The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish

MOD-03365; No of Pages 8 Mechanisms of Development xxx (2015) xxx–xxx

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The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish Jiaojiao Zhang a, Shipeng Yuan a, Aleksandr Vasilyev a,b,1, M. Amin Arnaout a,c,⁎ a b c

Leukocyte Biology & Inflammation Program, Division of Nephrology, Department of Medicine, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, United States Department of Pathology, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, United States Department of Developmental and Regenerative Biology, Harvard Medical School, Boston, MA 02115, United States

a r t i c l e

i n f o

Article history: Received 19 September 2014 Received in revised form 27 July 2015 Accepted 1 August 2015 Available online xxxx Keywords: Taz Pronephros Patterning Kidney cysts Zebrafish

a b s t r a c t The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction The nephron, comprising a blood filter (the glomerulus) and an epithelial tubule, is the basic structural and functional unit of the vertebrate kidney. It regulates solute and water homeostasis, excretion of metabolic waste and maintenance of acid–base balance (Al-Awqati, 2012). These distinct functions are fulfilled by segmentation of the nephron into defined regions that differ in both cellular anatomy and function. Recent studies in zebrafish have provided insights into the molecular pathways that direct the development and differentiation of the glomerular podocyte lineage (Gerlach and Wingert, 2013), but the factors that pattern the pronephric progenitor field along the anteroposterior (AP) axis and regulate tubule morphogenesis remain largely unknown, with only a handful identified to date (Wingert and Davidson, 2011; Wingert et al., 2007). The zebrafish pronephros is an excellent model for studying nephron patterning during embryogenesis. It comprises a pair of nephrons, with a single glomerulus and two pronephric tubules that fuse posteriorly at the

⁎ Corresponding author at: Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, United States. E-mail address: [email protected] (M. Amin Arnaout). 1 Present address: Department of Biomedical Sciences, New York Institute of Technology, Old Westbury, NY 11568, United States.

cloaca. During zebrafish development, the bilateral stripes of a renal progenitor cell field emerge from the intermediate mesoderm. By the 15-somite stage, renal mesenchymal progenitors situated on either side of the trunk adopt an epithelial cell morphology and undergo tubulogenesis (Drummond et al., 1998). Pronephric segment boundaries are established by 24 hpf in zebrafish embryos (Gerlach and Wingert, 2013). The most rostral nephron precursors, at the level of somite 3, give rise to glomerular cells including podocytes, and the pronephric tubules develop from the remaining nephron precursors. Several studies suggest that AP patterning of the pronephric progenitors is critically dependent on retinoic acid (RA) signaling, which promotes development of the proximal nephron identities (Wingert and Davidson, 2011; Wingert et al., 2007). Taz (also known as the WW containing transcription regulator 1, WWTR1), is a pivotal regulator of cell proliferation, cell differentiation, and stem cell renewal, particularly during organ growth and regeneration (Hong et al., 2005; Hong and Yaffe, 2006; Piccolo et al., 2013; Zhao et al., 2011). Loss of Taz in mice (Hossain et al., 2007; Makita et al., 2008; Tian et al., 2007; Varelas et al., 2010) or its knockdown in zebrafish embryos (Tian et al., 2007) also caused kidney cysts, suggesting that Taz plays an important role in kidney development, but how this is achieved remains unclear. In this communication, we reveal that Taz plays essential roles in AP patterning of the pronephric progenitor field in zebrafish embryos.

http://dx.doi.org/10.1016/j.mod.2015.08.001 0925-4773/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

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J. Zhang et al. / Mechanisms of Development xxx (2015) xxx–xxx

