Introduction

Two decades ago, the first member of the signal-induced proliferation-associated (SIPA) protein family was discovered in proliferating lymphocytes as mitogen-induced nuclear protein (Hattori et al. 1995). This protein belongs to the SIPA protein family also known as spine-associated RapGAPs (SPAR) consisting of four members: SIPA1, SIPA1L1, SIPA1L2, and SIPA1L3 (Spilker and Kreutz 2010). This protein family is part of the Rap GTPase-activating protein (RapGAPs) superfamily that is known to be crucial for synapse morphology and function due to inactivation of Ras-related proteins. All SIPA members are large proteins with the following characteristic domains: an N-terminal RapGAP-, a PSD-95⁄Dlg⁄ZO-1 (PDZ) and a C-terminal coiled-coil (CC) domain carrying a leucin-zipper motif (Spilker and Kreutz 2010). SIPA1L1-SIPA1L3 contains an additional domain of unknown function (DUF3401) (Evers et al. 2015). It is well known that the PDZ domain is required for protein-protein interaction, whereas the function of the C-terminal coiled-coil domain is still unknown. Furthermore, SIPA1L1 contains two additional actin domains (Act1 and Act2) and a guanylate kinase-binding domain (GKBD).

All SIPA proteins are expressed in the central nervous system, and basically most of the studies focused on their function within the brain. In the past few years, however, several studies pointed to the role of SIPA1 in human diseases due to its potential pathological role in chronic myeloid leukemia as well as the lung, breast, or colon cancer. The role of SIPA1L1 has been investigated in dendritic spine formation and synapse plasticity mediated by the Rap-signaling pathway (Herrick et al. 2010). Furthermore, the SIPA1L1 protein could be linked to Ephrin4 receptor signaling. SIPA1L2 was found to be predominantly expressed in the granule cells of the gyrus dentatus in the hippocampus and the cerebellum (Spilker et al. 2008) and showed a potential interaction with ProSAPiP1 in the dendritic spines. In contrast, beside its expression in the rat brain (Dolnik et al. 2016), SIPA1L3 expression has been described in the embryonic lens and mutations in the SIPA1L3 gene could be associated with human congenital cataract (Evers et al. 2015; Greenlees et al. 2015; Lachke et al. 2012). Potential interaction partners of SIPA1L3 are ProSAPiP1, Lapser1, and PSD-Zip70 (Dolnik et al. 2016).

As expression data of the SIPA family members during early embryonic development are rare, we here present for the first time a wide and comparative expression analysis of all sipa family members during Xenopus laevis embryogenesis that provides a good basis for future functional studies.

Results and discussion

In silico analysis of X. laevis sipa protein family members

We started our study with an in silico analysis of all sipa gene family members. A synteny approach revealed each sipa family member on different chromosomes. Only in Rattus norvegicus, sipa1, and sipa1l3 were localized on the same chromosome. We found that all members of the sipa family displayed highly conserved neighboring genes in Homo sapiens, Pan troglodytes, Bos taurus, R.norvegicus, Mus musculus, and Xenopus tropicalis (Figs. 1a, 2a, 3a, and 4a). This is in line with our previous study already demonstrating sipa1l3 gene localization (Dolnik et al. 2016).

Fig. 1
figure 1

In silico analysis of sipa1. a Synteny analysis of the sipa1 gene and its neighboring genes in Homo sapiens, Pan troglodytes, Bos taurus, Rattus norvegicus, Mus musculus, and Xenopus tropicalis. Conserved genes are indicated by boxes with identical colors. The orientations of the open reading frames were depicted by arrowheads. Gene lengths are proportional to their actual size, but distances between the individual genes are not drawn to scale. b Phylogenetic tree of sipa1. Numbers demonstrate the genetic distance between the nodes of the phylogenetic tree which was generated by Clustal Omega and iTOL. c Scheme of the Sipa1 protein organization of X. tropicalis. The protein is composed of 1044 amino acids and contains an N-terminal RapGTPase-activating protein (RapGAP) domain, a PSD-95/Dlg1/ZO-1 (PDZ) domain and a C-terminal coiled-coil leucine zipper motif (CCLZ). d Homology of the full length (overall) amino acid sequence of Sipa1 and the conserved protein domains were compared across species. Numbers represent the similarities in percentage compared to Homo sapiens

