Evidence of Functional Redundancy Between MID Proteins: Implications for the Presentation of Opitz Syndrome

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Developmental Biology 277 (2005) 417–424 www.elsevier.com/locate/ydbio

Evidence of functional redundancy between MID proteins: implications for the presentation of Opitz syndrome

Alessandra Granata, Dawn Savery, Jamile Hazan, Billy M.F. Cheung, Andrew Lumsden, Nandita A. Quaderi*

MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Hospital Campus, London, SE1 1UL, UK

Received for publication 30 April 2004, revised 17 July 2004, accepted 8 September 2004 Available online 27 October 2004

Abstract

Opitz G/BBB syndrome (OS) is a congenital defect characterized by hypertelorism and hypospadias, but additional midline malformations are also common in OS patients. X-linked OS is caused by mutations in the ubiquitin ligase MID1. In chick, MID1 is involved in left–right determination: a mutually repressive relationship between Shh and cMid1 in Hensen’s node plays a key role in establishing the avian left–right axis. We have utilized our existing knowledge of the molecular basis of avian L/R determination to investigate the possible existence of functional redundancy between MID1 and its close homologue MID2. The expression of cMid2 overlaps with that of cMid1 in the node, and we demonstrate that MID2 can both mimic MID1 function as a right side determinant and rescue the laterality defects caused by knocking down endogenous MID proteins in the node. Our results show that MID2 is able to compensate for an absence in MID1 during chick left–right determination and may explain why OS patients do not suffer laterality defects despite the association between midline and L/R development. The demonstration of functional redundancy between MID1 and MID2 in the node provides supports for the hypothesis that partial functional redundancy between MID proteins in other developing structures contributes to the wide variability of OS phenotype. D 2004 Elsevier Inc. All rights reserved.

Keywords: Opitz syndrome; MID1; MID2; Functional redundancy; Phenotype rescue; Left–right asymmetry; Chick; Hensen’s node; Electroporation; Misexpression; Morpholinos

Introduction identical MID1 mutations (Cox et al., 2000), suggesting that other factors are modifying the OS phenotype. X-linked Opitz G/BBB syndrome (OS; OMIM 300000 Although OS is a rare disease, research into MID1 function and 145410), characterized by defective midline develop- is of significant clinical interest as MID1 represents one of ment, is associated with mutations in MID1 (Quaderi et al., the few genes associated with either Mendelian inherited 1997). The principal features of OS are hypertelorism mental retardation or cleft lip and/or palate. MID1 is a (abnormally wide distance between the eyes) and hypo- microtubule-associated member of the RBCC family of spadias (abnormal positioning of the opening of the proteins (also known as the TRIM family) characterized by urethra), but other midline defects, such as tracheoesopha- an N-terminal tripartite motif consisting of a RING finger, geal fistulas, cardiac malformations, and CNS defects, are one or two B-boxes, and a coiled-coil domain (Reymond et also common in OS patients (De Falco et al., 2003). There al., 2001). In common with other RING finger proteins is considerable clinical variability in the presentation of OS, (Joazeiro and Weissman, 2000), MID1 is a ubiquitin ligase: even among male patients from the same family sharing it specifically targets the microtubule-associated pool of the catalytic subunit of phosphatase 2A (PP2Ac) for degradation by binding to its alpha 4 (a4) regulatory subunit * Corresponding author. MRC Centre for Developmental Neuro- biology, King’s College London, 4th Floor New Hunt’s House, Guy’s (Trockenbacher et al., 2001). MID2, closely related to Hospital Campus, London, SE1 1UL, UK. Fax: +44 20 7848 6550. MID1, is also microtubule-associated (Buchner et al., 1999; E-mail address: [email protected] (N.A. Quaderi). Perry et al., 1999) and able to bind a4(Short et al., 2002;

