Smoc2 modulates embryonic myelopoiesis during zebrafish development

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Smoc2 Modulates Embryonic Myelopoiesis During Zebrafish Development Article in Developmental Dynamics · November 2014 DOI: 10.1002/dvdy.24164





5 authors, including: Camila V Esguerra

Ursula Hartmann

University of Oslo

University of Cologne





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DEVELOPMENTAL DYNAMICS 243:1375–1390, 2014 DOI: 10.1002/DVDY.24164


Smoc2 Modulates Embryonic Myelopoiesis During Zebrafish Development a

Hendrik Mommaerts,1 Camila V. Esguerra,1,2 Ursula Hartmann,3 Frank P. Luyten,1 and Przemko Tylzanowski1,4*


1 Laboratory for Developmental and Stem Cell Biology, Skeletal Biology and Engineering Research Center, Department of Development and Regeneration, KU Leuven – University of Leuven, Leuven, Belgium 2 Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven – University of Leuven, Leuven, Belgium 3 Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany 4 Department of Biochemistry and Molecular Biology, Medical University, Lublin, Poland

Background: SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. Results: In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Conclusions: Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. DevelopC 2014 Wiley Periodicals, Inc. mental Dynamics 243:1375–1390, 2014. V Key words: hematopoiesis; Sparc; Bmp; Alk8; Spi1b Submitted 10 January 2014; First Decision 14 June 2014; Accepted 2 July 2014; Published online 15 July 2014

Introduction SMOC2 is a member of the BM-40/osteonectin/SPARC (Secreted Protein Acidic and Rich in Cysteines) family of secreted proteins that do not contribute to extracellular matrix structures but rather regulate cell-matrix interactions (Bornstein, 2000). The SPARC family consists of several proteins (SPARC, hevin, fstl-1, testican 1/2/3, SMOC1, and SMOC2) and all members contain an extracellular calcium binding (EC) domain with 2 EF hands and a follistatin-like (FS) domain (Alliel et al., 1993; Charbonnier et al., 1998; Vannahme et al., 1999, 2002, 2003; Brekken and Sage, 2001; Hambrock et al., 2003, 2004; Schnepp et al., 2005). In contrast to other family members, SMOC1 and SMOC2 proteins have an additional putative SMOC-specific domain, flanked by 2 thy*Correspondence to: Przemko Tylzanowski, Laboratory for Developmental and Stem Cell Biology, Skeletal Biology and Engineering Research Center, Department of Development and Regeneration, KU Leuven – University of Leuven, Herestraat 49, 3000 Leuven, Belgium. E-mail: [email protected] Grant sponsor: European Commission Framework 7 program Grant; Grant numbers: 200800 TREAT-OA, KUL-GOA/2007/12, KUL-OT/99/ 27; Grant sponsor: Fund for Scientific Research Flanders; Grant number: G.0777.10; Grant sponsor: IUAP (InterUniversity Attraction Poles; DEVREPAIR); Grant sponsor: the Deutsche Forschungsgemeinschaft; Grant number: HA-2263/2-2

roglobulin domains (TY), separating the FS and EC domain (Vannahme et al., 2002, 2003). In mice, the earliest Smoc2 transcripts were detected in Reichert’s membrane at 8.5 dpc. At 12.5 dpc, Smoc2 mRNA was also detected in the developing facial prominences, limbs, and somites and at 14.5 dpc Smoc2 mRNA was additionally expressed in the heart, lung, and kidney (Maier et al., 2008; Feng et al., 2009). During later stages of development, SMOC2 was also detected in the spleen, thymus, ovaries, cartilage, muscle, and skin (Vannahme et al., 2003; Maier et al., 2008). Additionally, SMOC2 was predominantly found in nonbasement membranes (Vannahme et al., 2003; Maier et al., 2008). The biological function of SMOC2 has been addressed before. It has been shown that SMOC2 enhanced the angiogenic effect of basic fibroblast growth factor and vascular endothelial growth factor by stimulating mitogenesis and migration in human endothelial cells (Rocnik et al., 2006). In addition, SMOC2 mediates mitogenesis in mouse fibroblasts by stimulating cyclinD1 expression through regulation of integrin-linked kinase (Liu et al., 2008). Furthermore, SMOC2 was reported to interact with Article is online at: 24164/abstract C 2014 Wiley Periodicals, Inc. V