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Fig. 2
figure 2

In silico analysis of sipa1l1 . a Synteny analysis of the sipa1l1 gene and its neighboring genes in Homo sapiens, Pan troglodytes, Bos taurus, Rattus norvegicus, Mus musculus, and Xenopus tropicalis. Conserved genes are indicated by boxes with same colors. The orientations of the open reading frames were depicted by arrowheads. Gene lengths are proportional to their actual size, but distances between the individual genes are not drawn to scale. b Phylogenetic tree of sipa1l1. Numbers demonstrate the genetic distance between the nodes of the phylogenetic tree which was generated by Clustal Omega and iTOL. c Scheme of the Sipa1l1 protein organization of X. tropicalis. Sipa1l1 protein is composed of 1778 amino acids and the conserved domains are the RapGTPase-activating protein (RapGAP) domain, a PSD-95/Dlg1/ZO-1 (PDZ) domain, a presumed DUF3401 domain of unknown function and the C-terminal coiled-coil region that harbors a leucine zipper motif (CCLZ). d Homology of the full length (overall) amino acid sequence of Sipa1l1, and the conserved protein domains were compared across species. The numbers represent the similarities in percentage compared to Homo sapiens

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Fig. 3
figure 3

In silico analysis of sipa1l2. a Synteny analysis of the sipa1l2 gene and its neighboring genes in Homo sapiens, Pan troglodytes, Bos taurus, Rattus norvegicus, Mus musculus, and Xenopus tropicalis. Conserved genes are indicated by boxes with same colors. The orientations of the open reading frames were depicted by arrowheads. Gene lengths are proportional to their actual size, but distances between the individual genes are not drawn to scale. b Phylogenetic tree of sipa1l2. Numbers demonstrate the genetic distance between the nodes of the phylogenetic tree which was generated by Clustal Omega and iTOL. c Scheme of the Sipa1l2 protein organization of X. tropicalis. Sipa1l2 protein is composed of 1593 amino acids, and the conserved domains are the RapGTPase-activating protein (RapGAP) domain, a PSD-95/Dlg1/ZO-1 (PDZ) domain, a presumed DUF3401 domain of unknown function and the C-terminal coiled-coil region that harbors a leucine zipper motif (CCLZ). d Homology of the full length (overall) amino acid sequence of Sipa1l2, and the conserved protein domains were compared across species. The numbers represent the similarities in percentage compared to Homo sapiens

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Fig. 4
figure 4

In silico analysis of sipa1l3. a Synteny analysis of the sipa1l3 gene and its neighboring genes in Homo sapiens, Pan troglodytes, Bos taurus, Rattus norvegicus, Mus musculus, and Xenopus tropicalis. Conserved genes are indicated by boxes with same colors. The orientations of the open reading frames were depicted by arrowheads. Gene lengths are proportional to their actual size, but the distances between the individual genes are not drawn to scale. b Phylogenetic tree of sipa1l3. Numbers demonstrate the genetic distance between the nodes of the phylogenetic tree which was generated by Clustal Omega and iTOL. c Scheme of the Sipa1l3 protein organization of X. tropicalis. Sipa1l3 protein is composed of 1690 amino acids, and the conserved domains are the RapGTPase-activating protein (RapGAP) domain, a PSD-95/Dlg1/ZO-1 (PDZ) domain, a presumed DUF3401 domain of unknown function and the C-terminal coiled-coil region that harbors a leucine zipper motif (CCLZ). d Homology of the full length (overall) amino acid sequence of Sipa1l3, and the conserved protein domains were compared across species. The numbers represent the similarities in percentage compared to Homo sapiens

