1. Developmental Biology
  2. Human Biology and Medicine
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T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number

  1. Uchenna Emechebe
  2. Pavan Kumar P
  3. Julian M Rozenberg
  4. Bryn Moore
  5. Ashley Firment
  6. Tooraj Mirshahi
  7. Anne M Moon  Is a corresponding author
  1. University of Utah, United States
  2. Geisinger Clinic, United States
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Cite this article as: eLife 2016;5:e07897 doi: 10.7554/eLife.07897

Abstract

Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. Mechanisms whereby T-box3 regulates limb development are poorly understood. We discovered requirements for T-box at multiple stages of mouse limb development and distinct molecular functions in different tissue compartments. Early loss of T-box3 disrupts limb initiation, causing limb defects that phenocopy Sonic Hedgehog (Shh) mutants. Later ablation of T-box3 in posterior limb mesenchyme causes digit loss. In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor. Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.

https://doi.org/10.7554/eLife.07897.001

eLife digest

Mutations in the gene that encodes a protein called T-box3 cause serious birth defects, including deformities of the hands and feet, via poorly understood mechanisms. Several other proteins are also important for ensuring that limbs develop correctly. These include the Sonic Hedgehog protein, which controls a signaling pathway that determines whether a protein called Gli3 is converted into its “repressor” form. The hair-like structures called primary cilia that sit on the surface of animal cells also contain Gli3, and processes within these structures control the production of the Gli3-repressor.

Emechebe, Kumar et al. have now studied genetically engineered mice in which the production of the T-box3 protein was stopped at different stages of mouse development. This revealed that turning off T-box3 production early in development causes many parts of the limb not to form. This type of defect appears to be the same as that seen in mice that lack the Sonic Hedgehog protein.

If the production of T-box3 is turned off later in mouse development in the rear portion of the developing limb, the limb starts to develop but doesn’t develop enough rear toes. When T-box3 production is turned off in the front portion of the developing limbs, mice are born with too many front toes. This latter problem mimics the effects seen in mice that are unable to produce Gli3-repressor or that have defective primary cilia.

Further investigation unexpectedly revealed that T-box3 is found in primary cilia and localizes to the same regions of the cilia as the Gli3-repressor. Furthermore, T-box3 also interacts with a protein complex that controls the stability of Gli3 and processes it into the Gli3-repressor form.

In the future, it will be important to determine how T-box3 controls the stability of Gli3 and whether that process occurs in the primary cilia or in other parts of the cell where T-box3 and Gli3 coexist, such as the nucleus. This could help us understand how T-box3 and Sonic Hedgehog signaling contribute to other aspects of development and to certain types of cancer.

https://doi.org/10.7554/eLife.07897.002

Introduction

The T-box gene family encodes transcription factors that play critical roles during embryonic development, organogenesis, and tissue homeostasis. Mutations in TBX genes in humans cause multiple developmental dysmorphic syndromes and disease predispositions (Naiche et al., 2005; Showell et al., 2004). Heterozygous mutation of TBX3 causes Ulnar-mammary syndrome (UMS), initially described as a constellation of congenital limb defects, apocrine and mammary gland hypoplasia, and genital abnormalities (Pallister et al., 1976). Recently, heart and conduction system defects have also been described in mice and humans with abnormal Tbx3 (mice) and TBX3 (humans) function (Bakker et al., 2008; Frank et al., 2012; Linden et al., 2009; Meneghini et al., 2006; Mesbah et al., 2008). Germline deletion of Tbx3 in mice results in embryonic lethality with heart, limb, and mammary defects (Davenport et al., 2003; Frank et al., 2012; 2013). Tbx3 also regulates pluripotency and cell fate in early development (Cheng et al., 2012; Han et al., 2010; Kartikasari et al., 2013; Niwa et al., 2009; Weidgang et al., 2013).

TBX3 transcriptional repression controls expression of cell proliferation and senescence factors (Brummelkamp et al., 2002; Kumar et al., 2014a); abnormal TBX3 expression occurs in multiple cancers (Liu et al., 2011; Lu et al., 2010; Peres and Prince, 2013). TBX3 also regulates splicing and RNA metabolism (Kumar et al., 2014b). Although these studies highlight the important pleiotropic molecular functions of TBX3, little is known about the core pathways it regulates in developing structures that require its function, such as the developing limb.

UMS limb phenotypes are variable ranging in severity from hypoplasia of digit 5 to complete absence of forearm and hand (OMIM #181450). Mouse Tbx3tm1Pa/tm1Pa (Davenport et al., 2003) and Tbx3Δfl/Δfl (Frank et al., 2013) mutant forelimbs lack posterior digits and the ulna. Hindlimbs of Tbx3tm1Pa/tm1Pa and Tbx3Δfl/Δfl null mutants have only a single digit, but Tbx3Δfl/Δfl mutants also have pelvic defects (Frank et al., 2013). Embryonic lethality of both types of mutants has prevented elucidation of Tbx3’s limb-specific roles.

The Hedgehog pathway is a key regulator of limb development. Shh signaling in posterior mesenchyme promotes digit development and prevents processing of full length Gli3 (Gli3FL) to its repressor form, Gli3R, which constrains digit number (Litingtung et al., 2002). The balance of Gli transcriptional activation and repression is critical for proper digit number and patterning (Cao et al., 2013; Hill et al., 2007; Litingtung et al., 2002; te Welscher et al., 2002; Wang et al., 2000; 2007a; Zhulyn et al., 2014). In mammals, the limited, partial proteolytic processing of Gli3FL to Gli3R requires functional primary cilia, the ciliary protein Kif7 (Goetz and Anderson, 2010; Liu et al., 2005), as well as balanced activity of Sufu and the ubiquitin ligase adaptors βTrCP and Spop (Chen et al., 2009; Wang and Li, 2006; Wang et al., 2010; Wen et al., 2010)

In this study, conditional ablation of Tbx3 reveals discrete roles for Tbx3 during limb initiation and compartment-specific functions during later limb development to regulate digit number. We discovered a novel molecular function of Tbx3 in the primary cilia where it interacts directly with Kif7 and is in a complex with Gli3. Loss of Tbx3 decreases Kif7-Sufu interactions, resulting in excess Gli3 proteolysis and decreased levels of both Gli3FL and Gli3R. The resulting preaxial polydactyly phenocopies limb defects seen in Gli3 null heterozygotes and in mutants with abnormal structure or function of the primary cilia (Cheung et al., 2009; Endoh-Yamagami et al., 2009; Goetz and Anderson, 2010; Haycraft et al., 2005; Liem et al., 2009; Liu et al., 2005; Ocbina et al., 2011; Putoux et al., 2011). Our findings reveal a novel mechanism where Tbx3 in the anterior mesenchyme is required for proper function of the Kif7/Sufu complex that regulates Gli3 stability and processing.

Results

Loss of Tbx3 in the limb bud mesenchyme results in preaxial polydactyly and postaxial oligodactyly

Tbx3 is expressed in discrete anterior and posterior mesenchymal domains in the limb buds from embryonic day (E) 9.5 (Figure 1A,C,E, Figure 1—figure supplement 1). To assess the role of these domains during limb development, we generated conditional mutants using our Tbx3flox allele (Frank et al., 2012; 2013) and the Prx1Cre transgene (Logan et al., 2002) (genotype Tbx3flox/flox;Prx1Cre, henceforth referred to in the text as Tbx3;PrxCre mutants). This driver initiates Cre activity at ~14-somite stage (ss) in the forelimb-forming region of the lateral plate mesoderm (LPM) (Hasson et al., 2007). Its activity in the hindlimb is irregular, so our analysis focuses on the forelimb. In situ hybridization and immunohistochemistry confirm complete ablation of Tbx3 mRNA and protein in Tbx3;PrxCre mutant forelimb mesenchyme by E9.5 (Figure 1B–F, Figure 1—figure supplement 1). Expression in the apical ectodermal ridge (AER) is preserved (Figure 1B,D,F). We previously reported the specificity of the custom anti-Tbx3 antibody used here and loss of limb mesenchymal protein production in Tbx3;PrxCre mutant forelimbs (Frank et al., 2012; 2013).

Figure 1 with 2 supplements see all
Tbx3 regulates anterior and posterior digit development.

(A) Tbx3 expression assayed by mRNA in situ hybridization in E9.5 forelimb bud (black line from a-p shows anterior-posterior axis). Red arrow points to Tbx3 expression in apical ectodermal ridge (AER). Red ellipse encloses posterior mesenchymal expression domain. (B) Tbx3 transcripts are absent in the limb bud mesenchyme of E9.5 Tbx3fl/fl;PrxCre mutants. Tbx3 expression persists in the AER (red arrow) and adjacent posterior-lateral body wall (black arrowhead). (C, D) As in A and B except limb buds are E10.5. Red ellipses enclose anterior and posterior mesenchymal expression domains which are Tbx3 negative in the mutants. Red arrows highlight expression in AER. (E, F) Tbx3 immunohistochemistry on sectioned E10.5 limb. Tbx3 protein is lost in mesenchyme of Tbx3fl/fl;PrxCre mutants (F, red ellipses) but AER staining persists as expected (white arrowhead). Please see also Figure 1—figure supplement 1. (G–J) Skeleton preparations reveal preaxial polysyndactyly (duplicated/fused digit 1,red bracket, H, H’, J) and postaxial oligodactyly (absent digit 5, red arrows in H’ and J) in Tbx3fl/fl;PrxCre mutants at E15.5 (H, H’) and E19.5 (J). Note delayed ossification of the humerus (H, black arrowhead), loss of deltoid tuberosity (J, black arrowhead) and short, bowed ulna (J, black arrow) in mutant. s, scapula; h, humerus; oc, ossification center; dt, deltoid tuberosity r, radius; u, ulna; digits numbered 1–5 (KN) Sox9 mRNA expression shows evolving skeletal defects are already evident in Tbx3fl/fl;PrxCre mutants at E10.5- E11.5. Digit condensations are numbered. Bracket in L shows broadening of digit 1 forming region; red arrows highlight indentation in digit 5 forming region (L, N) and absence of Sox9 digit 5 condensation (N).

https://doi.org/10.7554/eLife.07897.003

Unlike mid-gestational lethality seen in constitutive Tbx3 mutants (Davenport et al., 2003; Frank et al., 2013), Tbx3;PrxCre mutants survive to adulthood with forelimb defects (Video 1): 100% have bilateral preaxial polysyndactyly of digit 1 (called PPD1 in humans [Materna-Kiryluk et al., 2013]), and 70% lack digit 5 (Figure 1G–J). Loss of digit 5 was bilateral in 6/18 and in the remaining, only affected the left forelimb (Table 1). This is not due to asymmetric activity of PrxCre because it is also observed in Tbx3Δfl/Δfl mutants (Δfl = recombined floxed conditional allele) where no Cre activity is involved (Frank et al., 2013). Delayed ossification of the humerus (Figure 1H) and loss of the deltoid tuberosity (Figure 1J) were also observed. Abnormal limb bud morphology is evident by E10.5 (Figure 1L) and evolving skeletal defects at E11.5 by the altered pattern of Sox9 expression (Figure 1N).

Table 1

Increased severity of left limb defects in Tbx3;PrxCre mutants.

https://doi.org/10.7554/eLife.07897.006
Skeletal phenotypes: E13.5-adult
Loss of digit 5BilateralLeft onlyRight only
Tbx3 fl/+ or fl/fl000
Tbx3fl/fl;PrxCre6120
Molecular phenotypes: gene expression
Expression pattern or levelLeft = RightLeft >RightRight > Left
Tbx3 fl/+ or f/flcontrolcontrolcontrol
Tbx3fl/fl;PrxCre 19301
Video 1
Adult Tbx3;PrxCre mutant mouse is healthy and mobile despite forelimb deformities.
https://doi.org/10.7554/eLife.07897.007

Tbx3;PrxCre mutant limb defects are less severe than constitutive nulls

Germline Tbx3 null mutants (genotype Tbx3Δfl/Δfl) have more severe forelimb defects than Tbx3;PrxCre conditional mutants: of the few Tbx3Δfl/Δfl mutants that survive to E13.5, 100% have agenesis of the ulna and digits 3–5 (Figure 1—figure supplement 2B). Their hindlimbs have a single digit and no fibula (Frank et al., 2013), phenocopying Shh and Hand2 null mutants (Galli et al., 2010). Variable timing of Tbx3 loss of function by PrxCre may account for the disparate forelimb phenotypes of Tbx3Δfl/Δfl and Tbx3;PrxCre mutants, however, our skeletal data and phenotypes of Tbx3Δfl/fl;PrxCre mutants indicate that such variability manifests as incomplete penetrance of the ulnar and digit 5 defects (Figure 1 H, H',J, and Colesanto et al., in preparation).

The AER is a critical signaling center, and Tbx3 expression is preserved in the AER of Tbx3;PrxCre mutants (Figure 1B,D,F; Figure 1—figure supplement 1Frank et al., 2013). We tested whether AER Tbx3 has a required function using two Cre drivers: RarbCre (active in AER and mesenchyme from E9.0 [Moon and Capecchi, 2000]), and a novel Fgf8mcm allele, which produces tamoxifen-inducible Cre in Fgf8 expression domains (Moon et al., in preparation). Tamoxifen induction at E8.5 induces robust Cre activity in the AER in RosaLacZ/+;Fgf8mcm/+ embryos (Figure 1—figure supplement 2E,F) and ablates Tbx3 from forelimb AER by at least E9.5 (Figure 1—figure supplement 2G–J). Tbx3fl/fl;Fgf8mcm/mcm mutants have normal limbs (Figure 1—figure supplement 2L) and phenotypes of Tbx3fl/fl;PrxCre and Tbx3fl/fl;RarbCre are indistinguishable (Figure 1—figure supplement 2D versus N). The results with both Cre drivers indicate that the severe phenotypes of Tbx3Δfl/Δfl mutants are not due to a required function of Tbx3 in the AER.

Tbx3 is required for normal limb bud initiation and Tbx5 expression in the LPM

We next tested whether discrepant forelimb phenotypes in Tbx3Δfl/Δfl and Tbx3;PrxCre mutants reflect a role for Tbx3 in an earlier expression domain than affected by RarbCre or PrxCre. Limb initiation in the LPM requires Tbx5 expression in the prospective forelimb territory as early as the 8ss (Minguillon et al., 2005), upstream of Hand2 (Agarwal et al., 2003). Tbx3 is expressed in the LPM from E7.5 (Figure 2A–C’). Lineage tracing with a novel Tbx3mcm allele (Thomas et al., in preparation) revealed that Tbx3-expressing progenitors in the LPM at E8-8.5 give rise to most E10 forelimb mesenchyme in Tbx3mcm/+;RosaLacZ/+ embryos (Figure 2D). Consistent with a role for Tbx3 in limb initiation, Tbx3Δfl/Δfl mutants have decreased LPM expression of Tbx5 (Figure 2E,F), visible defects in forelimb initiation and early limb bud morphology (Figure 2G,H), and disrupted expression of Hand2 (Figure 2I,J). In contrast, early stage Tbx5 expression and limb bud initiation are unaffected in Tbx3;PrxCre mutants (Figure 2—figure supplement 1B,B')

Figure 2 with 1 supplement see all
Tbx3 is required for normal limb bud initiation.

