, plus olf, cranial ganglia, nephric meso same, plus oto, cranial ganglia, heart

same, plus nephric meso

same, plus nephric meso

st 32–38

NOVEL SIX1 TARGETS 19

3. anteroventral ecto 3. AC ecto 3. not detected*

ISH expression patterns at different developmental stages for 10 genes for which there are no ISH expression data. ISH pattern also was completed for 1 gene for which there are available descriptions. For 2 genes (Socs3, Cnfn1-a) presence of expression (*) was detected only by Northern blot, RNase protection, RT-PCR or RNA-Seq. AC, animal cap; BA, branchial arches; BZ, border zone; cem, cement gland; E, early; ecto, ectoderm; epi, epidermis; L, late; meso, mesoderm; NC, neural crest; noto, notochord; NP, neural plate; NT, neural tube; OB, Open Biosystems; olf, olfactory placode/pit; oto, otocyst; PPR, pre-placodal region; ret, retina; spcd, spinal cord; st, stage; Abbreviations in bold refer to previous published expression patterns; bold number indicate references for previously described expression data: 1. Kuliyev et al. (2005); 2. Xenbase: http://www.xenbase.org/gene/expression.do?method¼displayGenePageExpression&tabId¼1&geneId¼5856331; 3. Wessely et al. (2004); 4. Suri et al. (2005).

3. antero-ventral ecto

same, plus olf, cranial ganglia, nephric meso brain, ret, spcd, oto, cranial ganglia, BA, somites brain, ret, spcd, lens, oto, BA, somites 3. antero-ventral ecto NT, NC, PPR, dorsal epi, somites

E: dorsal ecto L: NP, BZ, epi, dorsal meso 3. BZ, anteroventral ecto ecto, weak meso AC

st 10–11.5 Cleavage

animal

Dnaja4.2 BC046660.1 4930076 Foxi1 (Xema)

Larva Tail bud

st 32–38 st 18–22

Neural tube closure

st 8–9 OB Clone #

14–17

Neural plate

E: st 12–13 L: st Gastrula Blastula

Gene title

accession #

TABLE 8. Continued

DEVELOPMENTAL DYNAMICS

st 23–31

20 YAN ET AL.

Labrecque et al., 2013); we found that in addition to the published expression pattern (Bollerot et al., 2001), it is expressed in the neural plate, neural crest, PPR, olfactory placode, cranial ganglia, and somites (Fig. 6; Table 7). HoxA3 is a homeodomain transcription factor involved in axis and craniofacial patterning (Quinonez and Innis, 2014); we found that, in addition to the expression pattern posted on Xenbase (http://www.xenbase.org/ gene/showgene.do?method¼display& geneId¼482266&), it is expressed in a pattern very similar to the other up-regulated genes before neural tube closure, but is not subsequently expressed in the placodes (Fig. 6; Table 7).