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Moreover, phylogenetic analysis of each SIPA member demonstrated that all genes are evolutionary related in human, chimpanzee, bovine, rat, mouse, and frog (Figs. 1b, 2b, 3b, and 4b). All Sipa protein members of X. tropicalis contain an N-terminal RapGTPase-activating protein (RapGAP), a PSD-95/Dlg1/ZO-1 (PDZ) domain and a coiled-coil leucine zipper motif (CCLZ) which is present as a heptad repeat at the carboxy terminus. Sipa1l1, Sipa1l2, and Sipa1l3 harbors an additional domain of unknown function (DUF3401) (Figs. 1c, 2c, 3c, and 4c) first described in Evers et al. 2015. Full length proteins and all core domains are highly conserved across species (Figs. 1d, 2d, 3d, and 4d).

Temporal and spatial expression of the sipa family members

In order to analyze the temporal expression pattern of the sipa genes during early X. laevis embryogenesis, we performed semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments. We found that all four sipa genes were maternally supplied and expressed during early development (Suppl. Figure 1).

To elucidate the spatiotemporal expression pattern of all sipa genes, we performed whole mount in situ hybridization (WMISH) experiments using X. laevis embryos from stage 1 to 42. We cloned gene-specific Xenopus leavis complementary DNA (cDNA) fragments and generated antisense digoxigenin-labeled RNA probes. For validation purposes, we performed also WMISH approaches using sense digoxigenin-labeled RNA probes and Xenopus embryos at stage 31/32 (Suppl. Figure 2). For detailed tissue specific analysis, we performed vibratome sections of stained Xenopus embryos.

Endogenous sipa1 expression during early X. laevis development

Similar to the RT-PCR results, sipa1 transcripts could be detected at the animal pole of Xenopus embryos at early cleavage stages (Fig. 5(a–c)). During gastrulation, we found sipa1 positive cells at the animal side (Fig. 5(d–f), orange arrowheads) and in the invaginating mesoderm (Fig. 5(e–f), white arrowheads). At stage 13, sipa1 positive cells were accumulated at the dorsal side (Fig. 5(g), white arrowheads) and in the anterior neural plate (Fig. 5(h)) of the embryo. During neurulation at stage 15, we found sipa1 expression in the closing neural tube (Fig. 5(i–j), black arrowheads) and the profundal-trigeminal placodal area (pPrV). From stage 20 on, we noticed sipa1 transcripts predominantly in the cement gland (dotted circle), the olfactory placode, the developing eyes (red arrowheads), the migrating neural crest cells of the mandibular, hyoidal and branchial arches, and the neural tube as well (Fig. 5(k, l)). In addition, sipa1 messenger RNA (mRNA) molecules were visible in myeloid cells (Fig. 5(l, n, p)). Between stages 30 and 40, we detected sipa1 mRNA in the tailbud (Fig. 5(o), orange arrowhead), intersomitic arteries (Fig. 5(o–m)’), the posterior cardial vein (Fig. 5(o, q)), the vascular vitelline vain network (Fig. 5(q, l)’, black arrowheads), and external gills (Fig. 5(s)). Sections of stage 35 showed a strong expression of sipa1 in differentiated neurons of the brain (Fig. 5(a’, b’, e’)) and in several cranial ganglions such as the profundal (gPr; Fig. 5(b’)) and trigeminal ganglion (gV) (Fig. 5(c’)), as well as in the anterior (gAD) and fused ganglion of the glossopharyngeal and middle lateral line ganglion (IX/M) (Fig. 5(e’)). Moreover, we visualized sipa1 RNA in the internal gills (Fig. (5 f’)), the glomerulus adjacent to the pronephros (Fig. 5(i’, j’)), in the dorsal and ventral aorta (Fig. 5(n’)), as well as the dorsal (Fig. 5(k’), blue arrowheads) and ventral caudal lymph vessels (Fig. 5(n’)). Sipa1 mRNA was also notable in the second heart field (Fig. 5(r, t)) and endocardium of the heart tube (Fig. 5(g’, i’)). Vibratome sections through the developing eye at stage 35 revealed sipa1 expression predominantly in the ganglion cell layer of the retina and within the lens (Fig. 5(d’)).