(A, B) Tbx3 expression at E7.5 (A) and E8.5 (B). Anterior on left (A), posterior on right (P). NM (black arrow) indicates nascent mesoderm exiting primitive streak in panel A. (C, C’) Tbx3 expression in the LPM (black arrow) of sectioned E8.5 embryo. Plane of section indicated by line in B. Panel D is magnification of red-boxed area in C. (D) X-gal stained E10.0 Tbx3MCM/+; Rosa LacZ/+ embryo after Cre induction at E8.5. FL, forelimb; HL, hindlimb. (E, F) 21 somite stage (ss) embryos assayed for Tbx5 mRNA. White arrows denote forelimb bud. Left sided view. (G, H) Dorsal view of budding forelimbs of 24 ss embryo forelimbs (neural tube stained for Shh expression). Note abnormal shape and size of Tbx3Δfl/Δfl mutant forelimb buds indicative of disrupted initiation; white brackets are of equal size in both panels. (I, J) 22 ss embryos assayed for Hand2 expression. Black arrows denote emerging forelimb bud.

https://doi.org/10.7554/eLife.07897.008

TBX3 positively regulates posterior digit development via the Hand2/Shh pathway in posterior mesenchyme

Our data reveal required functions for Tbx3 in limb initiation (demonstrated by Tbx3Δfl/Δfl mutants) and in later limb bud morphogenesis (demonstrated by Tbx3;PrxCre mutants). Most Tbx3;PrxCre mutants lack digit 5, whose specification and formation depend on 'early phase' Shh signaling beginning at E9.5 (Harfe et al., 2004; Scherz et al., 2007; Zhu and Mackem, 2011; Zhu et al., 2008). Hand2 protein is required to activate Shh expression in the limb bud (Benazet and Zeller, 2009; Galli et al., 2010). We found that Hand2 transcripts are reduced in E9.5 and E10.5 Tbx3;PrxCre mutant forelimb buds (Figure 2—figure supplement 1D,D'; Figure 3A, A',F) as is Shh expression (Figure 3B,B',F; Figure 3—figure supplement 1). Expression of two targets and effectors of Shh signaling, Ptch1 and Grem1, is also markedly reduced (Figure 3C',E'; Figure 3—figure supplement 2). Tbx3 expression in posterior limb mesoderm begins earlier than in the anterior compartment (Figure 1A,C) and is required for normal Hand2 in posterior mesoderm (Figure 3A, Figure 2J and Figure 2—figure supplement 1D’) (Rallis et al., 2005). Thus, intact Tbx5 expression in Tbx3;PrxCre E9.5 forelimbs (Figure 2—figure supplement 1B’) indicates that post-initiation, Tbx3 functions downstream of Tbx5 and upstream of Hand2.

Figure 3 with 5 supplements see all
Loss of mesenchymal Tbx3 disrupts Shh signaling in the posterior limb bud and decreases Gli3 protein stability.

(AE’) In situ hybridization of control and mutant forelimb buds with probes and at embryonic stages as labeled. (F) qPCR of E10.75 (36-39ss) limb buds for transcripts listed confirms findings by detected by in situ. (GI’) In situ hybridization for Zic3, Epha3 and Hoxd13 transcripts in forelimb buds of Tbx3 fl/+controls (KM) and Tbx3;PrxCre mutants (G’I’) at ages noted on panels. J) qPCR assay of Zic3, Epha3, Hoxd13 transcript levels confirms findings detected by in situ. (KL’) Representative images of E10.5 forelimb buds stained for DAPI (blue), pHH3 (green), TUNEL (red). K is Tbx3 fl/+ control and K’ is digital zoom of posterior mesenchymal boxed area in K. Panel L is Tbx3;PrxCre mutant and L’ is digital zoom of boxed area in L. This experiment is representative of data obtained from five biologic replicates. (M) Quantification of proliferating cells in anterior and posterior mesenchymal regions encompassing digit 1 and digit 5 progenitors from 20 control and 15 mutant sections. *p=0.02. Control anterior limb (CA), Tbx3;PrxCre mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP). (NO’) Representative images of E11.5 whole mount forelimb buds stained for DAPI (blue) and pHH3 (green). N is Tbx3 fl/+ control and N’ is digital zoom of boxed area. Panel O is Tbx3;PrxCre mutant and O’ is digital zoom of boxed area in O. Note decreased pHH3+ cells in mutants, particularly cells in prophase and anaphase, which have the faint and speckled patterns compared to the bright staining of highly condensed S-phase chromatin. (P) Quantification of proliferating cells in anterior and posterior mesenchymal regions encompassing digit 1 and digit 5 progenitors from 50 control and 44 mutant sections at E11.5. *p<0.1. Control anterior limb (CA), Tbx3;PrxCre mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP).

https://doi.org/10.7554/eLife.07897.010

To obtain a more comprehensive view of the transcriptional consequences of loss of Tbx3 in limb bud mesenchyme, we assayed gene expression of E10.25 limb buds (32–34 somite stage) by microarray. Shh and other hedgehog pathway members and target genes (Lettice et al., 2003Probst et al., 2011; Vokes et al., 2008; McGlinn et al., 2005)(Lewandowski et al., 2015) were present among the significantly dysregulated transcripts (Supplementary file 1) consistent with the previous report of decreased Shh expression in Tbx3tm1Pa/tm1Pa mutant limb buds (Davenport et al., 2003). In addition to decreased levels of Shh- activated transcripts (Gli1, Hand2, Osr1, Dkk1, Tbx2, Cntfr, Pkdcc), we noted increased Rprm, Zic3, Hand1, and Gli3 transcripts and confirmed these with qPCR and in situ hybridization at E10.5–11 (Figure 3, Figure 3—figure supplement 4). The increase in expression of these putative targets of the Gli3 repressor (Lettice et al., 2003; Probst et al., 2011; Vokes et al., 2008; McGlinn et al., 2005) was intriguing as it suggested the possibility of alterations in both Gli3 activator and repressor function in Tbx3;PrxCre mutant forelimbs.

The transcriptional profiles of anterior and posterior limb mesenchyme are quite different: alterations in gene expression in either compartment in response to Tbx3 could mask some changes in the other if assayed simultaneously. Thus, we proceeded to microdissect anterior and posterior limb segments and assayed them independently using RNA-sequencing on 38–42 somite stage (~E11; this later stage was needed in order to obtain sufficient RNA from accurately microdissected limb segments) wild type and Tbx3;PrxCre mutant forelimb buds (Figure 3—figure supplement 3). The resulting RNA-Seq data confirmed accurate dissection with the expected distribution of known compartment-specific transcripts (Figure 3—figure supplement 3 and Supplementary file 2). Shh, Fgf4 and anterior Hoxd family transcripts were over-represented in the posterior compartment, and Alx and Pax family members in the anterior.

Largely consistent with the previous microarray findings, we found evidence of aberrant cell differentiation/fate of posterior mesenchyme with downregulation of Shh-activated targets (Osr1, Dkk1, Tbx2, Cntfr, Ptch2, Supplementary file 3) that validated by qPCR (Figure 3—figure supplement 4). It is known that Gli3 expression increases with decreased Shh activity (Wang et al., 2000), as we see here (Figure 3D’, F). Although decreased levels of Hand2, Shh, Ptch1 and Grem1 are clearly evident by in situ and qPCR at this stage (Figure 3 and figure upplements), they were not detected on the RNA-Seq analysis for unclear reasons.

Shh signaling is required for proliferation to ensure sufficient cell numbers to form the normal complement of digits, and loss of Shh results in an increase in the number of cells in G1 arrest (Zhu et al., 2008). Assay of cell proliferation in E10.5 and E11.5 limb buds using anti-phosphohistone H3 immunohistochemistry revealed that at E10.5 there was a statistically significant decrease in the fraction of proliferating cells in the posterior mesenchyme (Figure 3K–M). At E11.5, proliferation was significantly decreased in both the anterior and posterior mesenchyme, indicating a global reduction in the number of mitotic cells in mutants (Figure 3N–P). This suggests that 5th digit agenesis is attributable, at least in part, to decreased cell number, as opposed to decreased proliferation specifically in digit 5 progenitors. Assay for apoptosis using TUNEL showed normal levels of anterior AER cell death at E10.5, as we have previously reported in this region (Moon and Capecchi, 2000) (Figure 3K,L).

Proliferation of limb mesenchyme depends on activity of FGF8 and FGF4 from the AER (Boulet et al., 2004; Moon and Capecchi, 2000; Sun et al., 2002) and integrity of this structure requires Shh activity in posterior mesenchyme (Chiang et al., 2001). Despite decreased Shh expression in Tbx3;PrxCre mutants, Fgf4 transcripts were increased in posterior mesenchyme while decreased in anterior (detected by RNA-Seq, Supplementary files 3,4 and qPCR, Figure 3—figure supplement 4), the latter consistent with an expanded digit 1 region. qPCR detected increased Fgf8 expression in the posterior AER (Figure 3—figure supplement 4). Despite these changes in transcript levels, there was no evidence of altered downstream FGF signaling as expression of Etv4, Etv5, Dusp6 and Sprys was unchanged (Figure 3—figure supplement 5, note these transcripts are not listed in Supplementary files 3 or 4 because they did not meet criteria for differential expression). We conclude that despite the decrement in Shh pathway activity in posterior mesenchyme of Tbx3;PrxCre mutants, the level is sufficient to maintain ectodermal FGF signaling, consistent with preserved limb outgrowth.

Tbx3 stabilizes Gli3 protein in anterior limb mesenchyme

To understand the cause of the anterior PPD phenotype in Tbx3;PrxCre mutants, we pursued molecular mechanisms known to cause this defect in humans and mice: ectopic Hedgehog pathway activity (Hill et al., 2007; 2003; Lettice et al., 2003); decreased Gli3R activity (Hill et al., 2009; Naruse et al., 2010; Wang et al., 2007a); and abnormal composition or function of the primary cilia (Goetz and Anderson, 2010).

RNA-Seq analysis of control versus mutant anterior compartments (Supplementary file 4) showed no evidence of ectopic hedgehog activity in Tbx3;PrxCre mutants, and this was confirmed by in situ hybridization and qPCR for Shh and Ptch1 (Figure 3B–C', Figure 3—figure supplements 1 and 2). Although Gli3 transcripts were increased in mutant limb buds (Figure 3D',F; Supplementary file 1), targets of Gli3R transcriptional repression such as Zic3, Epha3, Hoxd13 (McGlinn et al., 2005; Vokes et al., 2008) were overexpressed when assayed by microarray, RNA-Seq, qPCR and in situ hybridization (Figure 3 G-J, Supplementary files 1,3,4).

The discrepancy between Gli3 RNA levels and increased expression of some repressor targets prompted examination of Gli3 protein levels. Gli3R constitutes the vast majority of Gli3 protein species in the anterior limb bud (Wang et al., 2000) and Figure 4A). Gli3R protein was markedly decreased (7.4 fold on this representative immunoblot) with multiple bands of lower molecular weight than Gli3R present specifically in Tbx3;PrxCre mutant anterior mesenchyme (mutant anterior compartment: MA, Figure 4A, red box). Gli3FL was virtually undetectable in mutant anterior mesenchyme (Figure 4A’). This finding is not due to poor sample quality because it was reproducible (N=3), no degradation was present in simultaneously prepared posterior compartment lysates (mutant posterior, MP), and the β−tubulin control was intact. These findings indicate that Tbx3 is required for stability of Gli3FL and Gli3R proteins in the anterior limb mesenchyme, and are consistent with the PPD phenotype observed here and in other models of Gli3R deficiency (Hill et al., 2009; Naruse et al., 2010; Wang et al., 2007a).

Figure 4 with 2 supplements see all
Loss of Tbx3 results in Gli3 protein instability and aberrant localization of Kif7 in limb bud cilia.

(A) Representative immunoblot (N=3) blot of E10.75 forelimb bud lysates prepared from microdissected Tbx3fl/+ control anterior limb (CA), Tbx3;PrxCre mutant anterior limb (MA), control posterior (CP), and mutant posterior (MP) probed for Gli3 and βtubulin loading control. Note decreased level of Gli3FL and Gli3R, and multiple bands of lower molecular weight than Gli3R in MA sample. Densitometry of Gli3R bands in red box in N revealed that in this representative experiment, the level of Gli3R was 7.4 fold decreased in mutant anterior relative to control anterior. (A’) Longer exposure of top of blot shown in panel A to examine Gli3FL band. The control (CA) Gli3FL band is 31 fold more intense than mutant (MA, virtually undetectable). (B) Immunoblot of lysates from E10.5 forelimb buds immunoprecipitated (IB) with antibodies listed at top and immunoblotted (IB) for Tbx3. Lane 5 shows that immunoprecipitation with anti-Kif7 antibody co-IPs Tbx3. (C) As in panel B, but assayed for Kif7. (D) Co-IP assay of Myc-tagged Tbx3 and Flag-tagged GFP overexpressed in HEK293 cells. IP was performed with antibodies listed at top and immunoblotted for Tbx3. Myc-tagged Tbx3 co-IPs with Flag-tagged Kif7. Input lane was 5 s exposure (5” exp) while other lanes were 15 s (15” exp). (E) As in D, but in this case, blot probed for Kif7; confirms interaction of tagged, overexpressed proteins. (F, G) Representative images of anterior mesenchyme in sectioned forelimbs of control (F, Tbx3fl/fl) and mutant (G, Tbx3fl/fl;PrxCre) E10.5 embryos stained for the ciliary marker Arl13b (red), Kif7 (green) and DAPI (DNA, blue). White arrowheads highlight cilia with multiple punctae or streak of ciliary Kif7 immunoreactivity (yellow) indicating translocation of Kif7 within the cilia. Please also see Figure 5—source data 2 for z-stacks of additional Kif7 stained limb section. (H) Quantification of Kif7 staining pattern from multiple limb sections and three embryos of each genotype scored blinded to genotype. 10% fewer cilia have evidence of Kif7 translocation (multiple punctae or streak of Kif7 immunoreactivity) in Tbx3fl/fl;PrxCre mutants. N=1785 and 1792 cilia scored in controls and mutants, respectively. * p<0.001 There was no difference in the number of Kif7- cilia. Insets show digital zoom of cilia with representative pattern used for scoring. (I) Immunoblot assaying for Kif7 and b tubulin loading control in control anterior (CA), mutant anterior (MA), control posterior (CP) and mutant posterior (MP) e10.5 forelimb bud lysates. This is representative of four such experiments. (J) Western blot assaying for Sufu protein and b tubulin loading control in eE10.5 forelimb buds. Like Kif7 mutants,Tbx3;PrxCre mutants have increased Sufu protein. Sufu is increased 1.8 fold when normalized to loading control in this representative immunoblot; N=4. K) Western blot assaying for Spop protein and actin loading control in E10.5 forelimb buds. Spop is increased 1.5 fold when normalized to loading control in this representative immunoblot; N=3. Both Sufu and Spop protein levels are increased in mutant forelimbs although their transcript levels are unchanged (Figure 3—figure supplement 4).

https://doi.org/10.7554/eLife.07897.016

TBX3 interacts with Kif7 and is required for normal Kif7 trafficking

In an independent experiment to identify Tbx3 interacting partners, we performed Tbx3 co-immunoprecipitation (co-IP) on E10.5 mouse embryo lysates, followed by mass spectrometry (Kumar et al., 2014b). Surprisingly, Kif7 was among the Tbx3 interacting proteins identified. Kif7 is a ciliary protein that modulates activity of the Hedgehog pathway (Hui and Angers, 2011). It is required for proper formation of a 'cilium tip' compartment that regulates Gli function (He et al., 2014), and for the regulated, partial proteolytic processing of GliFL to Gli3R (Chen et al., 2009; Cheung et al., 2009; Endoh-Yamagami et al., 2009; Law et al., 2012; Ryan and Chiang, 2012). As in Tbx3;PrxCre and Gli3+/- mutants (Hill et al., 2009), human and mouse Kif7 mutants have PPD (Cheung et al. 2009; Endoh-Yamagami et al. 2009; Putoux et al. 2011).