Trockenbacher et al., 2001). It has therefore been proposed RACE–PCR using the SMARTTM RACE cDNA amplifi- that the variability of the OS phenotype can be attributed to cation kit (Invitrogen) and following manufacturer’s partial functional redundancy between MID proteins in instructions. The cMid2-specific primer used (cMid2.race3) tissues where their expression domains overlap (Short et has the following sequence: 5V-TCG CTG AGC GAC GCC al., 2002). ACT TGG TGA TCC-3V. RACE amplification products Improper development of the axial midline in vertebrates were cloned with the TOPO-TA cloning kit for sequencing can cause both midline and laterality defects, and the (Invitrogen) following supplier’s recommendations. involvement of the Sonic Hedgehog (SHH) and Nodal pathways in both midline and left–right (L/R) development Expression constructs and antisense morpholinos provides a molecular basis for this association (Roessler and Muenke, 2001). The hypertelorism characteristic of OS is The mouse Mid2 coding region was PCR-amplified reminiscent of the hypertelorism displayed by patients with using BamHI and ClaI tailed primers and cloned into the mutations in PTCH and GLI3, two known antagonists of the BamHI/ClaI sites of the pCAB expression vector to SHH pathway (Hahn et al., 1996; Johnson et al., 1996; generate pCAB.mMid2. The DR expression construct Vortkamp et al., 1991). This observation suggests that MID1 contains the portion of the cMid1 cDNA coding for may act as an intracellular antagonist of the SHH pathway, residues 73–667 cloned into the BamHI/ClaI sites of the prompting us to investigate whether MID1 plays a role in L/ pCAB expression vector. The B-box expression construct R determination. We have previously shown that the chick contains the portion of the cMid1 cDNA coding for MID1 orthologue, cMid1, is involved in establishing the residues 111–165 cloned into the BamHI/ClaI sites of the avian left–right (L/R) axis (Granata and Quaderi, 2003). L/R pCAB expression vector. The DC expression construct determination in chick involves the establishment of contains the portion of the cMid1 cDNA coding for asymmetric gene expression in Hensen’s node (Capdevila residues 1–307 cloned into the BamHI/ClaI sites of the et al., 2000; Mercola and Levin, 2001): a mutually repressive pCAB expression vector. All the pCAB expression interaction between Shh and cMid1 maintains asymmetric constructs detailed above contain an IRES-GFP cassette. gene expression in the node by restricting Shh to the left, and pCAB.cMid1 and the morpholinos cMid1.mo (renamed cMid1 to the right, of Hensen’s node (Granata and Quaderi, cMid.MO) and control.mo have been previously described 2003). The distinct signaling pathways on the left and right (Granata and Quaderi, 2003). sides of the node transmit L/R information to the lateral plate mesoderm (LPM) and then on to the organ primordia, Embryo culture and electroporations resulting in asymmetric morphogenesis (Capdevila et al., 2000; Mercola and Levin, 2001). Incorrect patterning of the Chick embryos were staged in accordance with L/R axis is potentially life-threatening as it results in the Hambuger and Hamilton (1951). A modified EC culture abnormal positioning and patterning of the visceral organs technique was used to transfer stage 4 embryos, dorsal (Capdevila et al., 2000; Mercola and Levin, 2001). side up with the vitelline membrane removed, into a Here, we have used the well-characterized molecular small electroporation chamber containing a positive cascades involved in avian L/R determination to address the platinum electrode in its base. Embryos were positioned issue of functional redundancy between MID1 proteins. We such that this electrode was on the left or on the right of show that cMid2 expression overlaps with that of cMid1 in Hensens node, as appropriate. pCAB.mMid2, pCAB.c- Hensen’s node and that MID2 can mimic MID1 as a right Mid1, pCAB.DR, pCAB.B-box, pCAB.DC, and side determinant. Furthermore, we find that MID2 is able to pCAB.GFP were applied using a glass micropipette at rescue the L/R patterning defects caused by knocking down 2 Ag/Al in 0.1% fast green for visualization. cMid.MO endogenous MID proteins expressed in Hensen’s node. The and control.MO were applied at 1 mM in 0.1% fast green demonstration of functional redundancy between MID for visualization. Electroporations were performed by proteins during avian L/R determination supports the placing a negative tungsten electrode over the DNA (or hypothesis that the variability of the OS phenotype can be morpholino) solution and applying four square pulses (5 attributed to partial functional redundancy between MID V, 50 ms) using an Intracept TSS10 dual-pulse isolated proteins. Our results also suggest a possible explanation for stimulator (Intracel, UK). Embryos were then grown the absence of laterality defects associated with OS. ventral side up as EC cultures (Chapman et al., 2001) until the appropriate stage. GFP/fluorescein fluorescence was used to monitor the position and efficiency of Materials and methods electroporation.