In situ expression of down-regulated genes ISH analyses were performed for 10 down-regulated genes for which there are no ISH data available, and for 1 gene for which the data are incomplete (Foxi1/Xema) (Figs. 6, (10 and 11), Table 8). At the early developmental stages, the 10 uncharacterized genes share very similar expression patterns with each other and with the up-regulated genes. All are expressed maternally, with enhanced localization to the animal hemisphere at cleavage and blastula stages (Fig. 10); this localization was not due to poor probe penetration (Fig. 7). At blastula stages, expression remained in the animal half of the embryo (Fig. 10), in some cases extending into the equatorial region (Table 8; Fig. 8). At gastrula stages, all 10 genes are expressed throughout the ectoderm; sectioning the embryos showed that a few also had weak expression in the involuting mesoderm (IMAGE 4057931, IMAGE 7010138, Ralgds, Dnaja4.2) (Table 8; Fig. 8). The diffuse staining at early stages was not the result of nonspecific background, as demonstrated by processing embryos with sense directed RNA probes (Fig. 9). At early neural plate stages, ectodermal expression is enhanced on the dorsal side of the embryo, including the early neural plate and BZ. At later neural plate stages, all genes are expressed in the epidermis, neural plate, neural crest, PPR, and epidermis, and most were also expressed in the dorsal mesoderm. Cnfn1-a is not detected in the mesoderm (Fig. 8), and is uniquely expressed in scattered cells throughout the ectoderm. At neural tube closure stages, 9/10 of the genes are expressed in the neural tube, neural crest, PPR, dorsal epidermis and somites. At neural tube through tail bud stages, Cnfn1-a is uniquely expressed in the hatching gland and in the epidermis near the ventral midline and surrounding the cloaca (Figs. 10B, 11B). At later tail bud and larval stages, expression patterns diverge (Fig. 11; Table 8). However, all 10 genes are expressed in tissues whose development is known to be regulated by Six1, including at least one placode derived structure (10/10), neural crest (9/10), somites (10/10), and nephric mesoderm (8/10). These temporal patterns of expression are mostly congruent with the RT-PCR results (Fig. 3B), although in some cases the color reaction was allowed to develop for up to 48 hr due to the low abundance of the transcripts. For example, even though IMAGE 7010138 is mostly detected by RT-PCR only at the onset of gastrulation, it is clearly detectable by ISH at earlier and later stages, albeit, with less chromogenic intensity (Fig. 8). Foxi1-Xema is a transcription factor involved in epidermal development (Suri et al., 2005). We found that, in addition to tissues previously reported, at larval stages there is expression in neural tube-derived structures, placode-derived otocyst and cranial ganglia, neural crest-derived branchial arches, and in somites (Fig. 6; Table 8). One would expect down-regulated genes to be detected in tissues that do not also express Six1. For some stages and tissues,

DEVELOPMENTAL DYNAMICS

NOVEL SIX1 TARGETS 21

Fig. 12. A–F: Whole-mount in situ hybridization for Pbx1 from primitive streak to 14-somite stages. Rostral is to the top, caudal to the bottom of the images. Lines in C and E indicate the levels of sections shown in a–h; i–k are higher magnifications of the placode areal shown in c, f, and g, respectively. bz, border zone; eb, epibranchial placode; l, lens placode; nc, neural crest; np, neural plate; oto, otic placode; so, somite.

this expectation holds. For example, 11/11 are expressed in the cleavage and blastula precursors of the ectoderm, which do not express Six1 (Pandur and Moody, 2000). In addition, at later stages 11/11 are expressed in the epidermis and neural tube; these results are consistent with gain-of-function studies in chick and frog showing that when Six1 is ectopically expressed it represses genes characteristic of these tissues (reviewed in Grocott et al., 2012; Saint-Jeannet and Moody, 2014). However, inconsistent with this expectation, most of the putative down-regulated genes are also expressed in the PPR, placodes (including otocyst), neural crest, somites, and nephric mesoderm. This overlap with Six1 expression suggests that additional transcriptional pathways up-regulate these genes even in the presence of Six1.

In situ expression of homologous genes in chick To assess whether the expression of putative Six1 target genes is conserved in amniotes, we identified chick homologues using the Basic Local Alignment Search Tool. Out of 19 up-regulated and 17 down-regulated Xenopus transcripts we determined 17

and 11 chick mRNAs with high sequence identity. Of those, we selected five up-regulated genes (Pbx1, Arnt, XXYLT1, WDR67, Nrp1) and one down-regulated gene (DNAJA1) for expression analysis. Except Nrp1, all transcripts show similar expression profiles to their Xenopus homologues (Figs. 12–16; Table 9). Nrp1 is not expressed in the ectoderm at any stage investigated, but found in the endoderm underlying the PPR (not shown). In contrast, Pbx1, Arnt, Wrd67, and DNAJA1 are broadly expressed in all three germ layers from primitive streak to 11–14 somite stages. However, their expression is generally enriched in the ectoderm including the future neural plate and PPR, the developing neural tube, neural crest cells and placodal tissue, in particular the otic and epibranchial placodes. Hybridization with sense-directed RNA probes indicates that these diffuse patterns of expression are not the result of nonspecific background staining (Fig. 17). At neural tube stages, Six1 remains expressed in all placodes except for the lens (Sato et al., 2012). Of interest, Pbx1, Arnt, XXYLT1, and WDR67 are also absent from the lens, whereas DNAJA1, a down-regulated transcript, is present. Unlike the other transcripts, XXYLT1, starts to be expressed in

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22 YAN ET AL.