vitronectin and cell surface receptors of the integrin family and affect the migration of keratinocytes, the process of metastasis, and pulmonary function (Wilk et al., 2007; Maier et al., 2008; Novinec et al., 2008; Krol et al., 2010). Finally, exome sequencing, homozygosity mapping, microarray, and GWAS analysis associated human SMOC2 with vitiligo, as well as with dental and craniofacial defects. In zebrafish, these craniofacial defects were phenocopied after injection with smoc2 morpholino (Alkhateeb et al., 2010; Birlea et al., 2010; Bloch-Zupan et al., 2011; Alfawaz et al., 2013; Melvin et al., 2013). However, no early developmental function of Smoc2 has been investigated to date. Hematopoiesis in zebrafish occurs in successive waves in two anatomically distinct regions: the anterior and the posterior lateral plate mesoderm (ALPM and PLPM) (Ellett and Lieschke, 2010; Xu et al., 2012). Anteriorly, the mesoderm differentiates into cardiac progenitors (most caudal ALPM) and the rostral blood islands (RBI; most rostral ALPM). Posteriorly, the mesoderm fuses at the midline and forms the intermediate cell mass (ICM) (Al-Adhami and Kunz, 1977). Both the ALPM and PLPM contain bipotent hemangioblasts expressing multiple hematopoietic transcription factors such as tal1, lmo2, and gata2, and vascular transcription factors such as fli1 and flk1 (Detrich et al., 1995; Gering et al., 1998; Thompson et al., 1998). During primitive hematopoiesis (10–24 hpf), the anterior blood islands give rise to both endothelial (fli1) and myeloid progenitors (spi1b), whereas in the ICM erythroid progenitors (gata1) also develop (Detrich et al., 1995; Bennett et al., 2001; Lieschke et al., 2002). Due to the inhibitory action of spi1b on the expression of gata1 in the ALPM, erythropoiesis is repressed anteriorly (Galloway et al., 2005; Rhodes et al., 2005; Monteiro et al., 2011). Intermediate hematopoiesis (18 hpf) is the process when erythro-myeloid progenitors (EMPs) are formed in the most posterior part of the ICM, the posterior blood islands (PBI). Here, the EMPs are able to differentiate to both erythroid and myeloid cells (Bertrand et al., 2007). Anteriorly, myelopoiesis proceeds from the spi1b positive myeloid precursors and runx1, a spi1b transcriptional target, regulates the induction of the macrophage (irf8, csf1ra) or the neutrophil (c/ebp1, mpx) program (Rhodes et al., 2005; Su et al., 2007; Jin et al., 2012). At 30 hpf, multipotent hematopoietic stem cells appear in the ventral dorsal aorta, from where they migrate towards the caudal hematopoietic tissue (CHT). Around 4–5 days postfertilization (dpf) and after further migration into the kidney and the thymus, their final location, they are able to give rise to all hematopoietic lineages (Jin et al., 2009). Because smoc2 is expressed in the hematopoietically active domains during early zebrafish development (this study), we decided to investigate its function in the process of hematopoiesis. We carried out loss of function studies in zebrafish and could demonstrate that smoc2 is involved in myelopoiesis in the anterior lateral plate mesoderm.

Results Expression Analysis of smoc2 The mRNA expression analysis was performed using wholemount in situ hybridization (WISH) and reverse transcriptasepolymerase chain reaction (RT-PCR) (Fig. 1A–H). Using two nonoverlapping probes, smoc2 transcripts were detected from early stages onwards. Initially, smoc2 expression was not spatially restricted (Fig. 1A,B: shield). During segmentation and later

stages of development (9 ss - 30 hpf), the expression pattern became restricted to anterior structures, with a predominant expression in the retina, notochord, anterior somites, lateral epidermis, lateral hindbrain, cerebellum, dorsal midbrain, telencephalon, and diencephalon (Fig. 1C–G). Furthermore, an additional expression pattern was detected at 30 hpf in the epidermis of the tail (see inset in Fig. 1G). As zebrafish development progressed, the expression of smoc2 was in accordance with the available online data (data not shown and; (Thisse et al., 2001)). RT-PCR analysis indicated that smoc2 mRNA was present before the mid-blastula transition (MBT), suggesting the maternal contribution of the transcript in the early embryo (Fig. 1H).

Morphology of the smoc2 Morphant To investigate the function of smoc2 during early zebrafish development, we carried out the loss of function studies using two different morpholinos (MO): an ATG-MO, targeting the translation start site and a SPLICE-MO, targeting the first exon–intron boundary (Fig. 2A). As a control for the splicing of intron 1, an RT-PCR analysis was performed 24 hr after injection. With the first intron of smoc2 spanning over 50 kb, no amplicon could be generated, using the primers I and II annotated in Fig. 2A. Co-injection with in vitro synthesized smoc2 mRNA, however, did allow for the amplification of the fragment as in the control conditions (Fig. 2B). Morphological analysis of morphant embryos at bud stage did not reveal apparent phenotypic abnormalities (Fig. 2D,E). The first observable phenotypes were detected during somitogenesis. At 15 ss, posterior structures appeared compressed and reduced in length and the somites lost their typical chevron pattern (Fig. 2F,G). Additionally, the development of the morphants was delayed as compared to the control embryos. The time point when the first blood cells entered the circulation and started flowing through the dorsal aorta and the posterior cardinal vein was delayed by 2–3 hr (around 29–30 hpf in the morphants vs. 26–28 hpf in the control embryos). At that moment, the location of the heart, development of the ear and initiation of the pigmentation of the eye were the same in control and morphant embryos. Therefore, and to correct for heterochronicity, the time point when the first blood cells started circulating was chosen to be a proper landmark for staging the embryos and comparing their phenotypes. At the onset of blood cell circulation, the yolk sac extension (YSE) of the ATG-MO or SPLICE-MO injected embryos appeared reduced in length (Fig. 2H–J: colored lines) and the tail was kinked (Fig. 2H–J). Posterior from the YSE, the posterior blood islands (PBI) appeared to be enlarged and denser in the morphants as compared to the control embryos (Fig. 2H–J). Despite the likely maternal smoc2 mRNA contribution in the SPLICE morphants, no differences in phenotypes were detected. Closer investigation and measurement of the surface area of the PBI indicated a significant increase in their size in morphant embryos (Fig. 2K–M). As development progressed, the morphant defects became more pronounced with heart and yolk sac edema, heart defects similar to the heartstring phenotype (Garrity et al., 2002) and a compression of the anterior head structures along the anterior–posterior axis (data not shown). Co-injection of the smoc2 ATG-MO with smoc2 mRNA at the one-cell stage embryo resulted in a dose-dependent rescue of the morphant phenotype at the onset of blood cell circulation. Up to 63% of the embryos lacked morphant-specific defects (kinked tail, PBI expansion, reduced YSE-length) after injection with 200 pg of smoc2 mRNA (Fig.