Cloning of 5V cMid2 cDNA Whole-mount in situ hybridization and immunostaining

5V Mid2 cDNA sequences were cloned from poly(A)+ A 758-bp cMid2 5V RACE clone was used as a template RNA derived from stage 13–15 chick embryos by 5V for riboprobe generation. Whole-mount in situ hybrid- A. Granata et al. / Developmental Biology 277 (2005) 417–424 419 ization and immunostaining were performed using standard expression is evident until stage 7 (Fig. 1F; Granata and techniques. Quaderi, 2003). The anterior crescent-shaped cMid1 expression domain (Fig. 1C; Granata and Quaderi, 2003) is not shared by cMid2 (Figs. 1B, D), and only cMid2 is expressed Results in the anterior primitive streak and extraembryonic tissue (Figs. 1B, G). At stage 8, both cMid1 and cMid2 are A single EST representing the chick MID2 orthologue, expressed in the neural plate, but cMid1 has a more cMid2, was identified in the BBSRC ChickEST Database posterior limit of expression (Figs. 1G and H). cMid1 (www.chick.umist.ac.uk; clone ID ChEST660F7); how- expression is excluded from the most medial (ventral) ever, this clone covers only part of the Mid2 coding region of the neural plate (Fig. 1H), but cMid2 expression is region and lacks both the 5V and 3V ends. Screening of a at a uniform level throughout the width of the neural plate stage 12–14 chick embryo cDNA library using the cMid2 (Fig. 1G). cMid1 expression in the somites and lateral plate EST clone only identified clones representing cMid1.5V mesoderm (Fig. 1H) is not shared by cMid2 (Fig. 1G). RACE PCR was performed on cDNA derived from stage We have previously shown that misexpressing MID1 on 13–15 chick embryos, and clones covering the cMid2 5V the left side of Hensen’s node results in ectopic expression coding region and UTR were recovered. This 5V cMid2 of right pathway markers (e.g., Fgf8; Boettger et al., 1999) cDNA sequence has been deposited in GenBank (acces- and repression of left pathway markers (e.g., Shh; Levin et sion number AY546077). We were unable to clone the 3V al., 1995)(Granata and Quaderi, 2003). To investigate cMid2 coding region. Whole-mount in situ RNA hybrid- whether misexpression of MID2 could also disrupt normal ization was used to determine the cMid2 expression pattern L/R molecular asymmetries in an analogous manner, we during early development (Fig. 1). Comparison of the focally electroporated an expression vector carrying a cMid1 and cMid2 expression patterns shows that they are mouse Mid2-IRES-GFP insert (pCAB.mMid2) into the left both expressed symmetrically in Hensen’s node at stage 4 side of Hensen’s node in stage 4 embryos (Fig. 2A). Mouse (Figs. 1B, C). cMid2 expression is not detected before this Mid2 was used as we were unable to clone the entire stage. cMid1 is first detected at stage 2 in a small, cMid2 coding region. Embryos were cultured to stage 6 symmetrical anterior domain (Fig. 1A). By stage 3, cMid1 andthenassayedforeitherShh or Fgf8 expression. expression is more intense and has expanded to form a Embryos electroporated with pCAB.mMid2 showed an crescent-shaped domain (Fig. 1E). cMid1/2 expression is absence of Shh expression in the node (n = 5/5; 100%; Fig. down-regulated on the left side of the node by stage 5 (Fig. 2C) or bilateral Fgf8 expression in the node (n = 6/7; 86%; 1D; Granata and Quaderi, 2003), and this asymmetric Fig. 2E). Control embryos electroporated with a vector

Fig. 1. Comparison of cMid2 and cMid1 expression during early development. (A) cMid1 expression is first detected at stage 2. (E) cMid1 is expressed in an anterior crescent-shaped domain at stage 3. (B) cMid2 expression is first seen in Hensen’s node and the anterior primitive streak at stage 4. (C) cMid1 is expressed in Hensen’s node and in a crescent-shaped ectodermal domain at stage 4. (D) By stage 5, cMid2 expression is confined to the right side of the node. (F) cMid2 expression in the node is still asymmetric at stage 7. (G) By stage 8, cMid2 is down-regulated in the node. Expression is seen in the neural plate and extraembryonic tissue. A transverse section through the neural plate, at the level of the black line, is shown. (H) cMid1 is expressed in the neural plate, somites, and lateral plate mesoderm at stage 8. Transverse sections through the neural plate and somites, at the level of the black lines, are shown. High magnification inserts of the node show the small changes in gene expression. Embryos are shown dorsal side up. L, left; R, right. 420 A. Granata et al. / Developmental Biology 277 (2005) 417–424