Fig. 13. A–F: Whole-mount in situ hybridization for Arnt from primitive streak to 14-somite stages. Rostral is to the top, caudal to the bottom of the images. Lines in C and E indicate the levels of sections shown in a–h; i–k are higher magnifications of the placode area shown in c, f, and g, respectively. Abbreviations are as in Figure 12.

the posterior epiblast and primitive streak at gastrulation stages before being strongly up-regulated in the neural plate and forming neural tube. At placodal stages it is present in the otic and epibranchial placodes, neural crest cells and the intermediate and posterior lateral plate mesoderm. Thus, expression patterns of selected genes in chick are generally similar to those observed in Xenopus.

Summary The expression patterns of the genes identified in this study as potential targets of Six1 are consistent with previous reports showing that Six1 plays an important role in the development of vertebrate cranial sensory placodes, somites and kidney. The majority of up-regulated genes are expressed in tissues known to express Six1 (PPR, placodes, somites, nephric mesoderm), which is expected for putative activated targets. However, their expression in Six1-negative tissues, including neural plate, and epider-

mis, indicates that they also are regulated by additional factors. The down-regulated genes are expressed in Six1-negative tissues, which is expected for putative repressed targets; their expression in some Six1-positive tissues indicates that they also are upregulated by additional factors even in the presence of Six1. Because the majority of the genes described herein are expressed in the developmental precursors of two organs whose defects are diagnostic for BOS and BOR syndromes, the ear and the kidney, they are new candidates for potential involvement in these birth defects.

Experimental Procedures Frog Embryos, Animal Cap Explants, and Microinjections Fertilized Xenopus laevis eggs were obtained by either in vitro fertilization (for ACs) or gonadotropin-induced natural mating of

DEVELOPMENTAL DYNAMICS

NOVEL SIX1 TARGETS 23

Fig. 14. A–F: Whole-mount in situ hybridization for XXYLT1 from primitive streak to 14-somite stages. Rostral is to the top, caudal to the bottom of the images. Lines in B and F indicate the levels of sections shown in a–h; i–k are higher magnifications of the placode area shown in c, f, and g, respectively. Abbreviations are as in Figure 12.

adult frogs (for ISH) as described elsewhere (Moody, 2000). mRNAs were synthesized by in vitro transcription (Ambion, mMessage mMachine kit). mRNA encoding wild-type Six1 (400 pg) was injected into the animal poles of both cells at the two-cell stage. ACs were dissected from microinjected embryos and from uninjected siblings (controls) at stages 8.5–9.0, cultured in 1 Modified Barth’s solution and collected when sibling embryos reached stage 16. For assessment of Six1 levels on gene expression in whole embryos, previously characterized Six1-specific morpholino antisense oligonucleotides (Six1 MOs) or mRNAs encoding wild-type Six1 (400 pg), an activating Six1-VP16 fusion protein (100 pg), or a repressive EnR-Six1 fusion protein (100 pg) were injected into lateral-animal blastomeres of 16-cell embryos (Brugmann et al., 2004). Embryos for RT-PCR and in situ hybridization (ISH) assays were cultured in 100% Steinberg’s solution through blastula stages and in 50% Steinberg’s solution from blastula through larval stages.