2. Results 2.1. Defective anterior migration of the pronephric tubule in taz morphants Knockdown of taz (WWTR1) in zebrafish was achieved using the antisense morpholino oligonucleotide tazMO, 5′-CTGGAGAGGATTACCGCT CATGGTC-3′, directed against the translation initiation codon of taz in zebrafish (Tian et al., 2007). Pronephric cyst and pronounced ventral body curvature seen in taz morphants were both reversed by coinjection of 1–2 cell stage embryos with tazMO plus RNA encoding full length wild type human taz but not taz where the WW domain was deleted (Supplemental Table 1 and Supplemental Fig. 1). To dissect the basis for pronephric cyst formation, we began by examining PCT development in control and taz morphants. In situ hybridization with the PCT-specific slc20a1a probe (Wingert et al., 2007) showed that PCT is normally positioned in taz morphants at 24 hpf,

but failed to extend anteriorly at 48 hpf and hence convolute at 72 hpf (Fig. 1A). At 24 and 36 hpf, the pronephric neck segments in controls (Fig. 1B a, c) and in taz morphants (Fig. 1B b, d) aligned similarly along the AP axis. By 48 hpf, the neck region in all control embryos reoriented along the mediolateral axis, with a smooth transition at the junction between the neck and PCT segments (Fig. 1B e). In contrast, reorientation of the neck region along the mediolateral axis was largely absent in 48 hpf taz morphants (Fig. 1B f). This failure in lateral extension of the pronephric neck and hence tube convolution in 48 hpf taz morphants was not caused by a primary defect in cell proliferation in the neck region, as there were no significant differences in the number of BrdU+ cells in the neck region between taz morphants and control embryos at 24 hpf (Fig. 1B g–j, Table 1), and at 36 hpf (mean = 24.8 BrdU+ cells per taz morphant, n = 10, vs. mean = 20.5 per control embryo, n = 10, p = 0.12) or at 48 hpf (mean = 21.3 BrdU+ cells per taz morphant, n = 13, vs. mean = 21.2 per control embryo, n = 9, p = 0.97).

Fig. 1. Anterior migration of PCT and morphogenesis of the neck segment are impaired in taz morphants. (A) In situ hybridization of embryos with the PCT-specific probe, slc20a1a. Note the absence of anterior migration and convolution of PCT in taz morphants. (B) Neck region morphogenesis is arrested in taz morphants. In situ hybridization using pax2a probe (red), followed by anti-BrdU staining (green) to visualize the neck region morphologies and detect proliferating cells in the neck regions in control embryos (a, c, e, g, i) and taz morphants (b, d, f, h, j). Note in f, the neck regions remained aligned with the A–P axis in the taz morphant, rather than repositioned to align with the mediolateral axis as in the normal embryo (e). (g, h) Individual optical sections of stacks. (i, j) Projected images of stacks. Anterior is to the top. Bars, 20 μm.

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

J. Zhang et al. / Mechanisms of Development xxx (2015) xxx–xxx Table 1 Number of BrdU-labeled Na+/K+ ATPase+ cells in pronephric domains strongly expressing pax2a and in the ET11-9 segment and at 24 hpfa. Treatment

Domain

No. of embryos

BrdU+ cells in domain per embryo (mean)

s.d.

P

control tazMO control tazMO

pax2a pax2a ET11-9 ET11-9

12 14 15 11

33.8 31.5 6.5 0.09

4.98 3.99 4.40 0.30

0.21 0.0001

a A representative experiment is shown, one of three independent experiments carried out, each using separate batches of non-sibling embryos.

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Table 2 Position and length of the ET11-9 segments relative to somite blocks at 24 hpfa. Treatment

No. of embryos

Start somiteb

End somiteb

Length

Control RA DEAB tazMO

8 8 7 14

9.7 (9–10) 11.9 (10–13) 8.2 (8–9) 11.9 (10–13)

14 (14) 20.2 (20–22) 12 (12) 15.1 (14–17)

5.3 (5–6) 9.8 (9–11) 4.8 (4–5) 4.3 (3–6)

a This is a representative experiment, one of four independent experiments each using different batches of non-sibling embryos. b Start and end somites mean starting at the anterior border of the aligned somite block and ending at the posterior border of the aligned somite block along the A–P axis, with an accuracy of half a somite block. Numbers represent average positions, and those in brackets represent ranges.