Fig. 2. MID2 acts as a right-side determinant. (A) A Mid2 expression vector was focally electroporated into the left side of the node in stage 4 embryos. (B) Control GFP electroporations have no effect on Shh expression. (C) Mid2 electroporations result in a loss of Shh expression in the node. (D) Control GFP electroporations have no effect on Fgf8 expression. (E) Mid2 electroporations result in ectopic Fgf8 expression on the left side of the node. The node is outlined in black. High magnification inserts of the node highlight the small changes in gene expression. Embryos are shown dorsal side up. L, left; R, right. expressing only GFP (pCAB.GFP) showed no change in pCAB.mMid2 to try and rescue the laterality defects caused Shh (n = 4/4; Fig. 2B; also Granata and Quaderi, 2003)or by treatment with cMid.MO. cMid.MO was focally electro- Fgf8 (n = 4/4; Fig. 2D; also Granata and Quaderi, 2003) porated into the right side of Hensen’s node at stage 4 (Fig. expression. These results indicate that MID2 is able to 4A). Embryos were cultured to stage 6, fixed, and assayed mimic MID1 function as a right side determinant, with for either Fgf8 or Shh expression. Fgf8 expression was respect to Shh and Fgf8 expression, when misexpressed on absent from the node (n = 5/5; 100%; Fig. 4B; also Granata the left side of Hensen’s node. and Quaderi, 2003), and Shh was expressed bilaterally in the Knocking down right-sided MID1 expression in the node node (n = 5/5; 100%; Fig. 4H; also Granata and Quaderi, using an antisense morpholino, cMid1.mo, inhibits the 2003) in these embryos. No change in normal Fgf8 (n = 6/6; expression of right pathway markers and induces the ectopic 100%; Fig. 4C; also Granata and Quaderi, 2003)orShh (n = expression of the left pathway on the right side of the 5/5; 100%; Fig. 4I; also Granata and Quaderi, 2003) embryo (Granata and Quaderi, 2003). cMid1.mo was expression was seen in embryos electroporated with a designed to target the first 25 coding nucleotides of the control morpholino (control.MO). Co-electroporation of cMid1 cDNA sequence. Comparison of 5V cMid1 and pCAB.GFP and cMid.MO has the same effect as electro- cMid2 sequences shows that there are only 2/25 mismatches porations with cMid.MO alone. Co-electroporated embryos in this region between cMid2 and cMid1.mo (Fig. 3). At show an absence of Fgf8 expression in the node (n = 4/5; least 4/25 mismatches are required to prevent efficient 80%; Fig. 4D) and bilateral Shh expression in the node (n = binding of a morpholino to a target sequence (www.gene- 5/5;100%; Fig. 4J). However, co-electroporation of pCAB. tools.com). Therefore, it is probable that cMid1.mo (from mMid2 with cMid.MO results in the rescue of the molecular now on referred to as cMid.MO) binds to both cMid1 and laterality defects seen with electroporation of cMid.MO cMid2, thereby blocking the translation of both MID alone: Fgf8 expression is restricted to the right side of the proteins. Consequently, the laterality defects caused by node (n = 8/10;80%; Fig. 4E), while Shh expression is focally electroporating cMid.MO into the node could be the restricted to the left side (n = 8/9;89%; Fig. 4K). Co- result of knocking down both MID1 and MID2 in the node, electroporation of pCAB.mMid2 and control.MO on the rather than MID1 alone. If this is the case, and if MID1 and right side of the node in stage 4 embryos results in right- MID2 are functionally redundant with respect to L/R sided Fgf8 expression (n = 4/4; 100%; Fig. 4F) and left- determination, overexpression of MID2 on the right side sided Shh expression (n = 4/4; 100%; Fig. 4L) in the node at of the node should be able to rescue the L/R defects caused stage 6, indicating that overexpression of MID2 on the right by cMid.MO treatment. If, however, there is no functional side of the node in the presence of normal background levels redundancy between MID1 and MID2, overexpression of of MID1/2 does not disrupt correct L/R determination. The MID2 should have no effect on the L/R defects caused by typical extent of overlap between pCAB.cMid2 (or cMid.MO treatment. In order to investigate whether MID2, pCAB.GFP) expression and cMid.MO (or control.MO) in the absence of MID1, is sufficient for correct establish- transfection at stage 6 in co-electroporated embryos is ment of the L/R axis, we have used the expression vector shown (Fig. 4G). Co-electroporations using pCAB.cMid1, rather than pCAB.cMid2, with cMid.MO also showed normal Fgf8 and Shh expression, indicating that the presence of either MID protein in the node is sufficient for the correct establishment of the L/R axis (data not shown). Fig. 3. 5V cMid1 and cMid2 sequences are highly conserved. There are only MID1 and MID2 are members of the RBCC family of 2/25 mismatches in the first 25 coding nucleotides between cMid1 and proteins. In addition to the RING finger, B-boxes, and cMid2. The amino acid sequence is perfectly conserved in this region. coiled-coil domain that characterize this family, the MID A. Granata et al. / Developmental Biology 277 (2005) 417–424 421