Fig. 1. Expression analysis of smoc2. A–G: Expression pattern of smoc2 during the indicated stages of zebrafish development. From somitogenesis onward, transcripts were detected in anterior somites (as), anterior and ventral retina (r), dorsal diencephalon (d), telencephalon (t), dorsal midbrain (dm), lateral hindbrain (lh), cerebellum (c), lateral epidermis (le) and the notochord (n). H: RT-PCR analysis at indicated stages of development show smoc2 transcripts from early stages onward.

2C). Attempts to obtain a 100% rescue, with a higher concentration of smoc2 mRNA, were unsuccessful due to the high number of early gastrulation defects induced by the smoc2 mRNA (data not shown and Vuilleumier et al., 2010). Co-injection of the SPLICE-MO with smoc2 mRNA resulted in the same rescued phenotypes. In addition, a similar dose response was seen, although more embryos presented early gastrulation defects (data not shown). This early defect was probably due to the presence of maternal mRNA, which the SPLICE-MO is unable to target. In conclusion, injection of the smoc2 ATG-MO or SPLICE-MO resulted in similar mild ventralizations of the posterior tissues. The phenotypes of the morphants could be rescued by coinjecting smoc2 mRNA, indicating that both morpholinos are specifically targeting smoc2 mRNA.

Role of smoc2 During Hematopoiesis To confirm our morphological observation that the morphants had an expanded PBI, we performed WISH analysis at the onset of blood cell circulation using several PBI markers (gata1, ikaros, draculin (Fig. 3A–F), and hbbe1.1 [data not shown]) and could show that the transcription of these markers was apparently up-

regulated in the PBI of the morphants and confirmed the morphological observation of the increase in the size of the PBI. One explanation of this phenotype at a molecular level could be that an expansion of the PBI is the result of a modulation of Bmp signaling (Dal-Pra et al., 2006). Therefore, we analyzed the activation of the Bmp pathway in pregastrula smoc2 morphants by analyzing the expression of chordin, at this stage the dominant Bmp antagonist, vox and vent, two Bmp target genes, and the phosphorylation status of the Smad1/5/8 proteins. Yet, when comparing the smoc2 morphants to the control embryos, no change could be detected in either the expression domains or levels of chordin, vox, or vent, or the activation of the Smad1/5/8 proteins (Fig. 4A–P). Interestingly, mpx, a marker for both posterior and anterior or rostral blood islands (RBI), appeared down-regulated in smoc2 ATG morphants in both populations of blood cell progenitors (Fig. 3G,H) and co-injection with smoc2 mRNA rescued the loss of mpx transcripts in both the RBI and the PBI (Fig. 3J). This change in mpx expression was also seen in the SPLICE morphants (Fig. 3I,K) indicating that both smoc2 morpholinos act specifically on the same transcript resulting in similar morphological and molecular phenotypes. To limit the maternal contribution of smoc2 mRNA, we chose



Fig. 2. Morphological defects in smoc2 morphant zebrafish embryos. A: Diagram illustrating the design of the ATG-MO and the SPLICE-MO. B: RT-PCR at 24 hpf using primers I and II indicated in A. C: Dose response analysis after co-injecting smoc2 ATG-MO and smoc2 mRNA. D–L: Lateral view of control embryos (D,F,H,K) and smoc2 morphants (E,G,I,J,L) at bud stage (D,E; anterior to the top), 15 ss (F,G; anterior to the top) and at the onset of blood cell circulation (H–L; anterior to the left). smoc2 morphants showed a reduced axial length (F–J), loss of “v-shaped” somites (compare red lines in F and G), shortened yolk sac extension (compare colored lines in H–J), a downward curvature of the tail (H–J) and denser and enlarged blood islands (H–J and magnification of the dotted region in K and L). M: Quantification of the size of the PBI of control embryos (n ¼ 20), ATG morphants (n ¼ 29; *P < 0.05), and SPLICE morphants (n ¼ 18; *P < 0.05).



Fig. 3. Molecular defects in smoc2 morphant zebrafish embryos. A–K: Lateral view of control embryos (A,C,E,G), smoc2 morphants (B,D,F,H,I) and rescue condition (J,K), dorsal to the top at the onset of blood cell circulation. WISH analysis for gata1 (A,B), draculin (C,D), ikaros (E,F), and mpx (G–K).

to work with the ATG-MO in all further experiments, which prevents translation of both the maternal and the zygotic mRNA. As the PBI defect could be secondary to the defective anterior– posterior outgrowth of the embryo or due to changes in vascular wiring or blood flow, and as the morphants also presented an anterior phenotype—where smoc2 is predominantly expressed— we focused on the changes in the anterior domain.