Microarray and Statistical Analyses Four independent samples of Six1 mRNA-injected ACs were collected. Each sample was derived from one different male and two different females and contained 100 ACs. For each Six1-expressing sample, a control, uninjected sample from sibling embryos also was collected. All samples were processed in

parallel for cDNA labeling and chip hybridization to reduce inter-sample variations. Total RNAs were extracted from ACs with the RNeasy mini kit (Qiagen). Integrity of RNA was assessed using the Agilent 2100 bioanalyzer (Agilent Technologies) and only samples with an integrity number > 8.0 were used. Total RNAs were labeled and fragmented with the Ovation Biotin RNA Amplification and Labeling System (NuGEN Technologies, Inc.). Briefly, 50 ng of total RNA was used for first- and second-strand cDNA syntheses. The synthesized cDNAs were amplified, purified with a PCR purification kit (Qiagen), then labeled and fragmented following the Ovation kit instructions. The labeled cDNAs were purified with the Dye Ex kit (Qiagen). Chip hybridization and statistical analyses were performed by the NINDS-NIMH Microarray Consortium at The Translational Genomics Research Institute (T-GEN, Tempe, AZ). The Affymetrix R Xenopus laevis Genome Array (v1.0) bears 15,503 GeneChipV probe sets representing approximately 14,400 transcripts. The chips were washed and scanned as recommended by the Ovation Biotin RNA Amplification and Labeling System User Guide (version 1.0). GCOS software was used to determine signal intensities and detection calls for each gene. Five replicates of each sample were analyzed. The average replicate correlation values for the control samples and for the Six1-injected samples each were 0.94, indicating consistency between replicates despite random genetic variance between parental frogs. Using GeneSpring

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24 YAN ET AL.

Fig. 15. A–F: Whole-mount in situ hybridization for Wrd67 from primitive streak to 14-somite stages. Rostral is to the top, caudal to the bottom of the images. Lines in C and F indicate the levels of sections shown in a–h; i–k are higher magnifications of the placode area shown in c, f, and g, respectively. Abbreviations are as in Figure 12.

Fig. 16. A–F: Whole-mount in situ hybridization for DNAJA1 from primitive streak to 14-somite stages. Rostral is to the top, caudal to the bottom of the images. Lines in C and E indicate the levels of sections shown in a–h; i–k are higher magnifications of the placode area shown in c, f, and g, respectively. Abbreviations are as in Figure 12.

NP, NC, posterior paraxial mesoderm NP, NC, PPR, notochord, mesoderm, endoderm

NP, NC, PPR, somites, posterior ectoderm

NP, NC

NP, NC, PPR, head fold

NP, NC, PPR, lateral mesoderm, lateral endoderm, posterior ectoderm

NE, BZ

NE, BZ, mesoderm, endoderm, posterior ectoderm

NE, BZ, mesoderm, endoderm

Primitive streak, posterior epiblast

NE, BZ, mesoderm, endoderm, posterior epiblast

NE, BZ, mesoderm, endoderm, posterior epiblast

ARNT/ 374026/ ChEST814a24

XXYLT1/ 424896/ ChEST85d22

WDR67/ 420349/ ChEST325c2

DNAJA1/ 427376/ ChEST151p15

NP, NC, PPR, ectoderm, mesoderm, endoderm

NP, NC, PPR, mesoderm, endoderm

NP, NC, PPR, somites, epidermis

NP, NC, PPR, somites

HH9/HH10

NT, NC, lens placode, otic, epibranchial placode somites, lateral plate mesoderm, paraxial mesoderm, endoderm, blood islands

NT, NC, otic, epibranchial placodes, epi, notochord, somites, lateral plate mesoderm, endoderm, blood islands Fore-, mid- and anterior hindbrain, cranial NC, posterior paraxial mesoderm NT, otic, epibranchial placodes, somites, notochord, mesoderm, endoderm