Fig. 4. MID2, in the absence of MID1, is sufficient for correct L/R patterning. (A) Expression constructs and morpholinos were electroporated into the right side of the node at stage 4. (B) Electroporation of cMid.MO results a loss of Fgf8 expression in the node. (C) Electroporation of control.MO has no effect on Fgf8 expression. (D) Co-electroporation of pCAB.GFP and cMid.MO results in a loss of Fgf8 expression in the node. (E) Co-electroporation of pCAB.mMid2 and cMid.MO results in the rescue of right-sided Fgf8 expression in the node. (F) Co-electroporation of pCAB.mMid2 and control.MO does not disrupt normal Fgf8 expression in the node. (G) Overlap between cMID.MO and pCAB.mMid2 in stage 6 co-electroporated embryos. (H) Electroporation of cMid.MO results in ectopic Shh expression on the right side of the node. (I) Electroporation of control.MO has no effect on Shh expression. (J) Co-electroporation of pCAB.GFP and cMid.MO results in ectopic Shh expression on the right side of the node. (K) Co-electroporation of pCAB.mMid2 and cMid.MO results in the normal confinement of Shh expression to the left side of the node. (L) Co-electroporation of pCAB.mMid2 and control.MO does not disrupt normal Shh expression in the node. The node is outlined in black. High magnification inserts of the node highlight the small changes in gene expression. Embryos are shown dorsal side up. L, left; R, right. proteins also contain a conserved C terminal domain of degradation of PP2Ac are seen to be MID1-dependent unknown function (B30.2 domain) shared by a subset of (Trockenbacher et al., 2001). RBCC proteins (Reymond et al., 2001). Human MID1 and The vast majority of OS-associated mutations disrupt the MID2 show an overall level of 84% similarity and 77% MID1 C-terminus, leaving the N-terminal tripartite motif identity at the amino acid level (Buchner et al., 1999). intact. Conflicting theories exist as to whether OS is caused Experiments performed in vitro have provided insight into by gain-of-function mutations in MID1, through deregulated the roles of the various domains of the MID proteins. Most activity of the intact tripartite motif (Schweiger et al., 1999) OS-causing MID1 mutations lie within the C-terminal and or by loss-of-function mutations in MID1 (Cox et al., 2000). are associated with a loss of MID1 microtubule local- We have generated a MID1 expression construct lacking the ization, indicating that microtubule association is mediated C-terminus (pCAB.DC; Fig. 5) in order to test in vivo through the C-terminal and is essential for correct MID1 whether the isolated MID1 tripartite motif behaves as a function (Cainarca et al., 1999; Cox et al., 2000; De Falco gain-of-function isoform. We have also generated two et al., 2003; Gaudenz et al., 1998; Quaderi et al., 1997; further MID1 deletion constructs (pCAB.DRand Schweiger et al., 1999). The coiled-coil domain has been pCAB.B-box; Fig. 5) to test whether the in vivo function shown to mediate MID protein dimerization: both MID1 of MID protein domains is consistent with the observed in and MID2 are able to homodimerize, but there is vitro biochemical function. If the in vitro model for MID conflicting evidence regarding the ability of MID proteins function is correct, we can predict that both these constructs to heterodimerize (Cainarca et al., 1999; Short et al., will behave as dominant negatives when electroporated into 2002). The a4 regulatory subunit of PP2A selectively MID-expressing cells as they should both block the binds to the B-box 1 sequence of both MID1 and MID2 targeting of PP2Ac for ubiquitin-mediated degradation: the and does not bind to B-box sequences derived from any DR construct blocks recruitment of an E2 enzyme, and the other RBCC proteins (Liu et al., 2001; Short et al., 2002; B-box construct blocks binding to a4. The DR construct Trockenbacher et al., 2001). A variety of RING finger lacks the RING finger domain (Fig. 5). This construct containing proteins has been shown to act as E3 ubiquitin should be able to bind a4 (through B-box 1) and localize to ligases by binding to both E2 ubiquitin-conjugating microtubules (through the C terminal domain). However, as enzymes, via the RING finger domain, and the target this construct lacks a RING finger, it is unable to bind to an protein destined for degradation (Joazeiro and Weissman, E2 enzyme. In vitro binding studies have shown that the 2000). Although an E2 enzyme has yet to be identified that dnakedT B-box 1 sequence has a much greater affinity for a4 binds to MID proteins, the ubiquitination and subsequent than the B-box 1 sequence in the context of a MID protein 422 A. Granata et al. / Developmental Biology 277 (2005) 417–424

Fig. 5. MID protein domains. Cartoon showing the content of the three MID1 deletion constructs.

(Trockenbacher et al., 2001). Overexpression of the B-box function construct: Fgf8 expression is restricted to the right construct into MID-expressing cells is predicted to out- (n = 12/12; 100%; Fig. 6E), and Shh expression is compete both MID1 and MID2 in binding a4 and, in restricted to the left (n = 9/9; 100%; Fig. 6K). Embryos common with the DR construct, prevent the ubiquitination co-electroporated with DC and cMid.MO also show and subsequent degradation of PP2Ac. normal Shh and Fgf8 expression (data not shown). These The cMid1 expression vector, pCAB.cMID1, was results do not support a dominant negative role for the focally electroporated into the right side of Hensen’s node MID1 tripartite motif and instead are consistent with OS in stage 4 embryos (Fig. 6A). Embryos were cultured to being a manifestation of loss-of-function mutations in stage 6 and then assayed for either Fgf8 or Shh expression. MID1. Fgf8 expression was confined to the right side of the node Electroporation of the DR construct into the right side of (n = 4/4; 100%; Fig. 6B), while Shh expression was the node in stage 4 embryos results in defective L/R confined to the left side of the node (n = 4/4; 100%; Fig. patterning by stage 6: electroporated embryos lack Fgf8 6H). These results show that overexpressing MID1 within expression in the node (n = 12/14; 86%; Fig. 6C) or have the endogenous MID1/2 expression domain on the right bilateral Shh expression in the node (n = 14/16; 88%; side of the node has no effect on normal L/R determi- Fig. 6I). Similarly, embryos electroporated with the B-box nation. Electroporation of DC into the right side of the construct also lack Fgf8 expression in the node (n = 13/16; node in stage 4 embryos has no effect on normal L/R 81%; Fig. 6D) or have bilateral Shh expression (n = 16/18; patterning at stage 6, suggesting that DC is a loss-of- 89%; Fig. 6J). These results are consistent with our