Immunoprecipitation of protein lysates from E10.5 forelimb buds showed that Tbx3 co-immunoprecipitates (co-IPs) with Kif7, confirming interaction of Kif7 and Tbx3 in the developing limb (Figure 4B; specificity and efficiency of Kif7 IP in this experiment is shown in Figure 4C). We next tested whether these proteins directly interact by overexpressing Flag-tagged Kif7 and Myc-tagged Tbx3 in HEK293 cells and immunoprecipitating for either Flag or Myc, followed by immunoblotting for Tbx3 (Figure 4D) or Kif7 (Figure 4E). Both experiments confirmed direct interaction of the tagged proteins.

The interaction of Tbx3 with Kif7, and the shared PPD phenotypes of Kif7 and Tbx3;PrxCre mutants, suggest that Tbx3 may be required for normal Kif7 function in the anterior limb bud, and that loss of Tbx3 may disrupt Gli3 stability and processing in part via a Kif7-dependent mechanism.

We examined Kif7 localization in E11 control and mutant forelimb buds, co-staining for the cilia marker Arl13b. Confocal fields spanning the anterior mesenchyme of controls and mutants were imaged and Figure 4F and G are representative 40X fields (a higher magnification image and confocal z-stack, which also show Kif7 in the cytoplasm and nucleus are shown in Figure 4—figure supplement 1 and Figure 4—source data 1). Blinded to genotype, fields were scored for ciliary Kif7 immunoreactivity as a single puncta, multiple punctae/streak, or none (N=1785 and 1792 cilia scored in controls and mutants, respectively). In 16% of control cilia, Kif7 was detected in two punctae (presumed base and tip) or as a streak along the cilia, but this was only the case in 6% of mutant cilia (Figure 4H, p<0.001). There was no significant difference in the number of Kif7+ cilia rather, there were more single puncta cilia in mutants than controls (Figure 4H, 84% vs 71%, p<0.001). We did not detect any difference in the amount of Kif7 protein in control versus mutant limb bud compartments by western blot (Figure 4I, representative of four4 separate experiments). Levels of Kif7 mRNA in the anterior limb mesenchyme were unaffected by loss of Tbx3 (Figure 3—figure supplement 4). There was a decrease in the transcripts posterior compartment but as shown, the protein level was unchanged.

One feature of Kif7-/-mutants is excess Sufu due to increased protein stability (Hsu et al., 2011). Transcript levels of Sufu were unchanged in mutant forelimbs (Figure 3—figure supplement 4, no difference was detected by microarray or RNA-Seq). However, increased amounts of Sufu (and Spop) protein were present in mutant limb buds (Figure 4J,K; increased 1.5 fold in both cases).

Humans and mice with Kif7 mutations have abnormally long cilia because Kif7 reduces the rate of microtubule growth in the ciliary axoneme (He et al., 2014; Putoux et al., 2011). With 3D images obtained from 100X confocal z-stacks of Arl13b stained limb sections, we used the 3D object counter from ImageJ to calculate the volume and surface area to volume ratio to derive the length of cilia. Consistent with aberrant, but not absent, Kif7 function, we found that while the range of cilia volumes detected were the same between mutants and controls, the distribution was not: mutants have an increased fraction of larger cilia and an average volume 18% greater (Figure 4—figure supplement 2A, B; 475 and 575 cilia assayed in controls and mutants, respectively). Mutants and controls had superimposable surface area/volume ratios (Figure 4—figure supplement 2C), indicating that the shape of cilia was not different thus, the derived length was 6% greater in mutants.

Together, these findings indicate that loss of Tbx3 results in aberrant ciliary localization of Kif7 and are consistent with abnormal Kif7 function.

Tbx3 is present in primary cilia and translocates in response to Hedgehog pathway activity

Kif7 and other proteins required for Gli3 processing and function are present in, or translocate to, primary cilia in response to Hedgehog pathway activity (Goetz and Anderson, 2010; Ryan and Chiang, 2012). Dual immunostaining for Tbx3 and Arl13b on whole mount optically sectioned E10.5 forelimbs shows that Tbx3 is present in control limb anterior mesenchymal cilia (Figure 5 A-E, Figure 5—source data 1). Specificity of Tbx3 staining was confirmed by loss of signal in mesenchymal cilia of Tbx3;PrxCre mutant limbs (Figure 5F-J, Figure 5—source data 2). The digital image overlap calculator in Zen software showed that 18/50 anterior mesenchymal cilia were Tbx3+ (36%, Figure 5—figure supplement 1A,B). Of note, no epithelial cilia were Tbx3+ in control limb epithelium, providing an internal negative control for the signal in mesenchymal cilia. This same calculation in Tbx3;PrxCre mutant anterior mesenchyme showed only 2/54 (<4%) of mesenchymal cilia had background Tbx3 signal (Figure 5—figure supplement 1, C, D). These findings were reproduced with a commercially available anti-Tbx3 antibody (Abcam ab99302) which also showed Tbx3 ciliary staining on control limb mesenchymal cilia (24/87, 28%) that was virtually absent in Tbx3 mutant limbs (2/52, <4%; Figure 5—figure supplement 2).

Figure 5 with 2 supplements see all
Tbx3 localizes to the primary cilia in limb mesenchyme.

(A–D, FI) Confocal 100X single Z-plane immunofluorescence images from optically sectioned E10.5 control (top panels AD) and Tbx3;PrxCre (FI) anterior limb buds after immunostaining with: Hoechst (DNA, blue), C-terminal anti-Tbx3 antibody (green, Frank et al., 2013), anti-Arl13b (red, cilia). Arrowheads demarcate Tbx3 colocalization with cilia marker. Panels E and J are further digital zooms of white boxed cells in D and I. The entire z-stacks containing these planes are in Figure 5—source data 1,2.

https://doi.org/10.7554/eLife.07897.020

Murine embryonic fibroblasts (MEFs) are a robust system for studying ciliary proteins (Chen et al., 2009; Dorn et al., 2012; Liem et al., 2012; Ocbina and Anderson, 2008; Rohatgi et al., 2007), so we used them to further explore Tbx3 localization and trafficking. In untreated wild type MEFs, our custom C-terminal antibody detected Tbx3 in a subset of cilia (30%, N=56; Figure 6A and B, a1-a5, b1-b5; Figure 6—source data 1,2). No Tbx3+ cilia were detected in Tbx3 null MEFS (Figure 6C–F; Figure 6—source data 3). Treatment of wild type MEFs with the smoothened agonist SAG increased the number of Tbx3+ cilia from 30% to 75% (Figure 6G-J p<0.005, Figure 6—source data 4, Figure 6—figure supplement 1A). Lysates from control and SAG-treated MEFs showed no detectable difference in Tbx3 protein levels indicating the increased Tbx3 signal in cilia was due to trafficking rather than increased protein levels (Figure 6—figure supplement 1B). The presence of Tbx3 in cilia and response to Hedgehog pathway activity were also detected with a commercially available anti-Tbx3 antibody with both SAG and Shh stimulation (Figure 6—figure supplement 1C,D and Figure 6—source data 5,6). In total, these data show that Tbx3 is present at baseline in cilia, and is trafficked to cilia in response to hedgehog signaling.

Figure 6 with 1 supplement see all
Tbx3 is present in some cilia at baseline in Murine Embryonic Fibroblasts and trafficks to cilia in response to hedgehog pathway activation.

(A, B) Confocal, 100X single z-plane immunofluorescence images from two different fields of wild type MEFS after immunostaining for: DAPI (DNA, blue), Tbx3 (green, c-terminal anti-Tbx3 antibody; Frank et al., 2013), Arl13b (red, cilia). White boxed regions outline single cells that are shown at higher magnification in panels a1–a5 and b1b5. Please see Figure 6—source data 1,2 for z-stacks. a1a4, b1b4) Single cells from white boxed areas in panels A and B. Individual cilia are shown in a5 and b5. White arrowheads highlight Tbx3+ cilia. (CF) Tbx3 null MEFs show loss of Tbx3 immunoreactivity in cilia and other cellular locations. Please see Figure 6—source data 3 for z-stack. (GJ) As in A and B, but MEFs were treated with smoothened agonist (SAG). White arrowheads highlight Tbx3+ cilia. Please see Figure 6—source data 4 for z-stack.

https://doi.org/10.7554/eLife.07897.025

Tbx3 co-localizes with Gli3 in the primary cilia

The presence of Tbx3 in cilia and the known association of Gli3 and Kif7 in primary cilia (Endoh-Yamagami et al., 2009) led us to test for interaction between endogenous Tbx3 and Gli3. Co-immunoprecipitation of E10.5 forelimb lysates showed that Tbx3 co-IPs with endogenous Gli3 (Figure 7A, and with Kif7 as shown previously), and that both Gli3FL and Gli3R are complexed with Tbx3 in the limb bud (Figure 7B, lane 1). These interactions are also detected in whole embryos (Figure 7C; specificity/efficiency of Gli3 and Kif7 IPs are shown in Figure 7C’ and C”, respectively. Additional experiments showing this interaction are in Figure 7—figure supplement 1). Overexpression of Flag-tagged Gli3 and Myc-tagged Tbx3 in HEK293 cells followed by co-immunoprecipitation did not indicate direct interaction in this cellular context (Figure 7—figure supplement 2), suggesting that their association occurs within a larger complex that includes Kif7 (Figure 4).

Figure 7 with 3 supplements see all
Tbx3 interacts with Gli3 in the limb bud and trafficks with Gli3 in primary cilia.

(A, B) Immunoprecipitation (IP) of E10.5 forelimb bud protein lysates with antibodies listed at the top of panel and immunoblotted (IB) to detect Tbx3 (A) or Gli3 (B). Black arrowhead indicates IgG. Gli3FL and Gli3R (red arrowheads in B) both co-immunoprecipitate with Tbx3. (C) Immunoprecipitation (IP) of E10.5 whole embryo protein lysates with antibodies listed at the top of panel and immunoblotted to detect Tbx3. Tbx3 co-IPs with Gli3. Specificity and efficiency of anti-Gi3 and anti-Kif7 antibodies in whole embryo lysates tested are shown in panels C’ and C”. Additional experiments demonstrating Tbx3/Gli3 interactions are in Figure 7—figure supplement 1. Em, empty lane (D–I’) Confocal 100X single Z-plane immunofluorescence images of vehicle (DMSO) treated MEFS after immunostaining for: DAPI (DNA, blue), Tbx3 (green, Frank et al., 2013), Gli3 (red), Arl13b (pink, cilia). Panel H is merged image of (DG). Panel I is 2.5X digital zoom of the boxed cell in panel H, and I’ shows the pink (cilia) channel pixel shifted to permit visualization of colocalized Tbx3 and Gli3 (yellow) within the cilia. White arrowheads highlight Gli3/Tbx3 colocalization. Please see Figure 7—source data 1 for z-stacks. (JO’) As above, but MEFS were treated with SAG in DMSO. Please see Figure 7—source data 2 for z-stack. (P) Quantitation of Tbx3+ and Gli3+ cilia in MEFS -/+ SAG. SAG treatment causes the majority of cilia to become Tbx3+ and these ciliary Tbx3 signals all colocalize with Gli3.

https://doi.org/10.7554/eLife.07897.033

Immunofluorescence assay of untreated MEFs for Tbx3, Gli3, and Arl13b by triple immunocytochemistry showed that 90% of cilia were Gli3+; Tbx3 colocalized with 66% of the Gli3 signals (N=38 total cilia scored, Figure 7D–IFigure 7—source data 1). Treatment with SAG increased the fraction of Gli3+ cilia to >95% and all but two of those Gli3 signals colocalized with Tbx3 (N=34, Figure 7J–O’, Figure 7—source data 2). These results are quantified in Figure 7P.

Loss of Tbx3 compromises the Sufu/Kif7 complex required for Gli3FL stability and normal processing of Gli3R

To obtain further mechanistic understanding into how loss of Tbx3 results in decreased Gli3 proteins, we assayed levels and interactions of members of the Gli3 processing machinery. Gli3FL stability and partial processing versus complete degradation are regulated by opposing actions of Suppressor of fused (Sufu) and speckle-type POZ protein (Spop), respectively (Wang et al., 2010; Wen et al., 2010). In the absence of Smoothened activation, mammalian Sufu recruits GSK3β to phosphorylate Gli3FL downstream of PKA, allowing for its partial processing to Gli3R (which requires Kif7 and intact cilia) (Kise et al., 2009; Tempe et al., 2006; Wang et al., 2000). Spop is an adaptor for Cullin3-based E3 ubiquitin ligase that drives complete degradation of Gli3FL in the absence of Sufu, but facilitates its processing to Gli3R when Sufu is present (Humke et al., 2010; Wang et al., 2010; Zhang et al., 2006). In contrast to Kif7 and Gli3, we did not detect interactions between Tbx3 and either Sufu or Spop (Figure 7—figure supplement 3). The observation that Gli3FL is virtually undetectable in Tbx3;PrxCre anterior mesenchyme (Figure 4A') indicates that despite the increased amount of Sufu (Figure 4H, Figure 8—figure supplement 1), it fails to prevent degradation of Gli3FL in the absence of Tbx3.

We next tested whether decreased levels of Gli3 proteins in the anterior mesenchyme reflected perturbed interactions between Sufu, Gli3 and Kif7. Limitations in sample quantity made it unfeasible to perform multiple co-IPs on isolated anterior forelimb buds, so we assayed lysates from control and Tbx3Δfl/Δfl E10.5 embryos. The altered levels of protein expression seen in mutant limb buds were recapitulated in whole embryos (Figure 8—figure supplement 1). As seen in mutant limb buds, the amount of both Gli3FL and Gli3R protein is reduced in Tbx3Δfl/Δfl embryos (Figure 8A, lanes 1–4; Figure 8—figure supplement 1; Figure 8—figure supplement 2A and B). Notably, the interaction between Gli3 and Sufu is reproducibly decreased in excess of the decrement in Gli3 proteins; this is evident by quantitating and comparing the ratio of Gli3 proteins detected in control versus mutant to that complexed with Sufu. For example, in the representative experiment shown in Figure 8A, the Gli3 FL band intensity ratio is ~1.6 fold greater in controls (Figure 8A, lanes 1 and 3) than in mutants (Figure 8A, lanes 2, 4), while the amount of Gli3FL protein that was immunoprecipitated with Sufu is 4.6 fold greater in controls than mutants (Figure 8A, lane 9 versus 10). Mean band intensity ratios of 3 replicate experiments are shown in Figure 8A’. The decreased Gli3/Sufu interaction in the absence of Tbx3 was also evident when assayed in the opposite direction, that is, by IP of Gli3 and immunoblotting for Sufu (Figure 8B, panel B’ shows relative band intensities for the experiment shown). Sufu/Gli3R interactions were also affected (Figure 8A’; Figure 8—figure supplement 2A–B). These findings indicate that normal stoichiometry of the interaction of Gli3 proteins with Sufu requires Tbx3.