2.2. The PST-DE (ET11-9) segment is shortened in taz morphants In zebrafish embryos, anterior migration of pronephric tubules, which is followed by PCT convolution, is coupled to proliferation of cells in the distal ET11-9 (DE) segment (Vasilyev et al., 2012). We found that this segment, marked by GFP in ET(krt8:EGFP)sqet11-9 transgenic fish, is shortened in 24 hpf taz morphants, in association with an overall reduction in the trunk region (Fig. 2 and Table 2). In addition, the shortened ET11-9 segment shifted posteriorly relative to the adjacent somite blocks in 24 hpf taz morphants, as judged by double immunostaining using anti-GFP and MF20 antibodies that mark the ET11-9 segment and somites, respectively (Table 2). At 24 hpf, the ET11-9 segment in control embryos was 5 somites long, extending from the 10th to the 14th somite (Fig. 2A,B, Table 2). In contrast, the ET11-9 segment was 4 somites long in 24 hpf taz morphants, extending from the 12th to the 15th somite (Fig. 2C,D, Table 2). These data also suggested that shortening of this segment is caused by an intrinsic pronephric defect.

2.3. Decreased principal cell numbers in the ET11-9 segment in taz morphants Shortening of the ET11-9 segment in 24 hpf and 48 hpf taz morphants (Fig. 3A) could be caused by impaired cell proliferation. The ET11-9 segment contains two types of ciliated cells-monociliated principal cells (Na+/K+ ATPase+ CD41−) and multiciliated (CD41+ cells) (Vasilyev et al., 2009). We therefore counted the number of proliferating (BrdU+) principal cells (Na+/K+ ATPase+ BrdU+) in this segment. We found that there was a significant reduction in the numbers of these cells in 24 hpf taz morphants compared to controls (Fig. 3B, Table 1) (Supplemental movies 1–3). While the number of principal cells progressively increased in control embryos at 26 hpf (mean = 11.4 BrdU+ cells per embryo, n = 10) and at 28 hpf (mean = 19.3 BrdU+ cells per embryo, n = 10), they remained undetectable in the majority of taz morphants

Fig. 2. Length and position of the ET11-9 segments relative to their corresponding somite blocks in 24 hpf embryos. Anti-GFP staining of the ET11-9 segments (red) was followed by MF20 staining of somites (green). (A, B) Control embryo; (C, D) 24 hpf taz morphant. White arrows indicate position of the cloacae.

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

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Fig. 3. The ET11-9 segment is shortened in 24 and 48 hpf taz morphants. (A) The relative lengths of the ET11-9 segment in the normal pronephric tubule. The ET11-9 segment was stained first with anti-GFP (red), followed by α6F antibody (green). The α6F antibody stains the pronephric tubule and duct, but the red fluorescence staining of the ET11-9 segments blocked subsequent α6F green fluorescence staining, so the α6F appeared to stain the two tubular domains flanking the ET11-9 segment. The ET11-9 segments were shortened and PCT segments lengthened in 24 hpf taz morphants. The PCT segment also became dilated and the ventrally bent DL segments were slightly shortened in the 48 hpf taz morphant. (B) Reduced cell proliferation in the ET11-9 segments of the taz morphant at 24 hpf. (a, b) Individual optical sections; (c, d) projected images of stacks. Embryos were stained with anti-GFP (red), followed by anti-BrdU (green) staining of proliferating cells. Bars, 20 μm.

at 26 hpf (mean = 0.60 BrdU+ cells per embryo, n = 10) and at 28 hpf (mean = 0.62 BrdU+ cells per embryo, n = 13). To determine if the reduction in cell number is limited to principal cells, we also used tg(CD41:GFP) transgenic embryos stained with anti-GFP and anti Na+/K+ ATPase (α6F) antibodies to assess the number of multiciliated cells in the pronephric tubule. We found that the number of these cells per embryo at 48 hpf was not decreased in taz morphants, and they clustered in the shortened ET11-9 segment (Fig. 4, Table 3). In addition, pronephric cilia in the multiciliated cells of taz morphants appeared to function normally, as judged by their beating rate (mean ± sd = 22.2 ± 11.4 bps in taz morphants vs. 28.0 ± 5.8 in controls, p = 0.36). Thus, the overall reduction in BrdU+ cells in the ET11-9 segment first detected in 24 hpf taz morphants primarily affects the monociliated CD41− Na+/K+ ATPase+ principal cell population, and may be responsible for the shortening of this segment. As anterior migration of the pronephric tubule normally starts at 28 hpf, these data suggest that defective anterior migration and convolution of