Fig. 6. Effect of MID1 deletion constructs on L/R patterning. (A) Expression constructs were electroporated into the right side of the node at stage 4. (B) Overexpression of cMid1 has no effect on Fgf8 expression in the node. (C) Expression of DR results in a loss of Fgf8 expression in the node. (D) Expression of B-box results in a loss of Fgf8 expression in the node. (E) Expression of DC does not effect Fgf8 expression in the node. (F) a4 is expressed symmetrically in the node at stage 5. (G) a4 expression is restricted to the right side of the node by stage 6. (H) Overexpression of cMid1 has no effect on Shh expression in the node. (I) Expression of DR results in ectopic Shh expression on the right side of the node. (J) Expression of B-box results in ectopic Shh expression on the right side of the node. (K) Expression of DC does not effect Shh expression in the node. The node is outlined in black. High magnification inserts of the node highlight the small changes in gene expression. Embryos are shown dorsal side up. L, left; R, right. A. Granata et al. / Developmental Biology 277 (2005) 417–424 423 prediction that the DR and B-box constructs act to block cMid.MO blocks the translation of either endogenous MID1 endogenous MID function in a dominant negative manner or MID2. However, the set of control electroporations and support the in vitro model for MID1 domain functions. performed strongly indicates that the laterality defects caused These two constructs will provide a valuable alternative to by cMid.MO are the specific result of knocking down cMid.MO for investigating the consequences of blocking endogenous MID1/2 expression in the node and that the MID function in other developing organ systems. rescue of these defects is through the specific action of Given that correct MID function in vitro appears depend- exogenous MID2. ent on association with a4, we have investigated whether a4 The DC MID1 deletion construct, containing the N- is expressed in Hensens node. Several EST clones represent- terminal tripartite domain but lacking the C-terminus, is ing the chick a4 orthologue were identified in the BBSRC representative of the vast majority of MID1 mutations ChickEST Database (www.chick.umist.ac.uk; template associated with OS. DC acts as a nonfunctional MID1 sequence 339799.6). EST clone ChEST463i11 was used as construct, indicating that OS is likely to be a manifestation a template to generate an antisense RNA probe for use in of loss-of-function, rather than gain-of-function, mutations whole-mount in situ hybridization. Weak a4 expression is in MID1. The observation that the DR and B box constructs first seen on both sides of Hensens node at stage 4 (data not both block endogenous MID activity in a dominant negative shown). At stage 5, when cMid1 and cMid2 are already manner indicates that the in vivo function of the MID1 confined to the right, a4 expression is still symmetric in the protein domains is consistent with their biochemical activity node (Fig. 6F), but its expression is restricted to the right side observed in vitro. Furthermore, the results of blocking MID by stage 6 (Fig. 6G). Expression remains asymmetric at stage function using the dominant negative DRandBbox 7 and is down-regulated by stage 8 (data not shown). constructs are consistent with the results obtained from blocking MID function using antisense morpholinos. The demonstration of functional redundancy between Discussion MID proteins during avian L/R determination suggests that functional redundancy between MID proteins could exist in The chick homologue of the Opitz syndrome gene, other systems where they are co-expressed. It is notable that cMid1, is involved in the establishment of asymmetric gene we have used mouse MID2 to compensate for an absence of expression in Hensen’s node: a key step in patterning the chick MID1, as this indicates that MID function is well avian L/R axis (Granata and Quaderi, 2003). We have conserved both between species (mouse and chick) and utilized our existing knowledge of the molecular pathways homologues (MID1 and MID2). Our results support the involved in avian L/R determination to investigate whether hypothesis that the phenotypic variability in OS can be functional redundancy exists between MID proteins. We attributed to functional redundancy between MID1 and have also used the L/R system to determine whether OS is MID2, although further studies are required to prove this associated with loss-of-function or gain-of-function MID1 hypothesis. In addition to the node, we have seen partial mutations and whether MID1 function in vivo is consistent overlap between cMid1 and cMid2 expressions in the with in vitro biochemical data. anterior neural plate. However, we have also identified cMid1 and