smoc2 Morphants Present Reduced Hemangioblast Marker Expression To further explore the anterior myeloid defect, we investigated the status of the hemangioblast in smoc2 morphants by analyzing the expression of tal1, lmo2, and gata2, the molecular markers for these precursor cells (Fig. 5A–I,S–U). At 12 ss, WISH and qPCR analysis indicated a reduced expression level without an apparent change in the size of the domain of tal1. This reduction could be

rescued by co-injection with smoc2 mRNA supporting the notion that the tal1 phenotype was specifically induced upon knockdown of smoc2 (Fig. 5A–C,S). The expression of lmo2 was also significantly reduced upon decrease of smoc2 levels (Fig. 5D–F,T). Although no significant change in expression levels of gata2 was detected (Fig. 5G–I,U), the significant reduction in tal1 and lmo2 expression in smoc2 morphants supports the requirement for Smoc2 during hemangioblast development in the ALPM.

smoc2 Morphants Display a Reduction in Myeloid Progenitors Within the myelopoietic lineage, the hemangioblasts develop into spi1b positive granulocyte-myeloid progenitors. In the ALPM, spi1b induces its own repressor, runx1, thereby creating a negative regulatory loop (Jin et al., 2012). WISH and qPCR analysis for spi1b and runx1 at 12 ss showed a reduction in the expression



Fig. 4. smoc2 morphants do not display early dorsoventral patterning defects. A–F, I–P: Animal view, dorsal to the right. G,H: Lateral view, dorsal to the right. A–D, I–L, M–P: WISH analysis for the extracellular Bmp inhibitor chd (A–D) and the Bmp target genes vox (I–L) and vent (M–P). E–H: Immunostaining for phosphorylated Smad1/5/8 proteins.

domain and levels of both markers upon injection with smoc2 MO (Fig. 5J–O,V–W). For spi1b and runx1, the expression could be restored by co-injecting smoc2 mRNA. The expression of another early myeloid marker, c-myb, apparently changed when analyzed by WISH but the quantification by qPCR did not support this observation (Fig. 5P–R,X). At 18 ss, granulocyte-macrophage progenitor specific spi1b expression was still down-regulated in smoc2 morphants (Fig. 6A–C,S), as was lmo2 (Fig. 6D-F,T). In addition, runx1 and c-myb

showed a significant reduction in their expression in the smoc2 morphants both by WISH and qPCR (Fig. 6G–L,U,V). Co-injection with smoc2 mRNA resulted in a significant restoration of the expression levels of all markers, except for lmo2 (Fig. 6A–L,S–V). The expression of tal1, however, did not change in the smoc2 morphants at this stage (Fig. 6M–O,W). A reduction of Spi1b levels was previously shown to induce an ectopic expression of gata1 in the ALPM at 24 hpf (Rhodes et al., 2005; Monteiro et al., 2011). However, our WISH and qPCR



Fig. 5. smoc2 morphants display defects in hematopoiesis at 12 ss. A–R: Dorsal view of anterior lateral plate mesoderm; anterior to the top. WISH analysis for tal1 (A–C), lmo2 (D–F), gata2 (G–I), spi1b (J–L), runx1 (M–O), and c-myb (P–R) of control embryos, ATG morphants and rescue embryos at 12 ss (n  12). S–X: Quantification of changes in expression levels by qPCR. Values plotted as mean 6 SEM; n  4; *P < 0.05.

analysis did not indicate an ectopic anterior expression of this erythroid marker at the onset of blood cell circulation (Fig. 3A,B and data not shown). In summary, during the initial stages of hematopoietic development, the knockdown of smoc2 resulted in defective myeloid progenitor development.

A prerequisite for a smoc2-dependent regulation of the development of the hemangioblast and myeloid tissues is the expression of smoc2 in the proximity of the genes regulating the development of these tissues. An expression analysis of smoc2, tal1 and spi1b in relation to krox20, the marker for rhombomere



Fig. 6. smoc2 morphants display defects in hematopoiesis at 18 ss. A–R: Dorsal view of anterior lateral plate mesoderm; anterior to the top. WISH analysis for spi1b (A–C), lmo2 (D–F), runx1 (G–I), c-myb (J–L), tal1 (M–O), and irf8 (P–R) of control embryos, ATG morphants and rescue embryos at 18 ss (n  11). S–X: Quantification of changes in expression levels by qPCR. Values plotted as mean 6 SEM; n  4; *P < 0.05.

3 and 5, at the 5 ss, indicated a weak but detectable smoc2 expression in the anterior lateral epidermis in the proximity of the tal1 and spi1b expression domain (arrow in Fig. 7A–C).

smoc2 Morphants Show Defective Myelopoiesis At the end of segmentation stages, spi1b positive progenitors give rise to irf8 and csf1ra-expressing macrophages and c/ebp1-


smoc2 Morphants Display a Reduction in Bmp Target Gene Expression


Fig. 7. Expression pattern of smoc2 in relation to tal1 and spi1b. A– C: Dorsal view, anterior to the top at 5ss. Dual WISH for tal1 (A), spi1b (B), and smoc2 (C) with krox20, the marker for rhombomere 3 and 5 (n  20).