the proximal segments are secondary to the shortening of the ET11-9 segment. 2.4. Defective proximodistal patterning of the pronephric tubule in 24 hpf taz morphants The primary abnormality in the length of the ET11-9 segment suggested that Taz, like RA, is involved in patterning the pronephric tubule along the AP axis. To determine if the two pathways interact functionally, transgenic ET(krt8:EGFP)sqet11-9 control embryos and taz morphants were treated with RA or DEAB starting at 90% epiboly, and nephron segmentation and segment boundaries were examined by double immunostaining of embryos using anti-GFP and α6F antibodies. RA treatment of control embryos caused proximalization of the pronephric tubule, manifested by lengthened PCT as well as ET11-9 segments, and shortened DL at 24 hpf (Fig. 5, compare C, D with A, B, Table 2). Treatment with the RA antagonist DEAB produced the opposite

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

J. Zhang et al. / Mechanisms of Development xxx (2015) xxx–xxx

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Fig. 4. Multiciliated cells in pronephric tubules of control embryos and taz morphants. Anti-GFP staining of the multiciliated cells (red) followed by α6F staining of the pronephric tubules (green). Note that in the 48 hpf taz morphant, the multiciliated cells were clustered in the shortened regions.

effects (lengthened DL at the expense of the proximal segments) (Fig. 5I, J vs. A, B and Table 2). At 24 hpf, taz morphants showed shortened ET11-9 and lengthened PCT (Fig. 5E, F, Table 2). With excess RA treatment of taz morphants, the PCT segment lengthened further to occupy most of the pronephric tubule by 24 hpf (Fig. 5G, H). DEAB treatment of taz morphants lengthened DL and shortened PCT at 24 hpf (Fig. 5M, N vs. K, L). The lengthened PCT in 24 hpf taz morphants became dilated at 48 hpf (Fig. 5T, Fig. 2A). A slight dilation of PCT was also observed in RA-treated control embryos at 24 hpf (Fig. 5, compare B and D). PCT dilation was dramatic in RA-treated taz morphants even at 24 hpf (Fig. 5H). DEAB treatment distalized the pronephric tubule in both control embryos and taz morphants (Fig. 5Q, R and U, V compared with O, P and S, T, respectively). Interestingly, it also reversed PCT dilation in taz morphants at 48 hpf (Fig. 5V vs. T). 2.5. Expression of taz in normal zebrafish embryos RA mediates AP patterning of the pronephric progenitor field near the end of gastrulation (Wingert and Davidson, 2008; Wingert et al., 2007). If Taz regulates AP patterning, as suggested in the above data, then it should also be expressed during the same period. RNA in situ hybridization of zebrafish embryos with antisense taz RNA showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors (Fig. 6A, B). taz mRNA was abundantly expressed in the developing somites (Fig. 6C), accounting for the shortened somite blocks in taz morphants. 3. Discussion Loss of taz has been shown to cause cystic kidney disease in zebrafish (Tian et al., 2007) and in mice (Hossain et al., 2007; Makita et al., 2008;

Table 3 Numbers of CD41+ multiciliated cells in a pronephric tubule at 48 hpf a. Treatment

No. of embryos

CD41+ cells in tubule per embryo (mean)

s.d.

P

Control tazMO

8 8

26.25 28.25

7.67 5.18

0.28

a

One representative experiment is shown, one of two independent experiments each using separate batches of non-sibling embryos.