Fig. 5
figure 5

Tissue specific expression of sipa1 during Xenopus embryogenesis. Upper part: WMISH to visualize sipa1. Embryonic stages are indicated. White dashed circles depict the cement gland; white dashed lines represent section levels shown in the lower part of the figure. a–d Sipa1 at the animal pole. e–f Vegetal view (e) and sagittal section (f) of a Xenopus embryo at stage 11. Sipa1 is weakly expressed in the ectoderm (orange arrowhead) and the invaginating mesoderm (white arrowheads). g + h Sipa1 in the dorsal side (arrowhead) and anterior neural tissue (an). i + j Sipa1 in the neural tube (arrowheads) and profundal-trigeminal placode (pPrV). k + l Sipa1 in the cement gland, olfactory placode (op), optic vesicle (arrowheads), mandibular- (ma), hyoidal- (ha), branchial (ba) arches, and myeloid cells (my). m, o, q, s Lateral views, anterior to the right. n, p, r, t Ventral views, anterior to the top. m–t Sipa1 in the eye (e), neural crest cells (ncc), intersomitic arteries (ia), posterior cardial vein (pcv), tailbud (orange arrowhead), otic vesicle (ov), vascular vitelline veins (vvn), second heart field (SHF), heart tube (h), and external gills (eg). Scale bars 500 μm (a–m), 250 μm (n, p, r, t), 1 mm (o, q, s). Lower part: transversal (a’–g’, i’–l’, n’), horizontal (h’, m’) sections stage 35 embryos. Dorsal view on top (b’–g’, i’–k’, n’). Posterior view on top (h’, m’). a’ Dorsal view left. Sipa1 in the forebrain (fb). b’–d’ Sipa1 in the profundal (gPr) and trigeminal ganglion (gV), midbrain (mb), ganglion cell layer (GCL), and lens (le). e’–g’ Sipa1 in the anterior (gAD) and fused ganglion of the glossopharyngeal and middle lateral line ganglion (IX/M), hindbrain (hb), branchial arches (ba), internal gills (ig), and endocardium (e). i’ + j’ Sipa1 in the brain (b), glomerulus (g), branchial arches (ba), and endocardium (e). k’ + l’ Sipa1 in the dorsal caudal lymph vessels (k’, blue arrowheads), vascular system (l’, black arrowheads) and neural tube (nt). h’ + m’ Sipa1 in the neural tube (nt) and intersomitic arteries (ia). n’ Sipa1 in the ventral caudal lymph vessels (vclv), dorsal (da), and ventral aorta (va). Nc indicates notochord; pt pronephric tubules, RPE retinal pigmented epithelium

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Not much is known about SIPA1 expression during early embryogenesis in other vertebrates. It has been shown to be expressed in adult mouse tissues including the spleen, bone marrow, brain, heart, lung, and testis. In addition, the thymus, liver, and lymphohematopoietic tissues revealed SIPA1 in adult and fetal mice (Hattori et al. 1995). Beside its expression, it has been reported that SIPA1 plays a role in the colon (Ji et al. 2012), breast (Zhang et al. 2015), and lung cancer (Gdowicz-Klosok et al. 2015). It would be important to elucidate the function of sipa1 during vertebrate embryonic development in future studies.