Figure 8 with 2 supplements see all
Altered stoichiometry of interactions between Gli3 and members of its processing/degradation complex.

(A) Anti-Gli3 immunoblot (IB) on immunoprecipitates (IP) from antibodies listed at top on lysates from E10.5 control (wt) and Tbx3Δfl/Δfl (ko) embryos. Gli3FL and Gli3R are denoted by red arrowheads, IgG heavy chain with black arrowhead. Note decreased levels of IP’d Gli3FL and Gli3R in mutants compared with controls; the IPs in lanes 1–2 and 3–4 are two independent biologic replicates and the band intensity ratio of control to mutant for both GliFL and Gli3R was ~1.6. The interaction between Gli3 and Sufu is decreased more than can be explained by the overall decrement in Gli3 protein levels: in this representative experiment Sufu co-IPs 4.6X more Gli3FL in controls than in mutants (lane 9 versus 10). (A’) Bar graphs show the results of quantitation of band intensities from three3 replicate experiments measured with densitometry and presented as the ratio of signal detected in controls relative to mutants. (B) As in A but immunoblot probed for Sufu. Comparison of lanes 1 and 2 confirms decreased interaction between Gli3 and Sufu, despite preserved levels of Sufu in the mutants (lane 10). (C) Anti-Gli3 immunoblot with IPs as listed at top. Note increased interaction between Kif7 and Gli3 in mutants (lane 4), despite overall decreased level of Gli3 (lane 8). (C’) Quantitation of band intensities from three replicate experiments measured with densitometry and presented as a ratio of signal detected in control relative to mutant. Even though there is less total Gli3 protein, since there is increased interaction between Gli3 and Kif7 in mutants, the Kif7 co-IP control to mutant ratios are <1. (D) Anti-Kif7 immunoblot with IPs as listed at top. Confirms increased interaction between Gli3 and Kif7 in mutants. (D’) Quantitation of experiment in D. (E) Anti-Kif7 immunoblot with IPs as listed at top. The interaction of Kif7 and Sufu is decreased in the absence of Tbx3 (lane 4) despite preserved levels of both proteins (lane 6; Figure 8, panels B, D and F; Figure 8—figure supplements 1 and 2). (E’) Quantitation of band intensities from two replicate experiments measured with densitometry and presented as ratio of signal detected in control relative to mutant. There is 2 fold less interaction between Kif7 and Sufu in mutants. (F) Anti-Sufu immunoblot with IPs as listed at top. The interaction of Kif7 and Sufu is decreased in the absence of Tbx3 (lane 3 versus 4). (F’) Quantitation of findings in F.

https://doi.org/10.7554/eLife.07897.039

In wild type embryos, we detected only trace interaction between Kif7 and Gli3R assaying by co-IP in either direction (Figure 8C, lane 3; Figure 8D, lane 7; Figure 8—figure supplement 2C, lane1). However, there was a robust and reproducible interaction in mutants despite the decrease in the total amount of Gli3 proteins. In the representative experiment shown in Figure 8C, in which we IP’d for Kif7 and assayed for Gli3, the band intensity ratios of control (lane 3) to mutant (lane 4) were 0.1 for Gli3FL and 0.25 for Gli3R. Quantification of the band intensity ratios from three replicate experiments in which we IP’d for Kif7 and assayed for Gli3 is shown in Figure 8C’. Furthermore, the increased interaction of Kif7 with Gli3 was confirmed by IP of Gli3 and assay for Kif7, as shown in Figure 8D: the band intensity ratio of control (lane 7) to mutant (lane 8) was 0.1, indicating a marked increase in interaction in mutants (Figure 8D’ graphs relative band intensities in experiment 8D).

Lastly, the interaction between Sufu and Kif7 was reproducibly decreased when assayed by co-IP in either direction (Figure 8E, lane 3 versus 4; Figure 8F, lane 3 versus 4; Figure 8—figure supplement 2D, lane 7 versus 8). Quantification of the band intensity ratios from replicate experiments in which we IP’d for Sufu and assayed for Kif7 is shown in Figure 8C’. Figure 8F’ graphs relative band intensities in experiment 8F in which we IP’d for Kif7 and assayed for Sufu. Note that this decrease in Sufu/Kif7 interaction occurs despite increased and normal levels of these proteins, respectively. In total, these data indicate that Tbx3 is required for normal stoichiometry and function of the Sufu/Kif7 complex that stabilizes and processes Gli3 as modeled in Figure 9.

Model of compartment specific functions of Tbx3 in forelimb bud mesenchyme and altered interactions and stoichiometry of the Kif7/Sufu Gli3 processing complex in Tbx3;PrxCre mutants.

In posterior forelimb mesenchyme, Tbx3 is required for normal levels of Hand2 upstream of Shh. Shh pathway activity and other Tbx3-reponsive factors promote digit 5 formation. In the absence of Tbx3, there is decreased expression of Hand2 and Shh and other digit 5 promoting pathways. In anterior mesenchyme, Tbx3 is in a complex with Gli3 proteins, Kif7 and Sufu and required for the stability of Gli3 FL and Gli3R. In the absence of Tbx3, Sufu and Spop protein levels are increased yet there is decreased interaction between Sufu and Kif7, and Sufu and Gli3. In mutant anterior mesenchyme, Gli3FL is barely detected: it is either degraded or converted to Gli3R. Levels of Gli3R are abnormally low due to a combination of decreased amount of Gli3FL precursor and its processing to Gli3R, and excess degradation. These findings are consistent with decreased function of cilia and Kif7 (required for processing of Gli3FL to Gli3R), and of Sufu (required for stability of both Gli3FL and Gli3R).

https://doi.org/10.7554/eLife.07897.042

Discussion

Role of Tbx3 in limb bud initiation

Tbx3 expression in the LPM is required for normal Tbx5 expression (Figure 2A–C) implicating Tbx3 as one of the earliest limb initiation factors. This is consistent with our finding that Tbx3+ progenitors in the LPM give rise to the majority of limb bud mesenchyme. Determining the fate of these progenitors in the future will help reveal the mechanism for loss of ulna/fibula and digits 2–5 in Tbx3 null embryos.

Differential sensitivity of the left and right limbs to Tbx3

Intrinsic differences in anatomically bilaterally symmetric structures have long been suspected: acetazolamide teratogenizes only the right limb in rats (Layton and Hallesy, 1965; Wilson et al., 1968) and nitroheterocyclics such as valproate also induce unilateral defects (Coakley and Brown, 1986; Fantel et al., 1986). Directional asymmetry in limb size in human fetuses was recently reported (Van Dongen et al., 2014). Left/right identity differences in bilaterally symmetric structures such as the limbs may be upstream of secondary patterning events such as dorsal/ventral axis appropriate to body side. Shiratori and colleagues reported asymmetric expression of Pitx2c in the developing mouse limb and postulated functional left/right differences (Shiratori et al., 2014). Our finding that the left limb is more sensitive to Tbx3 than the right is consistent with this hypothesis and warrants further investigation in humans with UMS and other mouse models.

Discrete functions for Tbx3 in the anterior and posterior limb bud mesenchyme

A model of molecular mechanisms contributing to the different anterior and posterior limb phenotypes of Tbx3;PrxCre mutants is shown in Figure 9.

Digit 5 formation is exquisitely sensitive to Shh activity and Grem1 (Harfe et al., 2004; Scherz et al., 2007; Zhu and Mackem, 2011; Zhu et al., 2008), so the altered expression of these genes in posterior mutant mesenchyme helps to explain loss of this digit. Shh null heterozygotes (Shh+/-) have normal digit number, suggesting that the loss of digit 5 in Tbx3;PrxCre mutants is not solely due to decreased Shh expression and pathway activity in posterior mesenchyme. Importantly, it is not known if compensatory mechanisms preserve normal Shh protein levels or activity in Shh+/- mutants to support normal limb development. Indeed, a 'buffering system' to modulate polarizing activity by Shh was proposed in the chick limb (Sanz-Ezquerro and Tickle, 2000).

We found evidence of dysfunction of other digit 5 promoting pathways in posterior mesenchyme of Tbx3;PrxCre mutants. Compound Hand2/Gli3 null mutants have more severe polydactyly than Gli3 mutants (Galli et al., 2010), indicating that the role of Hand2 in digit formation is not limited to regulation of Shh expression. Aberrant expression of other BHLH factors (Supplementary files 1,3: Hand1, Bhlhe40, Bhlhe41, Bhlha15) may also contribute to the phenotype because the stoichiometry and interactions of BHLH factors have complex roles in limb development (Firulli et al., 2005). Altered levels of BMP responsive targets (Dkk1, Noggin, Grem1, Grem2) in Tbx3;PrxCre mutants suggest disrupted BMP signaling. Overexpression of Gata6 decreases Shh and Grem1 expression and causes loss of posterior digits (Kozhemyakina et al., 2014); we detected increased Gata6 expression in the posterior mesenchyme of Tbx3;PrxCre mutants (Supplementary file 3). Additional studies are needed to determine if Tbx3 transcriptional or post-transcriptional functions directly regulate these pathways.

Although Tbx3 is also present in posterior mesenchymal cilia, we did not detect any evidence of altered stability or processing of Gli3 in the posterior mesenchyme (Figure 4A, representative of three replicates). Nonetheless, it is possible that decreased Shh signaling in the posterior of Tbx3;PrxCre mutant limb buds creates changes in Gli3 levels/ratios below our ability to detect. If so, decreased Shh activity would be predicted to increase Gli3R, decreasing digit number.

It is notable that decreased expression of Tbx2 observed in Tbx3;PrxCre mutants would be predicted to result in increased Grem1 expression (Farin et al., 2013) however, Grem1 expression is decreased (Figure 3). Decreased Shh signaling and Grem1 expression in the posterior mesenchyme would both be predicted to result in decreased Fgf4 and Fgf8 expression in the posterior AER (Khokha et al., 2003; Michos et al., 2004). Rather, the finding that these transcripts are increased in the posterior (Figure 3—figure supplement 4) indicates that loss of Tbx3 in posterior mesenchyme disrupts the Shh-Grem1-FGF signaling loop.

In wild type anterior mesenchyme, our data support the model that Tbx3 is part of a Kif7/Sufu complex that drives processing of the majority of Gli3FL to Gli3R and also prevents complete degradation of Gli3FL by Spop (Wang et al., 2010). Note that our model and data show a marked decrease in Gli3FL (virtually undetectable by western blot in anterior mesenchyme), but residual Gli3R in mutants. This is consistent with the phenotype of Gli3 deficiency, and digit 1 polysyndactyly is also seen with decreased function of Kif7 or Sufu. Residual Gli3R in the mutants is sufficient to prevent the extreme polydactyly seen in complete absence of Gli3, Kif7 or Sufu. In Tbx3;PrxCre mutant limb buds, Kif7 and Sufu proteins are present at normal and increased levels, respectively, but their interaction with each other, and that of Sufu with Gli3, are decreased. Because Spop facilitates processing of Gli3FL to Gli3R in the presence of Sufu, increased levels of Spop in mutants drives complete degradation of any Gli3FL not converted to Gli3R, resulting in undetectable levels of Gli3FL in anterior mesenchyme. The decreased levels of Gli3R in the anterior likely result from a combination of decreased levels of Gli3FL precursor, inefficient processing due to altered Kif7/Sufu function, and excess Gli3R degradation, as evident by the lower molecular weight species present in Figure 3A.

This synthesis is consistent with the large body of published data indicating that anterior digit number is tightly regulated by the balance of Gli3A and Gli3R, in turn controlled by Kif7 and Sufu (Cao et al., 2013; Wang et al., 2000; 2007a; 2007b; Zhulyn et al., 2014). Kif7-/- and other ciliary mutants maintain robust levels of Gli3FL but have a marked decrease in processing it to Gli3R and their PPD phenotypes are consistent with decreased Gli3R (Cheung et al., 2009; Endoh-Yamagami et al., 2009). Furthermore, recent studies from Chi Chung Hui’s group demonstrate that Kif7;PrxCre mutants have anterior PPD and that increasing Gli3R or decreasing Gli activators rescues all but digit 1 duplication (Zhulyn and Hui, 2015).

Tbx3 mutant limbs also display evidence of Sufu dysfunction with markedly decreased levels of Gli3R and absent Gli3FL. Levels of Gli2, Gli3FL and Gli3R are all drastically reduced in Sufu mutants, even in the absence of cilia, consistent with the fact that Spop is not a ciliary protein and drives degradation of the full length proteins in the absence of Sufu (Chen et al., 2009; Jia et al., 2009; Svard et al., 2006; Wang et al., 2010). However, βTrCP and Spop can only mediate processing of Gli3FL to Gli3R in the presence of both Sufu and Kif7/intact cilia/intraflagellar transport (Endoh-Yamagami et al., 2009; Law et al., 2012; Liu et al., 2005; Wang et al., 2010; Wen et al., 2010). This processing is believed to occur at the basal body/centrosome (Ryan and Chiang, 2012; Wang et al., 2013; Wen et al., 2010; Wigley et al., 1999). Thus, our data strongly support that Tbx3 functions at the cilia or basal body, as a part of the complex that regulates Gli3 processing and stability (Ryan and Chiang, 2012; Wen et al., 2010).

Loss of Tbx3 could also influence stability and processing of Gli2. The anterior PPD phenotype of Gli3 heterozygotes is slightly more severe in a Gli2 null background (Bowers et al., 2012; Mo et al., 1997). Gli2 is also expressed in the posterior mesenchyme where it regulates digit patterning but not digit number (Bowers et al., 2012). Bowers’ study also demonstrated that the role of Gli activators is in AP patterning of the posterior limb, whereas Gli repressors regulate digit number and anterior limb AP patterning. This is consistent with our findings that Tbx3 affects anterior digit number by regulating Gli3 repressor stability in the anterior mesenchyme.

The functional significance of increased interaction between Kif7 and Gli3R that we detect in mutants requires additional study nonetheless, since the anterior phenotype of Tbx3;PrxCre mutants is one of Gli3R deficiency rather than absence, we conclude that either there is a pool of Gli3R unbound by Kif7 and/or Gli3R complexed with Kif7 still has repressor function.

In addition to its function as a Gli3R co-repressor, Zic3 also regulates cilia morphogenesis and function (Sutherland et al., 2013). Since decreasing Zic3 levels in Gli3+/- mutants rescues their preaxial polydactyly (Quinn et al., 2012), Zic3 overexpression in Tbx3;PrxCre mutants may contribute to the PPD phenotype.