Tian et al., 2007; Varelas et al., 2010).However, the nature of the defects in kidney development caused by taz deficiency has not been well defined. The main finding in this report is that taz is expressed at the bud stage in intermediate mesoderm where it plays an essential role in AP patterning of the zebrafish pronephric progenitor field, interacting functionally with the AP morphogen retinoic acid. Suppression of taz in the intermediate mesoderm, led to a marked shortening of the ET11-9 segment in 24 hpf taz morphants, which appears to result from a marked deficiency of principal cells in that segment, and proximalization of the pronephric tubule. Excess RA lengthened the proximal ET11-9 segment, and this effect required Taz, as it was seen when Taz was present but not when absent. Shortening of the ET11-9 segment in 24 hpf taz morphants was not only caused by the concomitant shortening of the somite blocks in the posterior trunk, but by a marked reduction in proliferating cells in this segment. The reduction in cell proliferation in 24 hpf taz morphants preceded the defect in the anterior migration of pronephric tubules, which normally starts after 28 hpf (Vasilyev et al., 2009), suggesting a cause and effect relationship. Impaired cell proliferation in the ET11-9 segment in 24 hpf taz was not symptomatic of a general reduction in cell proliferation, as cell proliferation in the proximal neck segment was not different from that in normal 24 hpf embryos. Two major epithelial cell types distributed in a mosaic pattern are found in the ET11-9 segment: monociliated solute transporter principal cells and multiciliated cells that facilitate forward luminal fluid flow. Differentiation of the two cell types into the respective cell fates in the intermediate mesoderm is dependent on the notch/jagged2a pathway, with knockdown of jagged2a in zebrafish driving progenitors into a multiciliated cell fate in 24 hpf morphants, at the expense of the principal cell fate (Liu et al., 2007; Ma and Jiang, 2007). We did not however find multiciliated cell hyperplasia in taz morphants (their number was similar in taz morphants and controls at 24 hpf), but rather a dramatic reduction in numbers of principal cells in 24 hpf taz morphants, suggesting that Taz promotes principal cell fate. This activity may involve interaction of Taz with the notch/jagged2a pathway, as has been shown for the Taz paralog YAP (Camargo et al., 2007), which also plays an essential role in early embryonic development (Hu et al., 2013; Loh et al., 2014). The pronephric progenitor field is patterned at the bud stage by an RA gradient, which is established by the anterior distribution of the rate-limiting RA biosynthesis enzyme retinaldehyde dehydrogenase 2 (Aldh1a2) in the presomitic mesoderm (Begemann et al., 2001) and the posterior expression of the RA metabolizing enzyme cytochrome

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

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J. Zhang et al. / Mechanisms of Development xxx (2015) xxx–xxx

Fig. 5. Effects of RA and DEAB on patterning of the pronephric tubule in control embryos and taz morphants. Visualization of pronephric tubule segments in 24 and 48 hpf zebrafish embryos by anti-GFP (red) staining of the ET11-9 segment, followed by α6F staining of the pronephric tubule (green). Panels show untreated (DMSO) control zebrafish embryos and taz morphants, as well as embryos treated with RA or DEAB in DMSO. RA treatment shortened the DL segments in both control (D) and 24 hpf taz morphant (H), but extended the ET11-9 segments in control embryo (D) but not in the 24 hpf taz morphant (H). DEAB extended the DL segments in both control (I, J) and 24 hpf taz morphants (M, N) as well as in control (Q, R) and taz morphants at 48 hpf (U, V), and reversed PCT dilation in 48 hpf taz morphants (V compared with T).

P450-26a1 (Emoto et al., 2005). A progressive reduction in the RA signal anteroposteriorly specifies the pronephric field into the proximal tubule components—neck, PCT and PST, with the very low levels posteriorly allowing DE–DL–PD progenitor specification (Wingert et al., 2007). RA deficiency, caused by genetic loss or chemical inhibition of Aldh1a2, disrupts the pronephric proximal fates with a concomitant expansion of the distal fates (Wingert and Davidson, 2011; Wingert et al., 2007). Conversely, exposure of zebrafish embryos to excess RA resulted in an expanded proximal domain at the expense of the distal domain (Wingert et al., 2007), as seen in our 24 hpf taz morphants. Exposure of taz morphants to RA further enhanced pronephric proximal fates, suggesting that Taz may normally act here as a negative regulator of RA signaling. Exposure of taz morphants to RA did not however extend

the ET11-9 segment at 24 hpf (compare Fig. 5G with control 5E), suggesting that Taz-dependent principal cell proliferation is necessary for RA pronephric patterning in this segment. Knockout of Taz in the mouse caused proximal tubule dilation (Hossain et al., 2007; Makita et al., 2008; Tian et al., 2007). Dilation of PCT, the main fluid secretory segment of the pronephros, in taz morphants likely results from proximalization of pronephric tubules. PCT dilation was observed in embryos treated with taz morpholino or exogenous RA, and was exaggerated when both were combined, at a time when the glomerulus has not yet formed, thus excluding the possibility that dilation is causally linked to glomerular filtration. Consistent with this interpretation is the observation that DEAB reversed pronephric proximalization and PCT dilation caused by the treatment

Fig. 6. taz mRNA expression in zebrafish embryos. In situ hybridization with antisense taz RNA. (A, B) Expression of taz in the pronephric progenitor field (white arrows in A and B) at the bud stage. (A) Lateral view, anterior is to the left, dorsal is up. (B) Posterodorsal view, anterior is to the left. (C) Expression of taz in somites but not in pronephric progenitor field at the 11somite stage.