Endogenous sipa1l1 expression during early X. laevis development

Sipa1l1 was detected at the animal pole shortly after fertilization (Fig. 6(a, b)). During gastrulation, we found sipa1l1 transcripts in the invaginating ecto- and mesoderm (Fig. 6(c–d), orange arrowheads). At the end of gastrulation, sipa1l1 mRNA was distributed in the dorsal (white arrowheads) and anterior neural tissue (Fig. 6(e–g)). At stage 20, sipa1l1 was strongly expressed in the head region, in particular in the cement gland (Fig. 6(h)), the brain, the neural tube (white arrowhead), and the eyes (red arrowheads). At stages 25 and 30, sipa1l1 was clearly visible in the otic vesicle (Fig. 6(i, b’)); the olfactory placode (Fig. 6(j)); the neural crest cells of the mandibular, hyoidal, and branchial arches (Fig. 6(i)); the somites (Fig. 6(i, k, m, e’, f’, j’)); and the neural tube (Fig. 6(f’)). Furthermore, at early tailbud stages, sipa1l1 mRNA was detected in the glomerulus adjacent to the pronephros (Fig. 6(c’)); the first and second heart fields (Fig. 6(l, n, h’)); and within the peri-, endo-, and myocardium of the heart tube (Fig. 6(i’)). Additionally, we strongly visualized sipa1l1 transcripts in the optic cup and the lens at stage 30 (Fig. 6(a’)). At stage 35, sipa1l1 expression became manifested in the lens but could also weakly be shown in the future retina (Fig. 6(g’)).

Fig. 6
figure 6

Spatiotemporal expression of sipa1l1 during Xenopus development. Upper part: WMISH to detect sipa1l1. Embryonic stages are indicated. White dashed circles depict the cement gland; white dashed lines represent section levels shown in the lower figure part. a + b Sipa1l1 at the animal pole. c–d Vegetal view (c) and sagittal section (d). Sipa1l1 is strongly expressed in the invaginating ecto- and mesoderm (orange arrowheads). e Sipa1l1 at the dorsal side (white arrowhead). f + g Anterior (f), dorsal view (g). Sipa1l1 in the anterior (an) and dorsal (arrowhead) neural tissue. h Sipa1l1 within the eye (red arrowheads), brain (b), cement gland (cg), and posterior neural tube (white arrowhead). i, k, m, o, p Lateral views, anterior to the right. j, l, n Ventral views, anterior to the top. i–p Sipa1l1 in the anterior-dorsal part of the embryo, otic vesicle (ov), somites, (so), mandibular- (ma), hyoid- (ha) and branchial (ba) arches, eye (e), olfactory placode (op), first (FHF), and second heart field (SHF). Scale bars 500 μm (a–j), 1 mm (k, m, o), 250 μm (l, n, p). Lower part: Transversal sections at stage 30 ( a’–c’ ) and 35 (d’, g’–j’ ). Dorsal view to the top. e’ + f’ Horizontal sections at stage 30. a’–c’ Sipa1l1 in the brain (b), optic cup (oc), lens (l), otic vesicle (ov), and glomerulus (g). d’ Sipa1l1 in head mesenchyme (hm). e’ + f’ Sipa1l1 in somites (so) and neural tube (nt). g’–j’ Sipa1l1 in lens (le), retina (r), second heart field (SHF), myo- (m), endo- (e) and pericardium (p), and somites (so). Hg indicates hindgut, nc notochord

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In humans, SIPA1L1 is also known as E6-targeted protein 1 (E6TP1) (Gao et al. 1999). Northern blot analysis using several human tissues and different cell lines revealed SIPA1L1 expression in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (Pak et al. 2001) in line with our data. Furthermore, it has been reported that the Casein kinase I epsilon (CKIε) interacts with SIPA1L1 to phosphorylate SIPA1L1 resulting in the activation of Rap1 (Tsai et al. 2007).