Our data exclude ectopic Shh pathway activity as a cause of the PPD in Tbx3;PrxCre mutants: using multiple methods at multiple stages, we did not detect anterior Shh, Gli1, or Ptch1 expression in the anterior mesenchyme. Notably, expression of the BMP antagonist Grem1 is normally increased in PPD associated with ectopic anterior Shh pathway activity (Lopez-Rios et al., 2012; Zhang et al., 2009), but in Tbx3 mutants, Grem1 expression is markedly decreased. This is additional evidence in support of unique Tbx3-dependent pathways regulating digit number.

Materials and methods

Mice

Experiments were conducted in strict compliance with IACUC/AALAC standards. The Tbx3flallele was detailed in Frank et al. (2013). Prx1Cre, RARCre and Rosa26LacZ were previously reported (Soriano, 1999; Moon and Capecchi, 2000; Logan et al., 2002). Generation of the Fgf8mcm and Tbx3mcm alleles will be described elsewhere.

β-Galactosidase detection

Males bearing Fgf8MCM or Tbx3MCMalleles were crossed with Rosa26LacZ/+ females. Females were gavaged with tamoxifen (10mg/gm body weight) at stages stated in text. β−galactosidase activity was assayed using established protocols (Park et al., 2006).

Skeletal preparations

E15.5 fetuses were fixed in 4% PFA overnight, rinsed in water for 2 days and alcian blue stained for 30 hr, then cleared in BABB. Older specimens were processed as in Moon et al. (2000).

Whole mount RNA in situ hybridization

Digoxigenin-labeled riboprobes were generated according to manufacturer’s instructions (Roche). Embryos were processed using a standard protocol (Park et al., 2006).

RNA isolation and reverse transcription–qPCR analysis

Total RNA preparation, cDNA generation and qPCR were carried out as described in Yu et al. (2010). Primer sequences are provided in Supplementary file 5. All qPCRs were performed on a minimum of three biologic replicates of pooled forelimb buds (Figure 3) or anterior and posterior forelimb bud segments (Figure 3—figure supplement 4).

Gene expression analyses by microarray

Total RNA was prepared from three pools of dissected E10.25 control (Tbx3 fl/+) and Tbx3;PrxCre mutant forelimbs using the RNAeasy Micro Kit (Qiagen 74004). The microarray and genomic analysis and bioinformatics core facilities at the University of Utah performed Agilent mouse whole-genome expression arrays and array image data analysis using Agilent Feature Extraction software. Subtle intensity-dependent bias was corrected with LOWESS normalization, with no background subtraction. Statistical analysis of normalized log-transformed data was performed in GeneSifter (www.genesifter.net). Differentially expressed transcripts were defined (adjusted for multiple testing using the Benjamini and Hochberg method) as p<0.05. The results presented in Supplementary file 1 show transcripts that were statistically differentially expressed +/-1.3 fold in the mutant limb buds; yellow highlighting indicates changes that were replicated by RNA-Seq.

RNA-sequencing to detect differential gene expression

Total RNA was isolated from pools of dissected anterior and posterior regions of E11 control (Tbx3fl/+) and Tbx3;PrxCre forelimbs using the RNAeasy Micro Kit (Qiagen). Each pool contained 12 forelimbs and two biologic duplicate pools were assayed. cDNA was generated, sequenced, and raw sequence reads were processed as described in Kumar et al. (2014b). Supplementary file 2 contains transcripts that are differentially expressed +/-1.3 fold (+/-0.38 in log base 2, column L) in control anterior (CA) compared to control posterior (CP) limb segments. The transcripts listed in Supplementary file 3 were the result of mining the data to detect differential expression +/-1.3 fold (+/- 0.38 in log base 2, column L) in control posterior (CP) relative to mutant posterior (MP) segments. Supplementary file 4 shows the result of mining the data to detect differential expression +/-1.3 fold (+/-0.38 in log base 2, column L) in control anterior (CA) relative to mutant anterior (MA) limb segments.

Note that transcripts that were not differentially expressed +/-1.3 fold are not listed on the tables. The complete unmined dataset is available on GEO.

Immunoblotting

Protein lysates were prepared from E10.5 dissected limb buds or embryos or cultured MEFs using Dignam buffer. 50 ug of total protein were then subjected to SDS-PAGE analysis followed by immunoblotting according to standard protocols. Primary antibodies: Sufu (#2522, Cell signaling), Spop (#PA5-28522), Myc (#SC-789, Santa Cruz), Flag (#F7425, Sigma), E2F1 (#137415, Abcam), Ubiquitin (#7780, Abcam), Gli3 (AF3690, R and D systems), Tbx3 C-terminal antibody (Frank et al., 2013); Kif7 (ab 95884, Abcam); β tubulin (Santa Cruz).

Immunoprecipitations

Immunoprecipitations were performed as described in Kumar et al., 2014a and 2014b. Briefly, protein lysates were prepared from limb buds or embryos at E10.5 or transfected HEK293 cells using Dignam buffer C. Cleared protein lysates were obtained by centrifugation at 12,000g for 10 min. Equal amounts of protein lysates were incubated with 5–10μg of respective antibodies over night at 4°C with gentle shaking. Immune complexes were isolated with Protein-G Dynal beads and washed three times with Dignam buffer C. Precipitates were eluted from the beads by boiling in SDS-loading dye for 10 min, and analysed by western blotting by standard procedures using indicated antibodies at a dilution of 1:1000.

Densitometry analysis

Immunoblot signals were quantified by densitometry using ImageJ64 software as per the procedure described by Luke Miller (http://www.lukemiller.org/ImageJ_gel_analysis.pdf).

Immunofluorescence of sectioned and intact limb buds

Sections: Samples were fixed in 4% PFA at 4°C for 2 hr then washed with 0.3% Triton-X100 (in PBST). Heat retrieval in citrate buffer (Vector Laboratories) was performed for 2 min in a pressure cooker. Slides were washed with PBST and incubated in PBST with 5% serum corresponding to the secondary antibody origin for 1 hr. Slides were incubated with primary antibody in PBST 5% serum overnight at 4°C. Primary antibodies and dilutions: Tbx3 C-terminal antibody 1/200 (Frank et al., 2013), Tbx3 N- terminal antibody 1/100 (Abcam ab99302), Tbx3 internal epitope antibody (Santa Cruz A-20), Arl13b 1/100 (USDavis/NIH NeuroMab Clone N259B/66), Gli3 1/100 (R&D systems AF3690), pHH3 (Ser10), Millipore 06–570(1:2000); (1:50); Kif7 (1:200, kind gift from Dr. C-c Hui). After washing in PBST for 15 min, slides were incubated with secondary antibody from either Invitrogen or Jackson Immunoresearch diluted 1/1000 in PBST 2% BSA with Hoechst 33,342 at 1ug/ml for 1h at room temperature. Final wash was with PBST for 15 min and mounted using Fluoromount-G from Southern Biotech. Secondary antibodies: Donkey anti-rabbit Alexa 488; Donkey anti-goat Alexa 488; Donkey anti-mouse Alexa 647; Donkey anti-goat Alexa 594.

Whole forelimb buds: samples were fixed in 4% PFA for 1 hr at room temperature with PBST. Heat retrieval in citrate buffer (Vector Laboratories) was performed for 5 min at 100°C followed by washing with PBST. Limb buds were incubated in PBST with 5% serum that correspond to the secondary antibody origin (Goat or Donkey) for 2 hr. Limb buds were incubated with primary antibody in PBST with 5% serum overnight at 4°C, then washed with PBST once and incubated in PBST with 2% BSA for 4 hr. Incubated with secondary antibody and Hoechst as above with overnight at 4°C. Prior to imaging, samples were washed with PBST for 4 hrs and incubated in PBS/50% glycerol overnight.

TUNEL: was performed as described in (Park et al., 2006)

Confocal microscopy and image processing of immunofluorescence on intact limb buds

Imaging was performed using a Zeiss confocal microscope LSM710 with Zen black imaging software http://www.zeiss.com/microscopy/en_us/downloads/zen.html. 100x objective was used.

To generate the Arl13b/Tbx3 colocalization maps, we used Zen to define pixels in each plane of z that exceeded an arbitrary threshold of 0.1 relative intensity in both Arl13b and Tbx3 channels using the imaging calculation subtab in the image processing menu. This was followed by calculation of the maximum intensity projection. To quantitate Tbx3 positive cilia, the 3D object counter in ImageJ (Bolte and Cordelieres, 2006) was used employing the “redirection” option to superimpose Arl13b+ objects into the Tbx3 channel.

Assaying cilia volume and length

Cilia volume was assayed by quantitating Arl13b signal on 100x images of sectioned forelimbs from four pairs of mutant and control embryos stained for Arl13b and analyzing resulting in ImageJ. Imaged were Gaussian blurred using radius equal to '1' and further 3D object counter was used to measure volumes and surface area with threshold of 900 out of 4000 range. Distributions and parameters of the volume distributions shown in Figure 4—figure supplement 2A and B were calculated in Excel.

We calculated that the surface area and volume of both WT and mutant cilia tightly fit the equation: Surface=8.01 X Volume0.69 (Figure 4—figure supplement 2D), which indicates that the cilia shapes are the same in the mutants and controls, and change size proportionally. In this case, the relative change in volume between mutant and control (Volume mutant)/(Volume control)=1.1747, which correspond to a change in length of 6%.

MEF Immunocytochemistry

MEFs from E10.5 embryos were plated on fibronectin coated Matteks. MEFs were cultured and processed as in Kumar et al. (2014a); cilia outgrowth was stimulated by culture in 0.5% FBS for 24 hr followed by incubation with 100 nM SAG or 100 nM SHH (Phoenix Pharmaceuticals) for 24 hr. Cells were washed 2x with PBS and fixed in 4% PFA for 20 min on ice, then washed 2X with PBS and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After blocking in 5% donkey serum + 3% BSA for 1 hr, cells were incubated with anti-TBX3 (custom C-terminal or Santa Cruz sc-17871) and anti-Arl13b (1:50 NeuroMAb) at room temperature for 2 hr. Cells were washed 5 X 5 min in PBS and then incubated in secondary donkey anti-goat-488 or anti-rabbit 488 and donkey antimouse -594 (Jackson Immunoresearch). After washing 3 x 5min in PBS, cells were incubated in DAPI for 20 min. Cells were then washed 3X with PBS and mounted in Dabco for imaging. Images were captured on a Zeiss LSM-710 confocal microscope and processed using Zen software as described above.

Assaying protein interactions with transfected, tagged proteins

HEK-293 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. Cells were maintained in 5% C02 incubator at 37°C. Myc- tagged full length TBX3 was generated as previously described (Kumar et al., 2014b). The Flag-tagged Gli3 construct was obtained from Addgene (51246) and the Flag-tagged Kif7 construct was a kind gift of Kathryn Anderson. Transfections of plasmids were performed with Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer’s protocol.

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Decision letter

  1. Anna Akhmanova
    Reviewing Editor; Utrecht University, Netherlands

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for choosing to send your work entitled "Tbx3 is a Ciliary Protein that Regulates Gli3 Stability to Control Digit Number" for consideration at eLife. Your full submission has been evaluated by Janet Rossant (Senior editor) and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

All referees agreed that the part of the paper describing the phenotype of Tbx3 loss in limb development is solid, and thus the comments concerning this part were relatively minor. However, the referees were much more skeptical about the strength of the experimental support for the most novel and interesting claim, the localization of Tbx3 to the cilia and the interaction of Tbx3 with the Hedgehog pathway components such as KIF7. Both the biochemical and subcellular localization experiments would need to be very significantly extended (for example, by using tagged proteins for immunoprecipitation assays), and more thorough controls for these experiments would need to be included. Also the strength and the value of the RNA-seq data presented in the paper were questioned, and these data thus need to be improved. Taken together, it appears that very significant additional work will be necessary to make the story convincing.

Reviewer #1:

This paper describes the role of Tbx3 in limb development and connects this function to cilia-dependent regulation of the Hedgehog pathway. The authors use conditional ablation of Tbx3 to characterize in detail its role in limb formation. The authors also show that Tbx3 localizes to cilia, interacts with Gli3 and Kif7 and affects the formation of protein complexes involved in Hedgehog signaling.

I think that the paper is interesting and potentially suitable for publication in eLife. However, while the phenotypic description of Tbx3 loss appears solid to me, the part describing the biochemical interactions and ciliary localization of Tbx3 misses important controls and is therefore less convincing.

1) Figure 5A: it is very surprising that Tbx3 precipitates with equal efficiency with Tbx3 and Kif7 antibodies. Anti-Kif7 blot must be included here to show the efficiency of Kif7 immunoprecipitation. In the legend, the% of input loaded must be indicated, so that the efficiency of immunoprecipitation can be estimated.

A similar comment applies to Figure 6O. A Tbx3 blot must be included in this panel, as well as the input lanes, so that the efficiency of immunoprecipitation can be estimated. The loading of IgG in the control lane seems to be lower than in the two other lanes, which makes the value of this control doubtful. It would be preferable, instead of IgG, to use as a negative control antibodies against another protein, which does not bind to Gli3 or Tbx3.

Similar comments also apply to the blots shown in Figure 7E, G and H. Arl13 blot should be shown in panel E, Kif7 blot in panel G and Sufu blot in panel H. Are the cut blots in panel E (including input lanes) derived from the same or different gels and do they show the same exposures? The observed differences in immunoprecipitation efficiency are not convincing and therefore need to be quantified using three independent experiments for all data shown in Figure 7.

2) Are there any indications that the interactions of Tbx3 with Kif7 and Gli3 might be direct? For example, do these proteins co-immunoprecipitate when overexpressed in cultured cells?

3) The specificity of the Tbx3 staining in cilia (Figure 6) must be confirmed using Tbx3 knockout cells.

Reviewer #2:

This is an extensive body of work using a range of genetic tools and molecular approaches to tease apart the function(s) of Tbx3 in the limb.

The first part of the paper describes an analysis of the phenotypes produced following conditional deletion of Tbx3 at different times and places during limb development using a variety of cre lines. I have relatively minor comments on this.

The second, and main part of the paper (Figures 58) covers all the work from which the title of the manuscript derive. I have several concerns here and in general remain skeptical (but with an open mind) about the data and particularly the interpretation.

In many instances I feel the authors have gone too far in their conclusions without the necessary experimental data to support the final conclusions.

First and foremost, immunostaining for Tbx3 and Arl13b on limb sections is interpreted as showing Tbx3 in limb mesenchymal cilia. Immunostaining of Tbx3 is throughout the entire cell and apparent co-expresssion with Arl13b could simply be explained by overlap in stain. Much higher resolution microscopy imaging that can circumvent these potential artifacts is required. The staining on MEFS do not clarify the issue. It is not clear why the Tbx3 staining pattern is so different in these cells from that shown in A-C. (Other issues such as the legend for panels H-K states 25/33 cilia were Tbx3+. I do not see more than 20 cells or so in this panel further compounding the problem) (It can also be argued that any patterns identified in MEFs may not have relevance to the limb.)