NT, NC, otic, epibranchial placodes, notochord, somites, lateral plate mesoderm, endoderm

neural tube

HH11/12

Brain, NC, otic, nephric mesoderm, posterior paraxial mesoderm Brain, NC, lens placode, otic, epibranchial placode, episomites, notochord, mesoderm, endoderm Brain, NC, lens placode, otic, epibranchial placode somites, lateral plate mesoderm, nephric mesoderm, endoderm, blood islands

Mid- and hindbrain, NC, otic, epibranchial placode, notochord, somites, lateral plate mesoderm, endoderm Brain, notochord, NC, otic, epibranchial placode, epi, somites, lateral plate mesoderm, endoderm

BZ, border zone; epi, epidermis; HH, Hamburger-Hamilton stage; NC, neural crest; NE, neural ectoderm; NP, neural plate; NT, neural tube; otic, otic placode or vesicle; PPRPre-placodal region

NE, BZ, mesoderm, endoderm, posterior ectoderm

NE, BZ, mesoderm, endoderm

NE, BZ, mesoderm, endoderm

HH8/HH9 neural folds

NE, BZ, mesoderm, endoderm

HH6/HH7 neural plate

PBX1/ 395505/ ChEST643j12

HH5

EST #

head process

HH4

gastrula

Gene symbol/ NCBI gene ID/

TABLE 9. Developmental Expression Patterns of Chick Homologous Genes

DEVELOPMENTAL DYNAMICS

NOVEL SIX1 TARGETS 25

26 YAN ET AL.

TABLE 10. RT-PCR and qPCR Primer Sequences Name RT-PCR (Figure 3) Nrp1 Arrb2–4740312 BG022063 LOC100037047 BQ399495 Hprt1–5047264

DEVELOPMENTAL DYNAMICS

LOC414689 Uncx4.1 Xxylt1–6639045 Ism1 Arnt HoxA3 Image 417414 Image 3399268 Image 4057931 Image 340051 Socs3 MGC 114680 Image 7010138 Cnfn1-a-6316571 Ralgds-4681426 Dnaja4.2–4930076 H4 (reference) qPCR (Table 3) Aldh7a1 Pdgfra AF546707 Hnrnpr BG885936 BJ02409 Arrb2–4740312

Primer direction

Primer sequence

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

50 GCC ATC TCC AAA GAA ACC AA 3’ 50 CAT GTA ACC GGA CGG AGT CT 3’ 50 GCC ATT CAG ACA GCT TAT GG 3’ 50 TGA CTC GCT GAC AAA CTT GG 3’ 50 CTT CTG CGT AGG AGG GAC AG 3’ 50 TCT TAT GGC CAT GCA TTT CA 3’ 50 AGT CAG GAA CCA CCT GGA TG 3’ 50 GGC AGA TGG CTG GAA AAA TA 3’ 50 AGC AGA ATT GGC AAA CAA CA 3’ 50 AGC AGA GCA ATA CGG CAA GT 3’ 50 GGA TGA CCT TTC CAC CTT GA 3’ 50 GCC TGT ATC CCA CAC TTC GT 3’ 50 TGG GGA GGT TCC TAT CTC CT 3’ 50 ATC TGG GCG ATT TTA ACG TG 3’ 50 ACG GCC GAT TAA GGT TTC TT 3’ 50 CTC CAT CAT CTC CCA GGG TA 3’ 50 GTT CCA GGT GGC ACT CAG TT 3’ 50 TTC CGT CAG TGT GGT GAC AT 3’ 50 GAA TTA CGG GGA CCA GGA TT 3’ 50 ACT GCA CAC TGA CCA ACT GC 3’ 50 CGA TGT AGG TCA AAG CAG CA 3’ 50 CCT GTG GCT GGT ATC CAA CT 3’ 50 AAC GCT TGA ACA GGG ACA TC 3’ 50 CCA ATA ACT GAA GGC CCA GA 3’ 50 TCT GCA GTG CCA TCA TTC TC 3’ 50 GGA ACA ACA GTT GGG TCT CC 3’ 50 ATG GTT CAG GGC TGT TGT TC 3’ 50 ATG CAG GAT GGA GGA CAA AC 3’ 50 TTT TCG GGA TGA AAG TGG TC 3’ 50 GGC AGT CAG CCT TAG CAA TC 3’ 50 AAA TTC CAG ATG GGC TGT TG 3’ 50 TGC AGT CGG CAA TAA CTC TG 3’ 50 CGA GTA CAA CCT CGT GCT GA 3’ 50 TGC CAG ACT CAG TTT TGA CG 3’ 50 GGT CTG AAC GCC AGA ATG AT 0 5 CCA GAC GAA AGA AAG CAA GG 3’ 50 GCT TGG GAT CTT TGT GAA GG 3’ 50 AAA TGA GCC TTG GGA CTG TG 3’ 50 TCA GGA TTT CGG AGA GTG CT 3’ 50 TTT AGT TCC CGA GCC ATT TG 3’ 50 TTG GCT GTA GAA CGC AAC AC 3’ 50 CAC GTT AGA GGC CGG AAA TA 3’ 50 GAC CAA TAG CGG AAC GAT GT 3’ 50 TCA GGA TTT CGG AGA GTG CT 3’ 50 CGG GAT AAC ATT CAG GGT ATC ACT 3’ 50 ATC CAT GGC GGT AAC TGT CTT CCT 3’