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

J. Zhang et al. / Mechanisms of Development xxx (2015) xxx–xxx

of embryos with taz morpholino. Fluid accumulation through tubular secretion along a lengthened PCT may have contributed to PCT dilation in 24 hpf taz morphants, at a time when multiciliated cells have not yet developed. In Taz null mice, kidney cysts were reported to first appear in the juxtamedullary nephrons, sparing the outer cortical nephrons until late in the process (Hossain et al., 2007). The juxtamedullary nephron has a long U-shaped segment (loop of Henle, HL), which is primarily involved in urine concentration. HL, present in birds and mammals but is lacking in fish, is positioned between the proximal straight tubule (PST) and thick ascending limb (TAL, homologous to DE in zebrafish). The ET11-9 segment in zebrafish spans the PST and DE segments (this work and Wingert et al., 2007). It would be interesting to see if the corresponding region in juxtamedullary nephrons is also abnormally patterned and shortened in Taz null mice. The reduction in urine concentrating ability reported in Taz null mice (Makita et al., 2008) may be one outcome of such a patterning defect. 4. Methods 4.1. Fish strain and maintenance Zebrafish (Danio rerio) were maintained as described (Westerfield, 1993). Transgenic zebrafish lines ET(krt8:EGFP)sqet11-9 and tg(CD41:GFP) were kindly provided by Drs. Vladimir Korzh (Choo et al., 2006; Parinov et al., 2004) and Robert Handin (Lin et al., 2005), respectively. 4.2. Morpholino injection, RA and DEAB treatment Knockdown of taz in zebrafish was achieved with the previously used antisense morpholino oligonucleotide tazMO, 5′-CTGGAGAGGA TTACCGCTCATGGTC-3′, directed against the translation initiation codon of taz in zebrafish (Tian et al., 2007). tazMO and the p53-specific morpholino, p53MO (Robu et al., 2007) were purchased from GeneTools LLC (Philomath, OR). We injected tazMO (2.4 pmol) and p53MO (3 pmol) into the yolk of one-cell stage embryos. For morpholino rescue experiments, cDNAs encoding full-length wild-type human taz, or taz in which the WW-domain was mutationally deleted (ΔWW), were directionally cloned into pCS2+ plasmid, and sense RNA transcribed from the respective linearized pCS2+–Taz using the mMessage mMachine Kit according to the manufacturer's protocol (Ambion). The human cDNA used for rescue did not have any sequence complementary to tazMO, as judged by a comparison using BLAST, and sequences of the translation initiation sites in zebrafish and human taz are not conserved. 24 pg of RNA was injected into 1–2 cell stage embryos immediately following tazMO injection, and pronephric cyst formation and body curvature were assessed in embryos from two independent experiments. In experiments using RA and its antagonist DEAB (4diethylaminobenz-aldehyde), E3 medium was removed at 90% epiboly stage, and replaced with E3 medium containing RA (10−7 M) (SigmaAldrich, St. Louis, MO), DEAB (2 × 10−6 M) (Sigma-Aldrich, St. Louis, MO), or DMSO (0.1% v/v, as control). During the late somitogenesis stages, embryos were placed into fresh E3 medium and somite numbers were used to determine the stages of each treatment group. Somite numbers were counted again at 24 hpf to confirm the developmental stage. Because their somites were not well developed at the tail, stages for RA-treated control embryos and taz morphants were determined based on the time-matched DEAB-treated embryos of the same clutch. Embryos were fixed at the desired stage with Dent's fixative for immunofluorescence (IF) staining, or with 4% paraformaldehyde for RNA in situ hybridization. 4.3. In situ hybridization In situ hybridization was done according to the protocol of Thisse and Thisse (2008). Fluorescence in situ hybridization was done as