I also could not see a clear effect on KIf7 localization form the panels C in Figure 5. Table E appears to be an extreme way to show an apparent 8% difference on cilia length (I could not see where these numbers came from). This section ends with the conclusion that the data show that Tbx3 is present at baseline in cilia and is trafficked to and within cilia in response to hedgehog signaling. Unfortunately, I remain unconvinced of any of this.

There are similar issues with the data in Figure 7. Fundamentally I don't see clear evidence of co-expression of Tbx3, Gli3 Arl13b although I appreciate that IP data is also included.

The data on effect on GLi3 stability are potentially interesting but perplexing

What is the effect on Gli3 expression in the mutant limbs? It might be expected following reduction of hand2 and Shh expression in the mutant that Gli3 expression would be expanded but the western data would suggest not. Is there more GLi3FLin the MP? It is perhaps surprising that apparently equivalent levels of Gli3R are present in CP and MP.

The Tbx3 cKO is close (but I would not say identical to or a phenocopy of) a Het Gli3 mutant. This is not conveyed in the final schematic which suggests a critical role of Tbx3 in Gli processing and that this in its absence all Gli processing is affected. I would expect a more profound polydactyly if this were the case. The actual 'polydactyly that presents lacks a structure that could be considered a true digit (1H, H').

A further question raised is that if Tbx3 is playing such a critical role in Gli processing/Shh signalling in the anterior is it or why is it not playing a similar role in the posterior. This is not discussed and does not appear to have been studied.

Some other minor issues from Figure 1 include:

1L: I do not see any loss of Sox9 activity in the region indicated with the red arrow head. This domain looks relatively normal to me.

I do not see clear evidence of a delay in ossification. In the text the authors conclude that this "reveals a role for Tbx3 in later aspects of Indian hedgehog regulated bone morphogenesis.” This has not been investigated to any extent and this type of comments goes far beyond what is actually shown in this paper. Also here the loss of the deltoid tuberosity need not have anything to do a direct effect on bone (cf Blitz, E, 2009).

Comparison of the phenotypes produced using the early acting (RARCre) and later acting (Prx1Cre) appears to show an effect of Tbx3 on the early stages of limb initiation that can be distinguished form later events of limb bud patterning through establishment of Shh in the posterior. A regulatory relationship between Tbx3, Hand2 and Shh has been reported in chick (Rallis, 2005).

Decreased expression of markers detected by RNAseq analysis is interpreted as evidence that fewer cells are undergoing condensation and chondrogenesis in posterior mesenchyme. This would be much better analyzing in a more direct way rather than making inferences from RNA-seq data.

Similarly, 5th digits agenesis is attributed to deceased cell number as opposed to decreased proliferation of digit 5 progenitors. These conclusions are drawn from a broad analysis of effects throughout the limb bud rather than focusing on the cells in question themselves.

The authors have some interesting results and observations. I remain open to the ideas they are suggesting but I do feel strongly that currently the data shown does not support their main conclusions.

Reviewer #3:

The study described the limb phenotype in several Tbx3 conditional and null mutants in careful detail. Most of the conclusions based on whole mount RNA in situs are convincing and new findings. The most novel and surprising finding of the study is that the transcription factor TBX3 is localized to the cilium and interacts with KIF7. HOX proteins have been shown to interact with and inhibit GLI3 in the limb, however cilium localization was not explored. Given that the vast majority of TBX3 protein is in the nucleus (Figure 6), it seems surprising to me that the tiny amount in the cilium can be detected with IP (Figure 7E-H). The interaction with GLI3 could be in the nucleus and/or cilium, but KIF7 should not be in the nucleus. To further bolster the most significant claim of the paper, I think that additional experiments are needed. In particular, the specificity of the antibodies must be proven (e.g. using Tbx2 mutant MEFs for the Tbx3 antibody) and ideally some results confirmed using tagged proteins. Tagged GLI3 is available and other proteins could be easily generated. A genetic test would be to determine whether heterozygosity for Kif7 in mice (or knockdown in cells) rescues the defect proposed to be due to increased KIF7. In addition, many of the claims of changes in gene expression based on the RNA-seq data in Tables 2 and 3 do not seem to be significant differences, and some are opposite to what is said in the Results. Thus, the value of the RNA-seq data, is not clear, especially as only two samples were analyzed for each genotype, although I appreciate it is a difficult experiment. Many of the additional points listed below are simple changes that would make the paper more accessible and data more convincing.

Figure 3A': The mutant limb looks smaller than the control limb in (A), therefore the domain is expected to be smaller. The text implies the domain is smaller relative to the size of the limb. Which is the case?

"[…]downregulation of posterior genes including Shh, Shh pathway members, Tbx2, Sall1, Dkk1, and Osr1[…]": The decrease is very little (e.g. Sall1 -0.3). More important, why are Grem1, Ptch1 and Gli1 not down in the RNA-seq, which would confirm the data in Figure 3C, D and address the question of whether the decrease is just due to the limb being smaller?

"[…]up regulation of (AFP, Ttr, Apob, Apoa1, Apoa4, Trf, Ttpa)[…](Table 2).”: Apob and Ttpa are actually down and the others are barely up in the anterior (Table 2).

Results: "more single puncta cilia in mutants than controls (Figure 5E 84% vs 71%, p<0.001). The levels of Kif7 mRNA[…] unaffected[…] Tables 2, 3[…]": In Table 3 Kif7 is -0.3, which is greater than many of the changes in expression level claimed elsewhere in the text.

Results: "The presence of Tbx3 in cilia and response to Hedgehog pathway activation was confirmed with a commercially available anti-Tbx3 antibody and both SAG and Shh stimulation (Figure 6M, N).": Tbx mutant fibroblasts should be shown to demonstrate specificity of the antibody.

Tagged proteins should be used to confirm the interactions thought to be detected with commercial antibodies.

Figure 7C, D: quantification is needed.

Results: "Consistent with our hypothesis, there was decreased interaction between Gli3FL and Sufu in mutants (Figure 6E, lane 8[…]": Do the authors mean Figure 7E? Also, since Gli3 FL and R are reduced, does this experiment say anything?

Discussion: "Future studies will determine if Tbx3 transcriptional or posttranscriptional activity regulates production of a factor that represses expression or stability of Hand2 mRNA.": In the posterior limb, wouldn't Tbx3 also interfere with processing of Gli2/3 into activators, like in cilia mutants, and this could explain the posterior limb phenotype by the same mechanism as the anterior limb bud?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Tbx3 is a Ciliary Protein and Regulates Gli3 Stability to Control Digit Number" for further consideration at eLife. Your article has been favorably evaluated by Janet Rossant (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) There are some remaining concerns about the quality of the biochemical data. Specifically, the composition and the order of loading of the lanes is not the same in Figure 4B, C and Figure 7A, B, creating the impression that the shown immunoprecipitations are not from the same experiment. Gli3 and KIF7 blots should be included in Figure 7C. Most importantly, error bars should be included in the plots showing the quantification of immunoprecipitation results in Figure 8 in order to illustrate that the experiment was performed more than once and the observed differences are significant.

2) Better discussion should be included to explain why the authors insist that TBX3 does not regulate GLI3 function in the posterior limb, and why they have ignored the highly related protein GLI2. If their hypothesis is correct, one would expect the same might be true for GLI2 since it also binds SuFu and requires cilia for proper processing. Also, if whole embryos reveal the same protein interactions as the anterior limb, why would the posterior limb be different? Gli2/3 double limb mutants could well have a worse posterior limb phenotype than Gli3 null or het mutants. Only a relatively late double conditional mutant of Gli2/3 has been published, and it indicated the posterior phenotype is worse than in single Gli3 mutants. Thus, some of the posterior phenotype in Tbx3 mutants could be due to the altered GLI processing and activity in this tissue, rather than just the decreased Shh expression, especially as Shh heterozygous mutants have normal limbs. Finally, it seems to be an over-interpretation of the data to say that the posterior limb is not dependent on cilia. In cilia mutants’ production of both activators and repressors is altered (diminished), but obvious phenotypes are only seen where the balance is greatly skewed. It would be nice to extend the Discussion of your paper to address these points.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "T-box 3 is a Ciliary Protein and Regulates Gli3 Stability to Control Digit Number" for further consideration at eLife. Your revised article has been favorably evaluated by Janet Rossant (Senior editor) and a reviewing editor. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) The scan of the Western blot shown in Figure 7C is not publication quality. Please substitute it for a high-resolution scan. Also, please make sure that in all cases where lanes of Western blots have been left out, a clear separator line is present (this currently seems not to be the case in Figure 7C, 7C' and 8E).

2. Please reconsider the title of your paper. "T-box3 is a ciliary protein and regulates.." doesn't read well. Furthermore, eLife encourages the authors to provide brief explanations for the acronyms used in the title and Abstract. For your paper, mentioning that Gli3 is a player in Sonic Hedgehog signaling would be appropriate.

https://doi.org/10.7554/eLife.07897.048

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

1) Figure 5A: it is very surprising that Tbx3 precipitates with equal efficiency with Tbx3 and Kif7 antibodies. Anti-Kif7 blot must be included here to show the efficiency of Kif7 immunoprecipitation.

We agree; we have repeated the co-IP a total of 4 times: a different experiment is shown (now in Figure 4B) than in the original submission and another example is in the new Figure 7A. The anti-Kif7 blot requested by the reviewer reveals that as predicted, Tbx3 is not as efficiently IP’d by anti-Kif7 antibody as Kif7 itself.

In the legend, the% of input loaded must be indicated, so that the efficiency of immunoprecipitation can be estimated.

% input added to the figures and legends as requested.

A similar comment applies to Figure 6O. A Tbx3 blot must be included in this panel, as well as the input lanes, so that the efficiency of immunoprecipitation can be estimated. The loading of IgG in the control lane seems to be lower than in the two other lanes, which makes the value of this control doubtful. It would be preferable, instead of IgG, to use as a negative control antibodies against another protein, which does not bind to Gli3 or Tbx3.

We have made the additions and changes as requested, and used an additional negative control antibody as suggested by the reviewer. These data are now presented in the current Figure 7.

Similar comments also apply to the blots shown in Figure 7E, G and H. Arl13 blot should be shown in panel E, Kif7 blot in panel G and Sufu blot in panel H. Are the cut blots in panel E (including input lanes) derived from the same or different gels and do they show the same exposures? The observed differences in immunoprecipitation efficiency are not convincing and therefore need to be quantified using three independent experiments for all data shown in Figure 7.

Because it requires that a much greater amount of protein be loaded to detect these proteins from non- IP’d material, the blots in panel E were from a different gel to avoid exposure and well distortion issues that arise from the different amounts of protein loaded. The experiments originally presented in Figure 7 have now been repeated a minimum of 3 times and examples are shown in Figure 8 and Figure 8—figure supplement 2.

2) Are there any indications that the interactions of Tbx3 with Kif7 and Gli3 might be direct? For example, do these proteins co-immunoprecipitate when overexpressed in cultured cells?

We have performed the co-IPs with tagged Kif7 and tagged Gli3 as requested (Figure 4 C, D and Figure 7—figure supplement 1) in HEK293 cells. We found that Flag-Kif7 and Myc-Tbx3 interact, but Flag-Gli3 and Myc-Tbx3 do not under these conditions.

3) The specificity of the Tbx3 staining in cilia (Figure 6) must be confirmed using Tbx3 knockout cells.

We agree and apologize for not including this critical control in our initial submission. We have now repeated these experiments and used Tbx3 mutant limb buds (Figure 5) as well as Tbx3 mutant MEFs (Figure 6) and confirmed the specificity of signal in the cilia. Please note that in addition to our custom antibody against the C-terminus of Tbx3, 2 different commercial anti-Tbx3 antibodies detected Tbx3 in cilia (Figure 5—figure supplement 2, Abcam anti-Tbx3 antibody; Figure 6—figure supplement 1, panels C, D SantaCruz A-20 anti-Tbx3 antibody).

Reviewer #2:

First and foremost, immunostaining for Tbx3 and Arl13b on limb sections is interpreted as showing Tbx3 in limb mesenchymal cilia. Immunostaining of Tbx3 is throughout the entire cell and apparent co-expresssion with Arl13b could simply be explained by overlap in stain. Much higher resolution microscopy imaging that can circumvent these potential artifacts is required. The staining on MEFS do not clarify the issue.

We agree; please see response to Reviewer 1 above and the new data provided in control and mutant limb buds, including 100X confocal maximum image projections in Figure 5 and the z-stacks provided in Videos 3 and 4.

It is not clear why the Tbx3 staining pattern is so different in these cells from that shown in A-C.

We have shown that the staining pattern of Tbx3 is very context/tissue dependent (Frank et al., 2012; Frank et al., 2013, Kumar et al., 2014a eLife). In this case, serum starved MEFs are very different than the rapidly proliferating mesenchymal cells in the limb bud. Even within the limb, cells in the AER have a different pattern of Tbx3 staining (mostly cytoplasmic, none in cilia) than those in the mesenchyme (cytoplasmic, nuclear and cilia). In some cell types, such as the dorsal root ganglia, all staining is nuclear. The MEF pattern shown here is very similar to what we demonstrated in MEFs in Kumar et al., 2014a, eLife.

(Other issues such as the legend for panels H-K states 25/33 cilia were Tbx3+. I do not see more than 20 cells or so in this panel further compounding the problem) (It can also be argued that any patterns identified in MEFs may not have relevance to the limb.)

We apologize for this error; the text was written to describe a larger, lower magnification panel that we subsequently chose to replace with the panel presented. In the revision, we have replaced all of this data with cleaner, higher magnification/better resolution confocal images in Figures 6 and 7 that can also be viewed orthogonally and through z-stacks using the free Zen software that we employed to obtain and process the images http://www.zeiss.com/microscopy/en_de/downloads/zen.html. In addition to viewing z-stacks through the limb or MEFs, this software also permits the entire stack to be viewed in 3D and orthogonally so that the overlap is visible at the pixel level. We have also employed the image calculator function of this software to objectively quantitate overlap at the pixel level between the Arl13b and Tbx3 signals in control versus mutant. This method is the most sensitive and objective available to us to define true colocalization. It confirms the presence of Tbx3 in cilia and clearly shows Tbx3 immunoreactivity in limb mesenchymal cilia that is virtually absent after Cre recombination in mutants.

I also could not see a clear effect on KIf7 localization form the panels C in Figure 5.

The goal of the panel as shown was to provide the overall picture of a 40X confocal field and to show that there is not an appreciable difference in the number of ciliated limb mesenchymal cells between control and mutant. However, as stated in the legend, the white arrowheads denote the cilia with multiple spots or streak of Kif7 signal. This is quantitated on a much larger scale from many such fields in the bar graph which represents the results from 3 independent reviewers blinded to genotype, scored the Kif7 immunoreactive pattern of the cilia on 20 fields from 3 mutants and 3 controls.

Table E appears to be an extreme way to show an apparent 8% difference on cilia length (I could not see where these numbers came from).

We have replaced this Table with more accurate quantitation based on 3D analysis of the cilia, as described in response to Reviewer 1 above; this is in Figure 4—figure supplement 2.

This section ends with the conclusion that the data show that Tbx3 is present at baseline in cilia and is trafficked to and within cilia in response to hedgehog signaling. Unfortunately, I remain unconvinced of any of this.