and mpx-expressing neutrophils. To investigate the effect of the early defects in smoc2 morphants on myeloid development, we performed WISH and qPCR analysis for these marker genes. The earliest myeloid marker, irf8, specific for developing macrophages, was significantly down-regulated in morphants at 18 ss (Fig. 6P–R,X). At the onset of blood cell circulation, the macrophage defect persisted, as the number of csf1ra-positive cells was reduced in the morphants compared with the control or rescue condition (Fig. 8A). No quantitative analysis was performed for csf1ra, as csf1ra at this stage also marks the neural crest-derived pigment cells, potentially masking the macrophage defect. The initial decrease in irf8 expression persisted, although at this stage it was not significant anymore (Fig. 8B,E). In addition, the neutrophils were affected by the reduction of the levels of smoc2. Specifically, the number of c/ebp1-expressing cells and mpx-expressing cells was reduced as was the overall expression level of c/ebp1 and mpx mRNA (Fig. 8C,D,F,G). This neutrophil defect persisted as the Tg(mpx:GFP)i114 transgenic reporter fish, used to study neutrophilic inflammation, still showed a significant reduction in the GFP positive cells as compared to the control embryos at 3 dpf (Fig. 8H,I) (Gray et al., 2011).

smoc2 Morphants do not Present Cardiovascular Defects During gastrulation, the interaction of multiple transcription factors (gata4, gata5, gata6, tal1, nkx2.5, and fli1 among others) induces differentiation of the ALPM into hematopoietic, vascular and cardiac progenitors (Peterkin et al., 2009). qPCR analysis of the smoc2 morphants for the mesodermal markers gata4, gata5, gata6, and ets1 did not indicate early mesodermal defects (Fig. 9). To assess possible defects in cardiovascular development, we examined the effect of reducing smoc2 levels on the expression of vascular and cardiac markers (Fig. 10A–R0 ). Consistent with our morphological observations, no ectopic or loss of expression was seen in the expression pattern of the cardiac marker nkx2.5 at 12 ss and 20 ss (Fig. 10A–D) or the vascular markers (fli, vegfc, flt4, flk1, dll4) at 12 ss, 20 ss and at the onset of blood cell circulation (Fig. 10E–P). Also, the analysis of the vascular Tg(fli:eGFP)y1 and the cardiac Tg(myl7:GFP) reporter line at 24 and 50 hpf, respectively, did not indicate a defect in cardiac or vascular development (Fig. 10Q,R0 and data not shown) (Lawson and Weinstein, 2002; Huang et al., 2003). In summary, smoc2 deficiency did not influence the differentiation or outgrowth of the cardiovascular system.

It has been reported that Alk8-mediated Bmp signaling is required for rostral spi1b expression and the subsequent specification of myeloid progenitor cells in the RBI (Hogan et al., 2006). Therefore we performed a qPCR analysis on the ALPM at 12 ss to evaluate the effect of smoc2 knockdown on members of the Bmp signaling cascade (Fig. 11A). The expression level of Bmp ligands bmp2b and bmp4 did not change significantly. Neither did the expression of the extracellular Bmp inhibitor, chd, nor the Bmp type I receptor alk8. However, the expression of Bmp target genes vox and ved, was significantly downregulated in smoc2 morphants. This reduction was partially rescued by co-injection of smoc2 mRNA supporting the observation that smoc2 knockdown could affect Bmp signaling without affecting the expression of the ligands, the receptor or its extracellular inhibitors. To confirm the qPCR result, we performed a WISH analysis for vox (Fig. 11B,C). At 12 ss, the expression of vox in the anterior, and medial structures appeared to be reduced upon injection with the smoc2 ATG-MO. Furthermore, the gradient at the posterior end of the embryo appears to be steeper when compared with the control condition (Fig. 11B,C). In summary, our results suggest that Smoc2 functions as a modulator of Bmp signaling as the expression levels and domains of Bmp target genes appear to be altered upon injection with a smoc2 morpholino.

Discussion In this study we investigated the role of smoc2 during zebrafish development. Our data show that smoc2 mRNA is predominantly expressed in the anterior structures of the postgastrulation zebrafish embryo and functions as a modulator of embryonic myelopoiesis in the ALPM. Previous studies on smoc2 morphants reported tooth and craniofacial defects at 5 dpf but did not report earlier developmental defects (Bloch-Zupan et al., 2011; Melvin et al., 2013). We have analyzed earlier stages of zebrafish development as smoc2 mRNA has a striking anterior expression pattern from gastrula stages onward and morphological defects in smoc2 morphants could already be detected at 15 ss. The difference in phenotypes could be due to different morpholino target sequences used in both studies. The morpholinos in the other studies targeted the 30 exon–intron boundaries, the 50 UTR or a splice variant with only the C-terminal EC domain of full-length smoc2 (Bloch-Zupan et al., 2011; Melvin et al., 2013). These strategies could result in a truncated, partially functional Smoc2 protein. Such a protein could have an altered activity or specificity, which could result in differential phenotypes. In our analysis we limited this possibility by designing morpholinos against the ATG translation start site or the first exon–intron boundary of full-length smoc2. Morphant analysis did not reveal gastrulation defects, whereas during segmentation stages the morphants were reduced in length and presented somite defects. At the onset of circulation, an apparent mild ventralization of posterior tissues was detected. Molecular analysis revealed anterior defects as early as 12 ss with a reduced expression of hemangioblast markers (tal1 and lmo2) and a defective specification of myeloid progenitors (spi1b). Later, at the onset of blood cell circulation, these defects translated into a reduction of macrophage- and neutrophil-specific markers (irf8, csf1ra, c/ebp1, and mpx).