Endogenous sipa1l2 during early X. laevis development

The first tissue-specific expression of sipa1l2 was detected early on at the animal side of the embryo (Fig. 7(a–b)). At stages 9–11, sipa1l2 transcripts were faintly detectable (Fig. 7(c–d)). After gastrulation, sipa1l2 mRNA was detected in the dorsal and anterior neural tissue (Fig. 7(f–g), white arrowheads). At stage 20, sipa1l2 was expressed in the olfactory placode, the profundal-trigeminal placodal area (pPrV), and the brain (Fig. 7(h), white arrowhead). During organogenesis, we found a wide distribution of sipa1l2 transcripts including the head region, neural crest cells, somites, the developing heart (first and second heart field), and intersomitic arteries (Fig. 7(i–p)). Moreover, vibratome sections showed sipa1l2 in the isthmus (Fig. 7(a’), black arrowhead), the differentiated neurons of the hindbrain (Fig. 7(c’)), the profundal ganglion (gPr) (Fig. 7(b’)), the lens as well as the inner nuclear cell layer (Fig. 7(e’)) of the developing eye (Fig. 7(i, o, j, k), red arrowheads) and the glomerulus nearby the pronephros (Fig. 7(d’)). Furthermore, sipa1l2 was located in the mandibular, hyoidal, and branchial arches at stage 35 (Fig. 7(f’)).

Fig. 7
figure 7

Sipa1l2 spatial expression pattern during Xenopus development. Upper part: WMISH to detect sipa1l1. Embryonic stages indicated. White dashed circles depict the cement gland; white dashed lines represent section levels shown in the lower figure part. a–c Sipa1l2 at the animal pole. d Vegetal view. Sipa1l2 is very weakly expressed in the invaginating ectoderm (white arrowhead) which is not visible on the sagittal section (e). f Sipa1l2 in the dorsal neural tissue (arrowhead). g Anterior view. Sipa1l2 in anterior neural tissue (arrowhead). h Sipa1l2 in the olfactory placode (op), profundal-trigeminal placode (pPrV), and brain (arrowhead). i, k, m, o Lateral views, anterior to the right. j Anterior view. l, n, p Ventral views, anterior to the top. i–p Sipa1l2 in the eye (e, red arrowhead), neural crest cells (ncc), somites (so), olfactory placode (op), brain (black arrowhead), the first (FHF) and second heart field (SHF), and intersomitic arteries (ia). Scale bar 500 μm (a–i); 1 mm (k, m, o); 250 μm (l, n, p), 100 μm (j). Lower part: Transversal sections at stage 30 (a’–d’ ) and 35 ( e’–g’ ). Dorsal view on top. a’–d’ Sipa1l2 in the isthmus (black arrowhead) and fore- (fb) and hindbrain (hb), profundal ganglion (gPr), presumptive retina (r), and glomerulus (g). e’ Sipa1l2 RNA molecules in the lens (le) and inner nuclear layer (INL). f’ Sipa1l2 in the branchial arches (m mandibular, h hyoidal, b branchial) and brain (b). Nc indicates nc notochord, ov otic vesicle, RPE retinal pigmented epithelium

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Nothing is known about the embryonic sipa1l2 expression in other organisms. In the adult rat, SIPA1L2 was found to be expressed in the brain and the heart. A weak expression could be visualized in the testis, the liver, and the lung (Nagase et al. 2000; Spilker et al. 2008).

Endogenous sipa1l3 during early X. laevis development

During early cleavage stages, sipa1l3 mRNA was observed in the animal pole of Xenopus embryos (Fig. 8(a–d)). During gastrulation, sipa1l3 transcripts were visible in the invaginating dorsal ecto- and mesoderm (Fig. 8(e–f), orange arrowheads). At stage 13, sipa1l3 mRNA was weakly expressed at the dorsal side (Fig. 8(g)). During neurulation, sipa1l3 was localized in the anterior (Fig. 8(h), white arrowhead) and dorsal neural tissue (Fig. 8(i), white arrowheads), as well as in the neural plate border (Fig. 8(h), black arrowhead). At stage 20, we found a sipa1l3 specific expression in the cement gland (Fig. 8, dashed circle), presumptive eyes (Fig. 8(j), white arrowhead) and within the profundal-trigeminal placodal area (pPrV) (Fig. 8(j)). During organogenesis, we found sipa1l3 transcripts in the olfactory, auditory, and the visual system, in particular the olfactory placode (Fig. 8(m, o, q, n, r, a’, c’, g’) (black arrowheads)), the otic vesicle (Fig. 8(p, r, b’)) and in the retina and lens (Fig. 8(i’, n’)) of the eye. Furthermore, we found a strong sipa1l3 localization in the neural crest cells of the developing mandibular, hyoidal, and branchial arches (Fig. 8(l, p, r, a’, m’)), the profundal (pPr), and trigeminal (gV) ganglion (Fig. 8(n, d’, e’, h’)), dorsal interneurons of the neural tube (Fig. 8(k, f’, k’), black arrowhead), and the isthmus as well (Fig. 8(g’), green arrowhead). Other sipa1l3 expression sites were the pronephros (Fig. 8(n, p, r, o’, p’)), somites (Fig. 8(l, n, r, l’)), the tailbud (Fig. 8(l)), the hindgut, hypaxial muscle cells, and external gills (Fig. 8(t)). In addition, a faint expression was detected in the dorsal caudal lymph vessels (Fig. 8(j’), blue arrowheads).