There are similar issues with the data in Figure 7. Fundamentally I don't see clear evidence of co-expression of Tbx3, Gli3 Arl13b although I appreciate that IP data is also included.

Please see response to Reviewer 1 and extensive new, appropriately controlled data in Figures 58. We are confident that the reproducible immunofluorescence and IP data we have now provided address the reviewer’s doubts.

The data on effect on GLi3 stability are potentially interesting but perplexing.

We agree that this is an extremely interesting finding and while discovering the exact mechanism whereby Tbx3 stabilizes Gli3 is beyond the scope of this manuscript, the fact that Tbx3 is in a complex with Kif7 and Sufu which regulates processing and stability of Gli3 provides the first step toward understanding this.

What is the effect on Gli3 expression in the mutant limbs? It might be expected following reduction of hand2 and Shh expression in the mutant that Gli3 expression would be expanded but the western data would suggest not. Is there more GLi3FLin the MP? It is perhaps surprising that apparently equivalent levels of Gli3R are present in CP and MP.

As shown by the graph of qPCR results in Figure 3, and the new in situ images provided there Gli3 mRNA levels are increased, as is expected with decreased Shh activity (Wang et al.,2000). We have now included an early microarrary experiment which also showed upregulation of Gli3 expression in mutants (new Table 2).

In the posterior mesenchyme, the level of Gli3R is very low compared to anterior and this has been published by numerous groups including (Wang et al.,2000). Although Shh signaling is reduced in Tbx3 mutants, the westerns all indicated that baseline processing of Gli3FL to Gli3R is intact in mutant posterior mesenchyme.

The Tbx3 cKO is close (but I would not say identical to or a phenocopy of) a Het Gli3 mutant. This is not conveyed in the final schematic which suggests a critical role of Tbx3 in Gli processing and that this in its absence all Gli processing is affected. I would expect a more profound polydactyly if this were the case. The actual 'polydactyly that presents lacks a structure that could be considered a true digit (1H, H').

The phenotype of Gli3 null heterozygotes or Gli3R deficiency varies depending on genetic background and protein level; and others have also reported polysyndactyly of digit 1 (Wang 2007;Hill et al., 2009; Lopez-Rios et al., 2012;Quinn et al., 2012). The polysyndactylous digit of Tbx3;PrxCre mutants has two phalanges, the primary criteria for digit 1 (thumb) identity. Even in our mutants, although the penetrance is 100%, the severity of the duplication varies. Tbx3;PrxCre mutants are Gli3 deficient and not completely lacking Gli3R, thus PPD is the observed (and expected) phenotype. In the Discussion we consider other molecular events occurring in these mutants that may contribute to the phenotype, including overexpression of Zic3.

Our schematic model shows that in the anterior mesenchyme, some Gli3FL remains complexed with Sufu, but most is processed to Gli3R (which is also the case in controls); some of this Gli3R is aberrantly degraded which is what our data show. Our model does not indicate that all Gli3 processing is affected. The very low levels of Gli3FL detectable in the mutants are consistent with excess degradation of Gli3FL.

A further question raised is that if Tbx3 is playing such a critical role in Gli processing/Shh signalling in the anterior is it or why is it not playing a similar role in the posterior. This is not discussed and does not appear to have been studied.

With due respect to the reviewer, this is not discussed because, unlike other regions of the embryo, Shh signaling in the posterior limb mesenchyme is not dependent on cilia or Gli3R. Mouse mutants of Kif7 and ciliary components have anterior PPD; posterior digits are apparently normal. Our data indicate that Tbx3 regulates ligand-dependent Shh signaling in the posterior mesenchyme by affecting the level of Hand2 expression upstream of Shh and its downstream activator functions. In the anterior, we show strong evidence that Tbx3 regulates ligand-independent pathway function by controlling the stability and processing of Gli3. Gli3 processing requires Kif7, Sufu and intact cilia. Although the interactions and processing may not occur within the cilia proper; the literature supports that they occur in the cytoplasm or centrosome/basal body adjacent to the cilia (reviewed in Ryan and Chiang 2012; Ingham and McMahon 2009; and Wang et al., 2013; Wen et al., 2010, Humke et al., 2005; Tukachinsky et al., 2010).

As the reviewers are aware, the cellular compartments in which different functionally relevant interactions are occurring is an ongoing and highly controversial area in the field for all components of the pathway. Elegant work has been done with tagged proteins and overexpression systems. Despite this, these issues have not yet been resolved, especially for endogenous interactions and functions of Kif7, Sufu or Gli3. What is clear is that even if the functionally relevant interactions are occurring in the cytoplasm adjacent to (or even far) from cilia, processing of Gli3 to Gli3R requires the cilia.

Some other minor issues from Figure 1 include:

1L: I do not see any loss of Sox9 activity in the region indicated with the red arrow head. This domain looks relatively normal to me.

We agree that Sox9 expression at E10.5 appears normal however, the morphology of the limb is not at this stage and is variable as is apparent from the images in Figure 3 as well. In Figure 1L, the red arrow denotes abnormal indentation/lack of posterior tissue while the red bracket highlights extra tissue in the anterior, digit 1 forming region. We have clarified the text on this point.

I do not see clear evidence of a delay in ossification.

We have now added a label for the ossification center (oc) to the figures and the legends to clarify. Please consider the images of E15.5 skeletal preps in Figure 1G, H and Figure 1—figure supplement 1 panels K-N. There is no ossification center in the humerus of the mutants, whereas all control and the Tbx3fl/fl;Fgf8mcm/mcmmutant (which have normal limbs) specimens have a well-defined ossification center.

In the text the authors conclude that this "reveals a role for Tbx3 in later aspects of Indian hedgehog regulated bone morphogenesis.” This has not been investigated to any extent and this type of comments goes far beyond what is actually shown in this paper.

We have removed this statement; it was a hypothesis that we did not test.

Also here the loss of the deltoid tuberosity need not have anything to do a direct effect on bone (cf Blitz, E, 2009).

We agree and did not make this statement.

Comparison of the phenotypes produced using the early acting (RARCre) and later acting (Prx1Cre) appears to show an effect of Tbx3 on the early stages of limb initiation that can be distinguished form later events of limb bud patterning through establishment of Shh in the posterior. A regulatory relationship between Tbx3, Hand2 and Shh has been reported in chick (Rallis, 2005).

We apologize for failing to cite this reference and have included it in the revision.

Decreased expression of markers detected by RNAseq analysis is interpreted as evidence that fewer cells are undergoing condensation and chondrogenesis in posterior mesenchyme. This would be much better analyzing in a more direct way rather than making inferences from RNA-seq data.

The lack of digit 5 chondrogenesis is shown in Figure 1 panel N and we have now confirmed the RNA-Seq data of decreased expression of Shh activated targets with qPCR (Figure 3 and Figure 3—figure supplement 4.

Similarly, 5th digits agenesis is attributed to deceased cell number as opposed to decreased proliferation of digit 5 progenitors. These conclusions are drawn from a broad analysis of effects throughout the limb bud rather than focusing on the cells in question themselves.

We have now specifically quantified proliferation in the posterior region containing d5 progenitors and added this data to Figure 3.

Reviewer #3: The study described the limb phenotype in several Tbx3 conditional and null mutants in careful detail. Most of the conclusions based on whole mount RNA in situs are convincing and new findings. The most novel and surprising finding of the study is that the transcription factor TBX3 is localized to the cilium and interacts with KIF7. HOX proteins have been shown to interact with and inhibit GLI3 in the limb, however cilium localization was not explored. Given that the vast majority of TBX3 protein is in the nucleus (Figure 6), it seems surprising to me that the tiny amount in the cilium can be detected with IP (Figure 7E-H). The interaction with GLI3 could be in the nucleus and/or cilium, but KIF7 should not be in the nucleus.

We and others have shown (using different antibodies) that Kif7 is present in both the cilia and in scattered punctate regions of unknown composition in the cytoplasm and nucleus (Figure 4; He et al., 2014) We now also provide a confocal 100X maximum image projection showing this in sectioned limb bud and the z-stack (Figure 4—figure supplement1 and Video 2).

The relative amounts of Kif7 in these different compartments have not been determined; most immunofluorescence images (including ours in Figure 4) highlight the signal in cilia. We have previously demonstrated (and do so again here) that Tbx3 is present in the cytoplasm and the nucleus, and that the relative abundance in these compartments is cell/tissue specific.

We do not claim that the IP’d material only reflects an interaction in the cilia and we think it likely that the relevant interactions between Tbx3, Gli3, Kif7 and Sufu are occurring at the ciliary base or centrosome as has been proposed by Ryan and Chiang and others (reviewed in Ryan and Chiang 2012; Ingham and McMahon 2009; and Wang et al., 2013; Wen et al., 2010, Humke et al., 2005; Tukachinsky et al., 2010). As discussed above, dissecting the cellular compartments in which different functionally relevant interactions has been a major, unsolved challenge in the field. What is clear is that processing of Gli3 to Gli3R requires the cilia. Our schematic specifically avoids indicating any localization for the Kif7/Tbx3/Gli3 complex (beyond anterior mesenchyme) because at this time, we can only hypothesize which subcellular compartment is functionally important. Determining this is clearly beyond the scope of this manuscript.

To further bolster the most significant claim of the paper, I think that additional experiments are needed. In particular, the specificity of the antibodies must be proven (e.g. using Tbx2 mutant MEFs for the Tbx3 antibody) and ideally some results confirmed using tagged proteins. Tagged GLI3 is available and other proteins could be easily generated.

We have performed these experiments, provide the data in the revised manuscript, and detailed the changes in response to Reviewers 1 and 2 above.

A genetic test would be to determine whether heterozygosity for Kif7 in mice (or knockdown in cells) rescues the defect proposed to be due to increased KIF7.

There is not an increase in the level of Kif7; we detected a change in localization in mutants.

In addition, many of the claims of changes in gene expression based on the RNA-seq data in Tables 2 and 3 do not seem to be significant differences, and some are opposite to what is said in the Results. Thus, the value of the RNA-seq data, is not clear, especially as only two samples were analyzed for each genotype, although I appreciate it is a difficult experiment. Many of the additional points listed below are simple changes that would make the paper more accessible and data more convincing.

We apologize for the inconsistencies between the text and the RNA-Seq tables; please see detailed explanations below.

Figure 3A': The mutant limb looks smaller than the control limb in (A), therefore the domain is expected to be smaller. The text implies the domain is smaller relative to the size of the limb. Which is the case?

The text states “Hand2 transcripts are reduced in E9.5 and E10.5 Tbx3;PrxCre mutantforelimb buds (Figure 2—figure supplement 1D; Figure 3 A,A’)”. Based on the in situ images, the domain is both smaller and the intensity of the signal throughout the domain is decreased, which is consistent with fewer transcripts. We now provide qPCR data that support this claim (Figure 3F). The mutant limbs are not in general smaller at E10.5-11.5; as noted above, the morphology and size varies, as it does among somite-matched wild type embryos, even within the same litter.

"[…]downregulation of posterior genes including Shh, Shh pathway members, Tbx2, Sall1, Dkk1, and Osr1[…]": The decrease is very little (e.g. Sall1 -0.3).

To address issues on the RNA-Seq data in general. Comments by both reviewers 2 and 3 led us to go back to the eLife website where we found that Tables 2 and 3 were duplicated and incorrectly labeled; the Table we referred to in the text was Table 2 when discussing the posterior expression analysis, but the table provided to reviewers labeled as Table 2 contained the anterior analysis. We sincerely apologize that the reviewers did not have access to files that were correct, and instead had files that were mislabeled relative to how they were discussed/referred to in the text, and that we failed to catch the error before the manuscript was sent for review.

Since initial submission, we have not had the time to generate sufficient animals to repeat the RNA-Seq experiment, but we have done additional validation of the datasets by qPCR (Figure 3, Figure 3—figure supplement 4). We have now labeled the tables with clear titles, provided methods that precisely describe how the changes contained in the tables were defined. Within the tables, we have labeled the data columns explicitly as mutant or control, and the compartments for the FPKM reads, as well as the pooled sample raw values (2 pools per genotype). We have also included additional data from an early microarray we performed in 2010 which in addition to the phenotypes, initially launched our investigation of the Shh pathway in the posterior and Gli3R in the anterior. This microarray analysis is in the present Table 2; changes that were replicated by the RNA-Seq experiment are highlighted in yellow in this table.

For the comparison of control posterior and mutant posterior (current Table 4), the fold changes in column L are given in log base 2, thus:

Tbx2 -0.61, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)

Dkk1 -0.77, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)

Osr1 -0.89, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)

Cntfr -0.93, (decreased greater than 1.5 fold, statistically significant; also detected by microarray and confirmed by qPCR)

On the current Table 4, Sall1 is not listed because it was not changed more than +/-1.3 fold which was the criteria for inclusion on the table.

"[…]up regulation of (AFP, Ttr, Apob, Apoa1, Apoa4, Trf, Ttpa)[…] (Table 2).”: Apob and Ttpa are actually down and the others are barely up in the anterior (Table 2).

Please note, the manuscript paragraph was describing changes in the posterior mesenchyme and Table 2 should have been also posterior; as noted above, the reviewer was unfortunately evaluating the anterior analysis and thus it is understandable that the reviewer did not agree with our claims.

In the posterior dataset contained in the present Table 4, log base 2, column L:

Apob +2.7 (increased greater than 4 fold and statistically significant)

AFP +2.1 (increased greater than 4 fold and statistically significant)

Apoa4 +1.7 (increased greater than 2.5 fold and statistically significant)

Ttr +1.4 (increased greater than 2 fold and statistically significant)

Apoa1 +1.3 (increased greater than 2 fold and statistically significant)

Trf +0.6 (increased greater than 1.5 fold and statistically significant)

Ttpa +0.5 (increased greater than 1.5 fold and statistically significant)

However, based on the reviewers’ comments and the fact that we do not pursue these changes further, we chose not to make any claims about this set of transcripts in the revised manuscript.

More important, why are Grem1, Ptch1 and Gli1 not down in the RNA-seq, which would confirm the data in Figure 3C, D and address the question of whether the decrease is just due to the limb being smaller?

The mutant limbs are not in general smaller at E10.5-11.5; as noted above, the morphology and size varies, as it does among somite-matched wild type embryos in the same litter.

We performed thein situs showing decreased expression of these transcripts long before performing the RNA- seq experiment (the entire series of experiments took over 7 years to complete) because the phenotype and wealth of published data led us to consider alterations in Shh pathway activity as at least contributory to the loss of digit 5, and because the 2010 microarray detected the decrements in Shh, Hand2 and Gli1 transcripts. We cannot explain the failure of the RNA-Seq experiment to detect these differences. As this and the other reviewers noted, RNA-Seq is a screening test and must be validated for specific genes; in the case of Shh, Grem1, Ptch1, Ptch2, Gli1, Hand2, and many others, the in situ and qPCR data are consistent and clear.