Fig. 8. smoc2 morphants display defects in embryonic myelopoiesis at the onset of blood circulation. A–D: Lateral views of the embryos, anterior to the left, dorsal to the top, at the onset of circulation. A: WISH analysis or the macrophage markers csf1ra (A) and irf8 (B), and the neutrophil markers c/ebp1 (C) and mpx (D). E–G: qPCR analysis for irf8 (E), c/ebp1 (F) and mpx (G). Values plotted as mean 6 SEM; n ¼ 4; *P < 0.05. H,I: Visualization of the mpx-positive cells using the mpx reporter embryos (Tg(mpx:GFP)i114) at 3 dpf (H) and the quantification of the number of neutrophils in the ATG morphants (n ¼ 42) as compared to the control embryos (n ¼ 12; *P < 0.05) (I).


smoc2 Modulates the Expression of Bmp Target Genes

Fig. 9. smoc2 morphants do not display early mesodermal defects. qPCR analysis of the mesodermal markers gata4, gata5, gata6, and ets1. Values plotted as mean 6 SEM; n ¼ 4.


smoc2 Affects Hemangioblast and Myeloid Progenitor Development It has been reported that the reduction of hemangioblast markers, tal1 and lmo2, resulted in cardiovascular and hematopoietic defects (Dooley et al., 2005; Patterson et al., 2005). In contrast, spi1b morphants presented only myelopoiesis defects, without cardiovascular abnormalities (Rhodes et al., 2005; Patterson et al., 2007). We showed that smoc2 morphants did not display cardiovascular defects, despite the significant reduction of early tal1 and lmo2 expression in the ALPM. Potentially, restoration of tal1 expression levels at 20 ss in smoc2 morphants could compensate for the early loss of tal1 expression and therefore ensure proper cardiovascular differentiation at later stages of development. This compensatory mechanism however, appears to be unable to rescue spi1b expression and the spi1b-mediated myeloid defects in smoc2 morphants. Studies on Smad proteins in zebrafish indicated that the hemangioblast markers, tal1 and lmo2, are transcriptional targets of the Bmp signaling pathway and redundantly regulated by smad1 and smad5 (McReynolds et al., 2007). The reduction of the expression level of tal1 and lmo2 may therefore be a result of the reduced expression of Bmp target genes in smoc2 morphants. As spi1b expression was down-regulated in smoc2 morphants, and spi1b and smoc2 morphants present comparable myeloid phenotypes, we hypothesize that the smoc2 associated myelopoietic defects could be attributed to the reduced spi1b mRNA levels. Previous studies have shown that the induction of spi1b in hemangioblasts was dependent on alk8-mediated Bmp signaling (Hogan et al., 2006; Sumanas et al., 2008). Alk8, related to the vertebrate Alk2, is a type 1 Bmp receptor that mediates dorsoventral patterning in pregastrula stage zebrafish (Mintzer et al., 2001). The maternal zygotic mutant for alk8, lost-a-fin, displays a strong dorsalized phenotype. However, despite the caudal embryonic tissue being the primary affected tissue in both lost-a-fin mutants and smoc2 morphants, hematopoietic marker gene expression in the PLPM appeared unaltered during early developmental stages. Similar to smoc2 morphants, the lost-a-fin mutant has a reduced expression of spi1b, runx1, and c-myb in the ALPM during somitogenesis. Yet, the lost-a-fin mutant does not have reduced expression levels of tal1, indicating that the Bmp-dependent induction of tal1 happens independently of Alk8-mediated Bmp signaling, in contrast to the induction of spi1b (Hogan et al., 2006). As development progressed, both the loss of Smoc2 and Alk8 resulted in a reduction of macrophage- and neutrophil-specific markers (Hogan et al., 2006). Together, this suggests that Smoc2 might function as a specific modulator of the initiation of the myelopoietic program in the ALPM during early zebrafish development, potentially by modulating Bmp signaling.