figure 8

Recently, SIPA1L3 has been described in the developing lens of mouse and zebrafish what is in line with our date (Greenlees et al. 2015; Lachke et al. 2012). Additionally, SIPA1L3 has been associated with human congenital cataract (Evers et al. 2015; Greenlees et al. 2015). In a previous study, we showed that SIPA1L3 is expressed throughout the postnatal rat brain in distinct regions and the postsynaptic density (PSD) to be consistent with our results (Dolnik et al. 2016). Further studies will be required to determine whether sipa1l3 expression and function in the early embryo is conserved across species.

Taken together, our study provides a detailed overview of the expression of all four sipa family members during early Xenopus development (Table 1). As first studies showed a crucial role for SIPA proteins in different organs and diseases, it would be of high interest to further investigate the function of all SIPA family members during embryonic development by performing loss and/or gain of function approaches.

Table 1 Comparison of gene expression of sipa family members during Xenopus development
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Experimental procedures

Embryo cultures

X. laevis embryos were generated according to previous published protocols (Sive et al. 2000). We cultured the embryos in 0.1 × MBSH buffer (1 mM HEPES, 8.8 mM NaCl, 0.1 mM KCl, 0.033 mM Ca(NO3)2 × (H2O)4, 0.041 mM CaCl2 × (H2O)2, 0.082 mM MgSO4 × (H2O)7, 0.24 mM NaHCO3) at 12.5 to 18 °C (Sive et al. 2000). We staged the embryos according to Nieuwkoop and Faber (Nieuwkoop 1956) and fixed with MEMFAT (0.5 M MOPS, 10 mM EGTA, 5 mM MgSO4 (H2O)7, 10 % formaldehyde, 0.1 % Tween) at the desired stage.

Cloning of X. laevis sipa1, sipa1l1, sipa1l2, and sipa1l3

For expression analysis of sipa members during Xenopus development, we cloned DNA fragments of 1037 bp (sipa1), 1097 bp (sipa1l1), 976 bp (sipa1l2), 1082 bp (sipa1l3, construct 1), and 903 bp (sipa1l3, construct 2) into the pSC-B vector (Stratagene, La Jolla, Ca). The primers were designed according to conserved sequences acrossed species and low homology within gene sequences. Following primers were used sipa1_forw: 5′-ATG CAG TCC GAT GAC CTC TTT G-3′; sipa1_rev: 5′- TGC TCA TCC AGT TTG AGG AGA GTT-3′; sipa1l1_forw: 5′- ATG ACC AGT TTG AAA AGG TCC CA −3′; sipa1l1_rev: 5′- GCA GCA GCA GAA GCA CCT GTT −3′; sipa1l2_forw: 5′- GCA GTG CCC AAA ATG GGT G − 3′; sipa1l2_rev: 5′- CCA TCT CCT TCA TCT GCT TCT GG −3′; sipa1l3_(1)_ forw: 5′- ATG CCA AAG ATG GGG GTG CGT −3′; sipa1l3 (1)_rev: 5′- GGG GTG CCA AGA GAT GCT TTA GAG AA −3′; sipa1l3_(2)_ forw: 5′-TAC CAG GAC TAC GAG ATA ATG TT-3′; and sipa1l3 (2)_rev:5′-CGC AGT GTC ATG TCC ACT GT-3′. For all PCR amplifications, we used cDNA from stage 25 and 37 X. laevis embryos and the proof reading PfuUltraTM II fusion HS DNA polymerase (Agilent Tech., Santa Clara, CA). Annealing temperatures were 54 °C for sipa1, sipa1l1, sipa1l2; 60 °C for sipa1l3 (construct 1); and 55 °C for sipa1l3 (construct 2). Insert orientation and sequences were confirmed by sequencing.