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward

50 CCG CCT TCA ATT TTC CTG TTG CTG 3’ 3’ 50 GGC ATT GCC AAT GTC TGC ACC AC 3’ 50 CCC TCG CAA ATG CCA CTA CAG AAG 3’ 50 CCA AAC ATG GGC TCC AGG TCA ATG 3’ 50 TCT CTC GGG ATG GGA TGA TAA ATG 3’ 50 CCA ACC CCG CTG TAA CCA AA G 3’ 50 CCG AGG CCG TGG AGG CAG AG 3’ 50 CGC ACA GGG TAG CCA TCG TCA TAG 3’ 50 CTG GGG ACT GGA AGA ATC ATT TCA C 3’ 50 AAG TCA CGT CAG CGG GGA ACA G 3’ 50 CAT GTG CGC CGC CAT TAT TTT C 3’ 50 CAC GGA TGC TTC GAG TTA GAG ATG 3’ 50 GCC ATT CAG ACA GCT TAT GG 3’

NOVEL SIX1 TARGETS 27

TABLE 10. Continued Name BQ399495 Hprt1–5047264 Xxylt1–6639045 Image 4174145 Dnaja4.2–4930076 Image 3399268

DEVELOPMENTAL DYNAMICS

Image 4057931 MGC 114680 Cnfn1-a-6316571 Ralgds-4681426 Eef1a (reference)

Primer direction

Primer sequence

Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

50 TGA CTC GCT GAC AAA CTT GG 3’ 50 AGC AGA ATT GGC AAA CAA CA 3’ 50 AGC AGA GCA ATA CGG CAA GT 3’ 50 GGA TGA CCT TTC CAC CTT GA 3’ 50 GCC TGT ATC CCA CAC TTC GT 3’ 50 GTT CCA GGT GGC ACT CAG TT 3’ 50 TTC CGT CAG TGT GGT GAC AT 3’ 0 5 GGG CCT TGG GTC CTT CAC TTC TC 3’ 50 TCG GAG GGT TCG TTT GAG GAC TG 3’ 50 GAC CAA TAG CGG AAC GAT GT 3’ 50 TCA GGA TTT CGG AGA GTG CT 3’ 50 TCA GCG CCC ATT TGC TAT TTC AC 3’ 50 GGC GAG ATG GCA GTT GGA ACA C 3’ 50 TTT TCG GGA TGA AAG TGG TC 3’ 50 GGC AGT CAG CCT TAG CAA TC 3’ 50 GGT CTG AAC GCC AGA ATG AT 50 CCA GAC GAA AGA AAG CAA GG 3’ 50 TCA GGA TTT CGG AGA GTG CT 3’ 50 TTT AGT TCC CGA GCC ATT TG 3’ 50 TTG GCT GTA GAA CGC AAC AC 3’ 50 CAC GTT AGA GGC CGG AAA TA 3’ 50 CAG GCC AGA TTG GTG CTG GAT ATG 3’ 50 GCT GCC TTC TTC TCG ACT GCC TTG 3’