Our analysis of the Bmp signaling pathway in the smoc2 morphants showed no apparent defects during the initial stages of development. At 12 ss, however, we showed a down-regulation of the Bmp target genes vox and ved in the ALPM of smoc2 morphants. This reduction in expression could not be attributed to changes in the expression levels of the Bmp ligands, bmp2b and bmp4, although it was reported previously that the expression of bmp2b in the pharyngeal teeth was reduced in smoc2 morphants at 56 hpf (Bloch-Zupan et al., 2011). Furthermore, no change in expression was detected for the extracellular inhibitor chd and the receptor alk8. In summary, this suggests that smoc2 might function as a modulator of the Bmp signaling pathway, which was reported to regulate myelopoiesis in the ALPM. Through what mechanism smoc2 affects the expression of Bmp target genes remains to be determined. Taking into account the importance of the Bmp signaling pathway during pregastrulation stages, the effect of the smoc2 knockdown is rather late and discrete. A possible explanation is that the morpholino approach is not 100% efficient. This suggests that some maternal smoc2 mRNA will be able to be translated into Smoc2 proteins during initial critical stages. Studies on murine SPARC, a SMOC2 family member, have shown that Sparc mRNA has a half-life of over 24 hr (Delany and Canalis, 1998). The half-life of zebrafish Smoc2 is unknown, but it is possible that, although smoc2 morpholinos are able to silence most of the smoc2 mRNA, the fraction of proteins that is translated in the morphants could persist for much longer and ensures proper initial development. This potentially also explains the lack of phenotypes seen in embryos with altered levels of Bmp signaling (Lieschke et al., 2002). As we have shown unaffected dorsoventral patterning in pregastrulation morphants, it is unlikely that the pregastrulation fate map, as suggested by the group of Layton is altered (Lieschke et al., 2002). We are, however, aware of the fact that the resolution of the techniques used in this study is limited and might not be sensitive enough to detect subtle differences that could alter cell fates. Previous studies on the D. melanogaster orthologue Pentagone (pent), the closest Smoc2 homologue in D. melanogaster showed that pent regulates the gradient of Bmp signaling by competing with the Bmp ligand Dpp, for binding to Dally, a heparan sulphate proteoglycan (HSPG). By doing so, Pent was suggested to promote ligand distribution, antagonize the co-receptor function of Dally, or regulate the interaction of Dpp and Dally (Vuilleumier et al., 2010, 2011). In zebrafish, overexpression of pent mRNA resulted in a range of dorsalizations with a loss of ventral markers and expanded expression of dorsal markers. As these processes were previously shown to be regulated by Bmp signaling, this supports the hypothesis that Pent modulates Bmp signaling (Vuilleumier et al., 2010). In addition, SMOC1 was previously shown to interact with heparan sulphate proteoglycans (HSPG) in mice, thereby regulating cell adhesion (Klemencic et al., 2013). Similar HSPG binding sites have been found in mouse and zebrafish Smoc2. This suggests that an interaction between HSPG and Smoc2 in zebrafish may exist and also that competition with Bmp ligands for HSPG could be possible. This hypothesis is strengthened by the fact that Pent functions upstream of the Alk8 receptor, as the overexpression of pent could rescue the bmp2b- but not the alk8-mediated ventralization. Of interest, zebrafish smoc2 gain of function resulted in similar dorsalized phenotypes, suggesting a functional conservation between pent



Fig. 10. smoc2 morphants do not display cardiovascular defects. Dorsal (A–D: anterior to the top; E–H: anterior to the left) and lateral view (I–R0 ; anterior to the left) of embryos at 12 ss (A,B,E,F), 20 ss (C,D,G,H), the onset of blood cell circulation (I–P) and 50 hpf (Q,R0 ). Analysis of the expression pattern of the early cardiac marker nkx2.5 (A–D), the vascular marker fli (E–H), the dorsal aorta marker vegfc (I,J), the marker for all venous and arterial cells flt4 (K,L), the endothelial marker flk1 (M,N) and the arterial marker dll4 (O,P) (n  14). Q–R0 : Visualization of the fli-positive vasculature using the (Tg(fli:eGFP)y1) reporter fish at 50 hpf.


It is tempting to speculate on how smoc2 regulates embryonic myelopoiesis in the anterior lateral plate mesoderm during zebrafish development. One possibility is that zebrafish Smoc2, just as Pent in D. melanogaster, modulates Bmp signaling by promoting the distribution of the ligand, antagonizing the co-receptor function of HSPGs, or regulating the interaction of Bmps and HSPGs. Hence, Smoc2 could affect the function of the Bmp ligands and shape the gradient of Bmp signaling that is required for proper specification of the hemangioblasts during early stages and the activation of the myeloid progenitor specific program at later stages of zebrafish development. However, more research is needed to investigate the relation between Smoc2 and Bmp, and to assess if there is a direct interaction, or if a secondary partner, like proteoglycans is involved.


Experimental Procedures Zebrafish Care and Manipulations 

Adult zebrafish (Danio rerio) were maintained at 28.5 C, on a 14/10 hr, light/dark cycle under standard aquaculture conditions as described ( All experiments were performed using embryos obtained from random matings of the wild type AB strain, the Tg(mpx:GFP)i114, the Tg(fli:eGFP)y1, or the Tg(myl7:GFP) transgenic line (Lawson and Weinstein, 2002; Huang et al., 2003; Gray et al., 2011). Embryos were kept in embryo medium and staged by hours postfertilization and the number of somites (somite stage or ss) according to defined criteria. For later stages, hours postfertilization or number of somites appeared to be unreliable to stage the embryos. Therefore, the initial appearance of circulating blood cells was chosen as a landmark of developmental progress. Diluted MO or diluted mRNA was injected in 1 cell stage embryos  in a volume of 1 nl. Injected embryos were maintained at 28.5 C and analyzed at the appropriate stage. Images were acquired with DMR Leica and Stereo Discovery V8 (Zeiss) microscopes. Fig. 11. smoc2 modulates Bmp signaling in the ALPM at 12 ss. A: qPCR analysis of the effect of the reduction of smoc2 levels on the expression of members of the Bmp signaling cascade (chd, bmp2b, bmp4, alk8, vox, and ved) in the ALPM. Values plotted as mean 6 SEM; n ¼ 4; *P < 0.05. B: Lateral view, anterior to the top; 12 ss. WISH analysis of the expression pattern of vox mRNA (n  25). C: Dorsal view, anterior to the left. WISH analysis of the expression of vox mRNA.

and smoc2 (our unpublished data and Hogan et al., 2006; Vuilleumier et al., 2010). Furthermore, the X. laevis orthologue, xsmoc1, was shown to act as a Bmp antagonist by means of Mapk-mediated phosphorylation of the receptor Smads. The mildly ventralized phenotype in the xsmoc1 morphants was shown to be rescued after co-injecting zebrafish smoc2 mRNA (Thomas et al., 2009), suggesting a functional conservation of the gene. However, as there is only one smoc gene in X. laevis, and two paralogues in zebrafish, chickens, mice and humans, it is difficult to make statements on conservation of function. It must be noted, however, that the phenotype of smoc1 morphant zebrafish is very different than that of smoc2 morphants, which are more similar to the ventralized xsmoc1 morphant defects (Thomas et al., 2009; Abouzeid et al., 2011; Rainger et al., 2011). In addition, gain of function of xsmoc1 and smoc2 result in similar dorsalized phenotypes as well (our unpublished results and Thomas et al., 2009; Vuilleumier et al., 2010).