RT-PCR

In order to analyze the temporal expression of sipa1, and sipa1l1-1l3, total RNAs were isolated from X. laevis embryos at different developmental stages (1–42) using the peqGOLD RNAPure Kit (Peqlab, Erlangen, D) and following the manufacturer’s protocol. Reverse transcription was performed using cDNA with random primers and superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). RT-PCR was performed with the same set of cDNA using the Phire Hot Start II DNA polymerase (Thermo Scientific, Waltham, MA). Primer sequences, annealing temperature, cycle numbers, and product lengths for each sipa gene are provided in Suppl. Table 1.

Synteny analyses, phylogenetic tree, and protein alignment

For synteny analysis of sipa family members, chromosomal location and genomic structure in human, chimpanzee, bovine, rat, mouse, and frog were compared using the NCBI GeneBank. The Clustal Omega program from the EMBL-EBI homepage was used for multiple sequence alignment and calculations of the homology (Sievers et al. 2011). The iTOL program (http://itol.embl.de/) was used for phylogenetic tree generation based on Clustal Omega calculations (Letunic and Bork 2016). For amino acid sequence comparisons, the following sequences were used SIPA1: human NP_006738.3, chimpanzee XP_003313181.2, bovine NP_001095365.1, rat NP_001004089.1, mouse NP_001158040.1, frog XP_002935408.2; SIPA1L1: human NP_001271174.1, chimpanzee XP_510040.2, bovine XP_005212152.1, rat NP_647546.1, mouse NP_766167.2, frog XP_002936929.2; SIPA1L2: human NP_065859.3, chimpanzee XP_009439940.1, bovine NP_001193149.1, rat NP_001009704.1, mouse NP_001074806.1, frog XP_012819119.1; and SIPA1L3: human NP_055888.1, chimpanzee XP_524247.2, bovine XP_010825601.1, rat XP_008757353.1, mouse NP_001074497.1, frog XP_004917086.1. For domain identification, SMART, InterPro, ProDom, NCBI database, and Uniprot were used. For comparative analysis of the RapGAP-, PDZ, and DUF3401 domain, predicted regions from the NCBI database were used and for comparison of the coiled-coil domain between the different species predicted regions from InterPro software was used (Mitchell et al. 2015).

Whole mount in situ hybridization and sectioning

To investigate the spatial expression profile of the different sipa genes sipa1, sipa1l1-sipa1l3 during X. laevis embryogenesis, digoxigenin (DIG)-labeled antisense RNA probes were generated by in vitro transcription using either T7 or T3 RNA polymerase (Roche, Basel). Whole mount in situ hybridization analysis were performed as previously described (Hemmati-Brivanlou et al. 1990) and subsequently stained with BM Purple (Roche) or NBT/BCIP (Roche). BM purple stained embryos were bleached with 30 % H2O2, and pictures were recorded using a SZX12 microscope (Olympus). For detailed tissue analysis, NBT/BCIP stained X. laevis embryos were equilibrated in 1 ml gelatine/albumin solution (2.2 g gelatine, 135 g BSA, 90 g sucrose ad 500 ml 1×PBS) overnight at 4 °C and embedded in 1 ml gelatine/albumin solution with 75 μl glutaraldehyde (Fluka, Switzerland). Using a vibratome (Technical Products International Series 100), we performed sections with a thickness of 25 μm.