Fig. 17. Examples of chick embryos hybridized with sense RNA probes to evaluate the extent of nonspecific staining (cf. Figs. 12–16). A,D,F,I: Primitive streak stages. G,H: Head fold stage. B,E,J: The 3–4 somite stage. C: The 14-somite stage.

software, all experimental arrays were normalized to their matched control array. Genes were then filtered for at least 2 present calls out of 8 calls. The remaining list was tested for significant changes by ANOVA (P < 0.05). The raw and processed data are available (GEO accession number GSE11144).

RT-PCR and qPCR For RT-PCR analysis, whole, wild-type Xenopus embryos were snap-frozen at a series of developmental stages. Total RNA was extracted the with RNeasy mini kit (Qiagen). Semi-quantitative RT-PCR within linear ranges was performed as previously

DEVELOPMENTAL DYNAMICS

28 YAN ET AL.

described (Yan et al., 2009). Some primer sequences were obtained from published papers, whereas the others were designed with the Primer3 software (Rozen and Skaletsky, 2000; Table 10). Reverse transcription was performed with 1.0 mg of RNA using the SuperScript first-strand synthesis system (Invitrogen). Standard PCR amplifications were performed with PCR Supermix (Invitrogen) including 1.0 mCi a-[32P]dATP (Amersham) in the reaction mixture. All PCR assays were repeated at least three times with independent samples. Bands were visualized with a Storm 860 phosphorimager (Molecular Dynamics). For qPCR analysis, three independent sets of ACs derived from wild-type Six1 mRNA-injected and sibling control embryos were obtained as above, cultured to neural plate stages, homogenized in TRIzol (Invitrogen, Carlsbad, CA), and total RNA was isolated. Residual genomic DNA was removed using DNAse (Turbo DNAfree, Invitrogen), and random-hexamer primed cDNA generated using ImPromp-II reverse transcriptase (Promega). Reactions were assembled using a EpMotion 5070 liquid handling system (Eppendorf) that combine forward and reverse gene-specific primers (0.3 mM final concentration, Integrated DNA Technologies, Table 10), with 7.5 ml of SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA) in a 14-ml reaction. qPCR analysis was performed using a CFX-384 Real-Time PCR Detection System. Statistical differences were assessed by student’s t-test of the normalized CT values (delta-CT) for each independent analysis.

Whole-Mount In Situ Hybridization Plasmids encoding a subset of the putative downstream genes identified in the microarray assay were either obtained from the laboratory in which they were originally reported (listed in Tables 7, 8), or purchased from Open Biosystems (Thermo Scientific) or Source Bioscience. Anti-sense and sense digoxigenin-labeled RNA probes were synthesized by in vitro transcription (Ambion, Megascript kit). Frog and chick embryos were fixed and processed for whole-mount ISH according to standard protocols (Sive et al., 2000; Streit and Stern, 2001). Because some transcripts were low abundance, for some developmental stages the color reaction was allowed to develop up to 48 hours. For cleavage stage frog embryos, some were bisected after fixation to facilitate penetration of the reagents into the yolky vegetal cells during the ISH procedure. To visualize internal expression patterns Xenopus embryos already processed for whole-mount ISH were either bisected (blastula through neural plate stages) or embedded in 4% agarose and sectioned at 70 mm with a Vibratome (neural tube through larval stages); chick embryos were processed for standard paraffin sectioning.

Acknowledgments We thank Newt Moore and Himani Datta Majumdar for their technical assistance, and our many colleagues for providing Xenopus plasmids.

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