cMid2 are co-expressed on the right side of regions, such as the primitive streak and the developing Hensen’s node between stages 5 and 7. We have shown that somites, where only a single cMid gene is expressed, when misexpressed on the left side of the node, MID2 can indicating that there are structures in the developing embryo mimic MID1 in repressing endogenous Shh expression and where MID1 and MID2 have a nonredundant function. inducing ectopic Fgf8 expression. Furthermore, we show that Further studies will focus on examining whether functional overexpression of MID2 on the right side of the node can redundancy between MID proteins exists in developing rescue the laterality defects caused by treatment with midline structures affected in OS. cMid.MO. The antisense morpholino cMid.MO was origi- Although there is an association between midline and nally designed to target the first 25 coding nucleotides of the laterality defects (Danos and Yost, 1995, 1996; Lohr et al., cMid1 sequence, and it was assumed that the L/R patterning 1997; Roessler and Muenke, 2001; Yost, 1998), L/R defects defects caused by cMid.MO were a result of knocking down are not a typical feature of OS. This may reflect the fact that MID1 alone (Granata and Quaderi, 2003). We now show that the mechanisms of early L/R determination are poorly the expression of cMid1 in the node is shared by cMid2 and conserved between species, and MID1 is not required for that there are only two mismatches between cMid1 and patterning the human L/R axis (Capdevila et al., 2000; Levin, cMid2 in the 5V region targeted by cMid.MO. As at least 4/25 2003). However, our results indicate that the absence of mismatches are recommended to prevent binding of antisense laterality defects in OS patients does not necessarily preclude morpholinos to their target sequences and up to 11/25 the involvement of MID1 in human L/R determination: it is mismatches are used in practice (Sheng et al., 2003), it is possible that MID2 might compensate for an absence of likely that cMid.MO binds to both cMid1 and cMid2.We MID1 during the establishment of the L/R axis in individuals have been unable to raise antibodies that reliably detect chick with OS. A recent study suggests that similarities in the MID proteins and so are unable to test directly whether anatomy of early embryos, rather than taxonomic similarities, 424 A. Granata et al. / Developmental Biology 277 (2005) 417–424 may be a key factor in shared mechanisms of early L/R Hahn, H., Wicking, C., Zaphiropoulous, P.G., Gailani, M.R., Shanley, S., patterning among species (Fischer et al., 2002): Fgf8 is a right Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A.B., Gillies, S., et al., 1996. Mutations of the human homolog of Drosophila patched determinant in chicks and rabbits, both of which develop as a in the nevoid basal cell carcinoma syndrome. Cell 85, 841–851. flat blastodisc, but is a left determinant in mouse, although Hambuger, V., Hamilton, H.L., 1951. A series of normal stages in the both mice and rabbits are mammals (Boettger et al., 1999; development of the chick embryo. J. Morphol. 88, 49–92. Fischer et al., 2002; Meyers and Martin, 1999). As human Joazeiro, C.A., Weissman, A.M., 2000. RING finger proteins: mediators of embryos also develop as a flat blastodisc, factors found to ubiquitin ligase activity. Cell 102, 549–552. Johnson, R.L., Rothman, A.L., Xie, J., Goodrich, L.V., Bare, J.W., Bonifas, play an early role in L/R determination in chick or rabbit, such J.M., Quinn, A.G., Myers, R.M., Cox, D.R., Epstein Jr., E.H., et al., as the MID1 proteins, may have a conserved function in 1996. Human homolog of patched, a candidate gene for the basal cell establishing the human L/R axis. nevus syndrome. Science 272, 1668–1671. Levin, M., 2003. Motor protein control of ion flux is an early step in embryonic left–right asymmetry. BioEssays 25, 1002–1010. Acknowledgments Levin, M., Johnson, R.L., Stern, C.D., Kuehn, M., Tabin, C., 1995. A molecular pathway determining left—right asymmetry in chick embryo- genesis. Cell 82, 803–814. We would like to thank Cliff Tabin for the Shh probe and Liu, J., Prickett, T.D., Elliott, E., Meroni, G., Brautigan, D.L., 2001. Ivor Mason for the Fgf8 probe. This work was supported by Phosphorylation and microtubule association of the Opitz syndrome a Wellcome Trust Research Career Development Fellowship protein mid-1 is regulated by protein phosphatase 2A via binding to to N.A.Q. the regulatory subunit alpha 4. Proc. Natl. Acad. Sci. U. S. A. 98, 6650–6655. Lohr, J.L., Danos, M.C., Yost, H.J., 1997. Left–right asymmetry of a nodal- related gene is regulated by dorsoanterior midline structures during References Xenopus development. Development 124, 1465–1472. Mercola, M., Levin, M., 