Cloning of smoc2 ORF and Design of smoc2 MO The full-length ORF of smoc2 was cloned in the pCS2þ expression vector using primers designed by aligning EST GW712100, CK127336, EB944477, EB786386, and EB948756. - smoc2 ORF-F: 50 -TATATAATCGATATGCGCGTATCGGTG-30 - smoc2 ORF-R: 50 -ATAGCCTTGTTTCTTTGACAGGTTCAG-30 An ATG and a SPLICE morpholino (MO) against smoc2 were designed based on the genomic sequences NW_003040138.2, NW_001877353.3, NW_003336778.1 and purchased from GeneTools. - smoc2 ATG-MO: 50 -CGCATCCTCGCA GCTCCCCAGAAGC-30 - smoc2 SPLICE-MO: 50 -AAGGTGTTGTGACCCACCGTGAGCG-30 Both MOs were tagged with fluorescein, which allowed us to screen for green fluorescent signal in the injected embryos and hence control for injection efficiency. For injection of the transgenic zebrafish embryo, a nonfluorescent ATG-MO was used. MOs were resuspended at a stock concentration of 2 mM. Both morpholinos were injected together with a p53-MO to prevent off-targeting effects and the associated activation of p53-


mediated apoptosis (Robu et al., 2007). The working solution consists of 0.4 mM (3.5 ng) smoc2 MO and 0.05 mM p53-MO (0.39 ng; p53-MO: 50 -GCGCCATTGCTTTGCAAGAATTG-30 ). Capped mRNA was generated in vitro using the mMESSAGE mMACHINE SP6 Kit (Ambion) according to manufacturer’s protocol. RT-PCR as a control for the splicing of the first intron was performed with the primers indicated in Figure 2A: - primer I: 50 -GATGCGCGTATCGGTGCTG-30 - primer II: 50 -CTCTGCAACACACTTGGGCG-30


Whole-Mount In Situ Hybridization Antisense probes were generated from linearized plasmids using SP6, T3 or T7 polymerase and a DIG DNA labeling mix (Roche Applied Science, manufacturers protocol). WISH was performed using the following probes: tal1, gata2, lmo2 (McReynolds et al., 2007), spi1b, mpx (Yamauchi et al., 2006), c-myb, ikaros, runx1 (Kalev-Zylinska et al., 2002; Peterkin et al., 2009), irf8, csf1ra, c/ ebp1 (Li et al., 2011), dll4, flt4, vegfc (Hogan et al., 2009), myoD, vox, vent, chd, gata1, nkx2.5, hbbe1.1, draculin, fli, flk1, krox20 (in-house probes). After fixation in 4% PFA, dehydration and rehydration with graded methanol, probes were hybridized as described by the group of Thisse (Thisse and Thisse, 2008).

Quantitative PCR Embryos at 12 or 20 ss were fixed gently with 1% trichloroacetic acid (TCA) on ice for 10 min, followed by dissection of the anterior part of the fish by cutting at the level of the most anterior somites (Law and Sargent, 2013). Older embryos were clipped at the most rostral part of the yolk sac extension. Total RNA was isolated from 10–15 dissected stage-matched pooled embryos using the High Pure RNA Tissue Kit (Roche Applied Science; manufacturers protocol). At least four RNA isolations were performed per condition and subsequently reverse transcribed using Primescript RT Reagent (Takara; manufacturers protocol). Realtime PCR was performed in duplicate with gene specific primers (Supp. Table S1, which is available online) with SYBR Premix Ex Taq II (Takara; manufacturers protocol) using Rotor-gene 6000 detection system (Corbett Research, Westburg) (Supp. Table S1). Samples were confirmed to be anterior by checking anterior six3b expression and posterior charon expression. Gene expression was normalized to the housekeeping gene b-actin and presented as a ratio to control embryos. The significance of the difference in expression was analyzed using the Student’s t-test.

Immunohistochemistry After O/N fixation in 4% PFA, embryos were dechorionated and further processed as described by Tucker with minor modifications (Tucker et al., 2008).

Acknowledgments The authors thank Kathleen Lambaerts, Frederic Henderickx, Joris Vandenbempt, and Sophie Louwette for outstanding fish care and maintenance, Inge Van Hoven for technical assistance, and the lab members for the helpful discussions. Furthermore, the authors thank Roger Patient, Stephen Devoto, Zilong Wen, Nobuyuki Itoh, Todd Evans, Kathryn Crosier, and Stefan Schulte-Merker for pro-

viding us with in situ probes. H.M. is the recipient of a fellowship from the Agency for Innovation by Science and Technology (IWT).

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