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described (Julich et al., 2005). Antisense probes used were taz, slc20a1a, and pax2a. After in situ hybridization with the taz probe, some embryos were embedded in JB4 (Polysciences, Warrington, PA) and sectioned with a Leica RM 2165 rotary microtome. Whole mount images of taz and slc20a1a in situ hybridization were taken with a Leica MZ16F microscope (Leica, Wetzlar, Germany), and section images were taken with a Nikon ECLIPSE E800 (Nikon, Tokyo, Japan). pax2a fluorescence in situ hybridization images were taken with a Zeiss Pascal LSM5 confocal microscope (Zeiss, Jena, Germany). 4.4. Immunofluorescence staining Embryos were fixed with Dent's fixative. The transgenic line ET(krt8:EGFP)sqet11-9 was used for staining the GFP+ tubular segment, ET11-9, which is at about the position of the PST-DE segment (Vasilyev et al., 2012). The transgenic line tg(CD41:GFP) was used for staining multiciliated cells (Vasilyev et al., 2009). Anti-GFP (Sigma-Aldrich, St. Louis, MO) was used as a primary antibody, followed by a horseradish peroxidase-labeled donkey anti-rabbit secondary antibody (Pierce, Rockford, IL). The fluorescence signal was developed with a TSA Plus Cy3 System (Perkin-Elmer, Boston, MA). For pronephric tubule and somite staining, α6F, directed against the α1 subunit of the Na+/K+ ATPase (DSHB, Iowa City, IA), and MF20, directed against the myosin heavy chain (DSHB, Iowa City, IA) were used respectively as the primary antibodies, followed by a donkey anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Images were taken with a Zeiss Pascal LSM5 confocal microscope (Zeiss, Jena, Germany). Numbers of multiciliated cells in the pronephric tubule domains were counted on the unprojected z stacks. Statistical analysis of the data was performed using the two-sample t-test. 4.5. BrdU labeling BrdU labeling was based on the method of Aman et al. (2011) with some modifications. Embryos of the desired stages were dechorionated manually, pre-cooled in ice cold E3 in an ice bath for 15 min, then soaked in ice cold E3 containing 15% DMSO and 10 mM BrdU (Sigma-Aldrich, St. Louis, MO) for 30, 35, or 40 min for the 24 hpf, 36 hpf, or 48 hpf embryos respectively. Embryos were rinsed with ice cold E3 three times, placed into pre-warmed E3 and incubated at 28.5 °C for 5 min, then fixed. Embryos were first subjected to fluorescence in situ hybridization or IF staining, and the fluorescent signals were developed with the TSA Plus Cy3 System (Perkin-Elmer, Boston, MA). Afterwards, DNA was denatured with 2 N HCl. The BrdU signal was detected with a rabbit anti-BrdU antibody (Rockland, Gilbertsville, PA), followed by a donkey anti-rabbit antibody conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR). Images were taken with a Zeiss Pascal LSM5 confocal microscope (Zeiss, Jena, Germany). The numbers of BrdU-labeled proliferating cells in the ET11-9 or the strongly expressing pax2a domains were counted by examining un-projected single planes in a stack layer by layer. Only a cell showing a positive correlation between BrdU and pronephric signal intensities in all the stack of planes harboring that cell was considered pronephric. The two-sample t-test was performed for data analysis. 4.6. Cilia beating rate The beating frequency of cilia was measured using a high-speed digital camera (Dragonfly Express, Point Grey Research Inc., Richmond, BC, Canada) fitted onto a Nikon ECLIPSE E800 microscope (Nikon, Tokyo, Japan). High-speed movies were analyzed in ImageJ by measuring mean pixel intensity within an elliptical region of interest around a cilia bundle. The entire stack measurement was performed using the “measure stack” plugin tool. The intensity values were imported into Excel, and average beat frequencies were calculated from 4 to 5 bundles using average inter-peak distances. The statistics were performed using the two-tail unpaired t-test with unequal variance.

Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001

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Please cite this article as: Zhang, J., et al., The transcriptional coactivator Taz regulates proximodistal patterning of the pronephric tubule in zebrafish, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.08.001