2001. Left–right asymmetry determination in Boettger, T., Wittler, L., Kessel, M., 1999. FGF8 functions in the vertebrates. Annu. Rev. Cell Dev. Biol. 17, 779–805. specification of the right body side of the chick. Curr. Biol. 9, 277–280. Meyers, E.N., Martin, G.R., 1999. Differences in left–right axis Buchner, G., Montini, E., Andolfi, G., Quaderi, N., Cainarca, S., Messali, pathways in mouse and chick: functions of FGF8 and SHH. Science S., Bassi, M.T., Ballabio, A., Meroni, G., Franco, B., 1999. MID2, a 285, 403–406. homologue of the Opitz syndrome gene MID1: similarities in Perry, J., Short, K.M., Romer, J.T., Swift, S., Cox, T.C., Ashworth, A., subcellular localization and differences in expression during develop- 1999. FXY2/MID2, a gene related to the X-linked Opitz syndrome gene ment. Hum. Mol. Genet. 8, 1397–1407. FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing Cainarca, S., Messali, S., Ballabio, A., Meroni, G., 1999. Functional protein that associates with microtubules. Genomics 62, 385–394. characterization of the Opitz syndrome gene product (midin): evidence Quaderi, N.A., Schweiger, S., Gaudenz, K., Franco, B., Rugarli, E.I., for homodimerization and association with microtubules throughout the Berger, W., Feldman, G.J., Volta, M., Andolfi, G., Gilgenkrantz, S., cell cycle. Hum. Mol. Genet. 8, 1387–1396. et al., 1997. Opitz G/BBB syndrome, a defect of midline develop- Capdevila, J., Vogan, K.J., Tabin, C.J., Izpisua Belmonte, J.C., 2000. ment, is due to mutations in a new RING finger gene on Xp22. Nat. Mechanisms of left–right determination in vertebrates. Cell 101, 9–21. Genet. 17, 285–291. Chapman, S.C., Collignon, J., Schoenwolf, G.C., Lumsden, A., 2001. Reymond, A., Meroni, G., Fantozzi, A., Merla, G., Cairo, S., Luzi, L., Improved method for chick whole-embryo culture using a filter paper Riganelli, D., Zanaria, E., Messali, S., Cainarca, S., et al., 2001. The carrier. Dev. Dyn. 220, 284–289. tripartite motif family identifies cell compartments. EMBO J. 20, Cox, T.C., Allen, L.R., Cox, L.L., Hopwood, B., Goodwin, B., Haan, E., 2140–2151. Suthers, G.K., 2000. New mutations in MID1 provide support for loss Roessler, E., Muenke, M., 2001. Midline and laterality defects: left and of function as the cause of X-linked Opitz syndrome. Hum. Mol. Genet. right meet in the middle. BioEssays 23, 888–900. 9, 2553–2562. Schweiger, S., Foerster, J., Lehmann, T., Suckow, V., Muller, Y.A., Walter, Danos, M.C., Yost, H.J., 1995. Linkage of cardiac left–right asymmetry G., Davies, T., Porter, H., van Bokhoven, H., Lunt, P.W., et al., 1999. and dorsal–anterior development in Xenopus. Development 121, The Opitz syndrome gene product, MID1, associates with microtubules. 1467–1474. Proc. Natl. Acad. Sci. U. S. A. 96, 2794–2799. Danos, M.C., Yost, H.J., 1996. Role of notochord in specification of cardiac Sheng, G., dos Reis, M., Stern, C.D., 2003. Churchill, a zinc finger left–right orientation in zebrafish and Xenopus. Dev. Biol. 177, 96–103. transcriptional activator, regulates the transition between gastrulation De Falco, F., Cainarca, S., Andolfi, G., Ferrentino, R., Berti, C., and neurulation. Cell 115, 603–613. Rodriguez Criado, G., Rittinger, O., Dennis, N., Odent, S., Rastogi, Short, K.M., Hopwood, B., Yi, Z., Cox, T.C., 2002. MID1 and MID2 A., et al., 2003. X-linked Opitz syndrome: novel mutations in the homo- and heterodimerise to tether the rapamycin-sensitive PP2A MID1 gene and redefinition of the clinical spectrum. Am. J. Med. regulatory subunit, Alpha 4, to microtubules: implications for the Genet. 120A, 222–228. clinical variability of X-linked Opitz GBBB syndrome and other Fischer, A., Viebahn, C., Blum, M., 2002. FGF8 acts as a right determinant developmental disorders. BMC Cell Biol. 3, 1. during establishment of the left–right axis in the rabbit. Curr. Biol. 12, Trockenbacher, A., Suckow, V., Foerster, J., Winter, J., Krauss, S., Ropers, 1807–1816. H.H., Schneider, R., Schweiger, S., 2001. MID1, mutated in Opitz Gaudenz, K., Roessler, E., Quaderi, N., Franco, B., Feldman, G., Gasser, syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for D.L., Wittwer, B., Horst, J., Montini, E., Opitz, J.M., et al., 1998. Opitz degradation. Nat. Genet. 29, 287–294. G/BBB syndrome in Xp22: mutations in the MID1 gene cluster in the Vortkamp, A., Gessler, M., Grzeschik, K.H., 1991. GLI3 zinc-finger gene carboxy-terminal domain. Am. J. Hum. Genet. 63, 703–710. interrupted by translocations in Greig syndrome families. Nature 352, Granata, A., Quaderi, N.A., 2003. The Opitz syndrome gene MID1 is 539–540. essential for establishing asymmetric gene expression in Hensen’s node. Yost, H.J., 1998. The genetics of midline and cardiac laterality defects. Dev. Biol. 258, 397–405. Curr. Opin. Cardiol. 13, 185–189.