Despite the failure of the RNA-Seq to detect some bona fide changes, we do not think that these RNA-Seq data have no value to us or to the field: aside from confirming and expanding on previously published anterior/posterior polarity datasets in the wild type limb (which we have now included in a separate Table 3: Differentially expressed transcripts detected by RNA-Seq of E11 control anterior forelimb versus control posterior forelimb buds), many expression changes detected in the control versus mutant datasets have been validated by multiple methods including in situ hybridization, microarray and qPCR and were also validated for effects on alternative splicing in our 2014 Plos Genetics paper (Kumar et al., 2014b, eLife).

Results: "more single puncta cilia in mutants than controls (Figure 5E, 84% vs 71%, p<0.001). The levels of Kif7 mRNA[…] unaffected[…] Tables 2, 3 […]": In Table 3 Kif7 is -0.3, which is greater than many of the changes in expression level claimed elsewhere in the text.

See notes on the mix-up of the tables above; the original Table 3 reviewed contained the posterior gene expression data. In the current anterior analysis (Table 5), Kif7 is not listed on this Table because Kif7 log base 2-fold change is only 0.1042 (1.07 fold, not statistically significant) and only transcripts with changes +/- 1.3 fold (0.38 log base 2) are included on the revised tables.

Results: "The presence of Tbx3 in cilia and response to Hedgehog pathway activation was confirmed with a commercially available anti-Tbx3 antibody and both SAG and Shh stimulation (Figure 6M, N).": Tbx mutant fibroblasts should be shown to demonstrate specificity of the antibody.

We agree and have responded as noted in response to other reviewers above. The specificity of our custom antibody has previously been published Frank et al., 2012, Frank et al., 2013 and is also shown in Figure 1 panel E/F and now, specifically relevant to ciliary localization in revised Figures 5 and 6.

Tagged proteins should be used to confirm the interactions thought to be detected with commercial antibodies.

These data are now provided for the interactions between Tbx3 and both Kif7 and Gli3. The interactions we detected between Kif7 and Sufu and Gli3 proteins are well established in the literature.

Fig7C, D: quantification is needed.

We have now replicated these experiments and quantitated the changes in interactions using densitometry and the appropriate input controls. These data are in Figure 8 and its supplements.

Results: "Consistent with our hypothesis, there was decreased interaction between Gli3FL and Sufu in mutants (Figure 6E, lane 8[…]": Do the authors mean Figure 7E? Also, since Gli3 FL and R are reduced, does this experiment say anything?

We agree that the overall decrease in amount of Gli3 proteins in Tbx3△fl/△fl mutants makes it difficult to detect any decrement in protein/ protein interactions. We have not only assayed the Sufu/Gli3 interaction in both directions, but quantitated the amount of interaction and compared it to the decrease in protein levels (all in current Figure 8 and its supplements). In general, control embryos have ~ 1.6 fold more Gli3R than Tbx3△fl/△fl mutants (Figure 8A, A’ lanes 1-4, and Figure 8—figure supplement 2A and B). However, the ratio of Gli3R interacting with Sufu in controls ranges from 4.3 to 9.5 fold higher than in mutants (Figure 8—figure supplement 2A-B’). The interaction with Gli3FL is harder to assess reliably/quantitatively because of variable transfer efficiency due to high molecular weight. In the experiment shown in Figure 8A, the ratio of Gli3FL in controls to mutants is 1.6-1.7, while the ratio of that IPd with Sufu is 4.6 fold greater in controls. When assaying for Sufu that coIPs with Gli3, the change is not as dramatic because it reflects the Sufu that IPs with all GLi3 species (Figure 8B).

Discussion: "Future studies will determine if Tbx3 transcriptional or posttranscriptional activity regulates production of a factor that represses expression or stability of Hand2 mRNA.": In the posterior limb, wouldn't Tbx3 also interfere with processing of Gli2/3 into activators, like in cilia mutants, and this could explain the posterior limb phenotype by the same mechanism as the anterior limb bud?

As discussed in detail above, Shh signaling in the posterior limb bud is not cilia dependent; no ciliary mutants described thus far have posterior limb defects. Cilia mutants fail to process Gli3FL into Gli3R but as discussed above and in the manuscript, cilia phenotypes affect anterior digit number. Our data do not reveal any decrease in GliFL in the posterior, Shh responsive compartment.

[Editors’ note: the author responses to the re-review follow.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: 1) There are some remaining concerns about the quality of the biochemical data. Specifically, the composition and the order of loading of the lanes is not the same in Figure 4B, C and Figure 7A, B, creating the impression that the shown immunoprecipitations are not from the same experiment. Gli3 and KIF7 blots should be included in Figure 7C.

We responded with this query on Jan. 26:

“We agree that it is not ideal that the order of the lanes is not consistent; nonetheless, the IPs were from the same experiment/limb bud lysates. These experiments were repeated at the request of the reviewer from the first submission. Since these IPs require literally hundreds of limb buds, and the results shown in the revision reproduce those originally presented, repeating them for the sake of changing the order of the lanes seems excessively burdensome and time/embryo consuming given that the conclusion is unchanged.”

We received this response from eLife:

1) Figure 4B, C and Figure 7A, B: Repeating the experiment just to present a consistent order of the lanes is indeed not necessary in this case since the material is difficult to obtain. The authors should state in the main text of the paper (if this is correct) that the lanes shown in these gels are from the same immunoprecipitations.

We apologize that the order of the lanes is not consistent; the IPs were from the same experiment and that is now stated in the text.

Control blots for Gli3 and KIf7 from the same experiment should be included in Figure 7C.

Now provided.

Most importantly, error bars should be included in the plots showing the quantification of immunoprecipitation results in Figure 8 in order to illustrate that the experiment was performed more than once and the observed differences are significant.

We sent this query on Jan. 26:

“To clarify, Figure 8 shows analysis of each protein-protein interaction performed once in each direction. The quantitation in the accompanying bar graph is for the blot shown and that is why the bar graphs do not have error bars. The repeat experiments showing that the altered interactions are reproducible, and their quantitations, are in Figure 8—figure supplement 2.

When considering these blots, it is important to realize that the IP and transfer efficiency do unavoidably vary from experiment to experiment, making it impossible to combine raw densitometry intensity data from all experiments to obtain the type of statistical analysis the reviewer is requesting. Furthermore, we think that the key point is that the changes in interactions in mutants are reproducible from experiment to experiment and when assayed in both directions.

As the reviewers are aware, it took 7 months to obtain sufficient numbers of embryos to provide the repeat experiments requested in the first review. We appreciate your reconsideration and additional clarification as to what it required for acceptance of our final submission.”

We received this response from eLife:

“Quantification of immunoprecipitation results in Figure 8: It is obvious that to draw any conclusions about protein quantities on Western blots, each experiment should be performed at least twice. It is also obvious that the raw densitometry data cannot be compared between different experiments. However, band intensity ratios, which are currently presented per experiment, should be easy to combine and average between two or more experiments/samples, generating error bars. The authors should also label these plots appropriately (for example, instead of "ratio wt to mutant, lanes 1:2" use a label "Gli3FL, anti-Gli3-IP, ratio wt to mutant", etc.). Basically, instead of leaving the job of comparing different experiments to the reader, the authors should combine the quantitative outcome of their repeated experiments in one plot and include it in the main figures of the paper ".

We have now remade Figure 8 and supplements in accordance with these requests. We have separated Figure 8 into sections entitled: Gli3/Sufu Interactions; Gli3/Kif7 Interactions; Kif7/Sufu Interactions

Within each of these sections, the upper blots are representative IP experiments that were repeated 3 times. Adjacent to these blots are bar graphs with the averaged band intensities and standard error of the mean. The lower blots in each section are the complementary IP/western performed in the “other direction”; these complementary experiments were done once, so the bar graphs only show the ratio of band intensities between control and mutant for that experiment.

So, for each interaction, we not only performed replicate experiments (some of which are presented in Figure 8—figure supplement 2), but also the complementary experiments. We hope the reviewers agree that this is a rigorous analysis for a methodology that is difficult to quantitate.

Please note that ratio of two bands cannot be negative (because if one positive value is divided by another positive value, the outcome is positive), so the values showing an increase in the mutant should be presented differently”.

Thank you for the correction. We have remedied this in Figure 8 and Figure 8—figure supplement 2.

2) Better discussion should be included to explain why the authors insist that TBX3 does not regulate GLI3 function in the posterior limb, and why they have ignored the highly related protein GLI2. If their hypothesis is correct, one would expect the same might be true for GLI2 since it also binds SuFu and requires cilia for proper processing. Also, if whole embryos reveal the same protein interactions as the anterior limb, why would the posterior limb be different? Gli2/3 double limb mutants could well have a worse posterior limb phenotype than Gli3 null or het mutants. Only a relatively late double conditional mutant of Gli2/3 has been published, and it indicated the posterior phenotype is worse than in single Gli3 mutants. Thus, some of the posterior phenotype in Tbx3 mutants could be due to the altered GLI processing and activity in this tissue, rather than just the decreased Shh expression, especially as Shh heterozygous mutants have normal limbs.

We agree, and have considered and investigated this further. While ShH+/- mutants do not have an abnormal digit phenotype, and they have half as much Shh mRNA in their limb buds, the amount of protein is normal (Brian Harfe, personal communication/manuscript in revision) revealing a compensatory mechanism at the translational or post-translational level. Sanz-Ezquerro and Tickle (2000) proposed a compensatory, buffering system to stabilize polarizing activity in chick limb stating: “A buffering system can account for several regulative features of polarising region signalling. It can explain why limbs with normal patterns develop after application of extra Shh polarising region cells (Tickle et al., 1975), Shh expressing cells (Riddle et al., 1993) or Shh beads (Yang et al.,1997) to the posterior margin of chick buds, and why normal patterned limbs also develop after most, but not all, of the polarising region is removed (Fallon and Crosby, 1975; Pagan et al., 1996).”

Additionally, the ShH+/- genotype is sensitized to other mutations. For example, Zeller’s lab showed that rescued digits in Grem1-/-;Bmp4+/- are more sensitive to reduced Shh level (Benazet et al., 2009) and Hui's lab showed that Irx3/5 KO (excess HH activity) is improved by Shh dosage reduction. In addition, the Wnt7a mutant (Parr and McMahon, 1995), which affects Shh activation, has loss of digit 5. Mackem’s group re-evaluated the Wnt7a mutant (Zhu and Mackem, 2009), and found delayed onset and decreased level of Shh expression – which is consistent with our results.

In consideration of this information and to address the reviewers’suggestions, we have modified the model presented in Figure 9 and added the following paragraphs to the relevant sections of the Discussion.

For the section discussing posterior phenotype and mechanisms:

“Digit 5 formation is exquisitely sensitive to Shh activity and Grem1 (Harfe et al., 2004; Scherz et al., 2007; Zhu and Mackem, 2011; Zhu et al., 2008), so the altered expression of these genes in posterior mutant mesenchyme helps explain loss of this digit. […] Nonetheless, it is possible that decreased Shh signaling in the posterior of Tbx3;PrxCre mutant limb buds creates changes in Gli3 levels/ratios below our ability to detect. If so, decreased Shh activity would be predicted to increase Gli3R, decreasing digit number.”

For the section discussing anterior phenotype and mechanisms we have added:

“Loss of Tbx3 could also influence stability and processing of Gli2. The anterior PPD phenotype of Gli3 heterozygotes is slightly more severe in a Gli2 null background (Bowers et al., 2012; Mo et al., 1997). […] This is consistent with our findings that Tbx3 affects anterior digit number by regulating Gli3 repressor stability in the anterior mesenchyme.”

Finally, it seems to be an over-interpretation of the data to say that the posterior limb is not dependent on cilia. In cilia mutants’ production of both activators and repressors is altered (diminished), but obvious phenotypes are only seen where the balance is greatly skewed. It would be nice to extend the Discussion of your paper to address these points.

We agree; in the manuscript, we discuss what is known with regard to ciliary function and limb phenotypes but do not exclude the possibility of posterior functions yet to be determined.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below: 1) The scan of the Western blot shown in Figure 7C is not publication quality. Please substitute it for a high-resolution scan.

We have replaced the image in 7C with that exported at 600 dπ from the scanner; we have shown the original image without any alterations to lane order or intensity. Because of the magnification at which the image was originally taken, the Biorad scanner gives a pixelated quality to the over exposed IgG bands and unfortunately, this is the lowest exposure we have of this experiment. We have provided additional experiments showing the Gli3/Tbx3 interaction in the new Figure 7—figure supplement 1.

Also, please make sure that in all cases where lanes of Western blots have been left out, a clear separator line is present (this currently seems not to be the case in Figure 7C, 7C' and 8E).

For Figure 7C, the new image shown was exported at 600 dπ from the scanner; we have shown the original image without any alterations to lane order or intensity, thus there are no lane rearrangements requiring separator lines. We have added more obvious separators to Figures 7C’and 7C” and presented these blots with lane order consistent with that of Panel 7C. We have added the requested separator to Figure 8E.

2. Please reconsider the title of your paper. "T-box3 is a ciliary protein and regulates.." doesn't read well. Furthermore, eLife encourages the authors to provide brief explanations for the acronyms used in the title and Abstract. For your paper, mentioning that Gli3 is a player in Sonic Hedgehog signaling would be appropriate.

We have revised the title to: “T-box 3 is a Ciliary Protein and Regulates Stability of the Gli3 transcription factor to Control Digit Number”. We think that including Sonic Hedgehog signaling in the title is too cumbersome and also, inaccurate because its function in the anterior is Shh independent, as stated in the Abstract. We are open to suggestions if the title is still not satisfactory.

We have revised the Abstract as requested to include additional reference to Gli3 as a transcriptional effector in the Hedgehog pathway:

“Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. […] Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.”

https://doi.org/10.7554/eLife.07897.049

Article and author information

Author details

  1. Uchenna Emechebe

    Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, United States
    Contribution
    UE, Acquisition of data, Analysis and interpretation of data
    Contributed equally with
    Pavan Kumar P
    Competing interests
    The authors declare that no competing interests exist.
  2. Pavan Kumar P

    Weis Center for Research, Geisinger Clinic, Danville, United States
    Contribution
    PKP, Acquisition of data, Analysis and interpretation of data
    Contributed equally with
    Uchenna Emechebe
    Competing interests
    The authors declare that no competing interests exist.
  3. Julian M Rozenberg

    Weis Center for Research, Geisinger Clinic, Danville, United States
    Contribution
    JMR, Conception and design, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  4. Bryn Moore

    Weis Center for Research, Geisinger Clinic, Danville, United States
    Contribution
    BM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  5. Ashley Firment

    Weis Center for Research, Geisinger Clinic, Danville, United States
    Contribution
    AF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  6. Tooraj Mirshahi

    Weis Center for Research, Geisinger Clinic, Danville, United States
    Contribution
    TM, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  7. Anne M Moon

    1. Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, United States
    2. Weis Center for Research, Geisinger Clinic, Danville, United States
    3. Department of Human Genetics, University of Utah, Salt Lake City, United States
    4. Department of Pediatrics, University of Utah, Salt Lake City, United States
    Contribution
    AMM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents
    For correspondence
    ammoon@geisinger.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9090-1093

Acknowledgements

We thank Drs. Chi-Chung Hui, Cliff Tabin and Kathyrn Anderson for generously providing the Kif7 polyclonal antibody, the Prx1Cre mouse line, and the Flag-tagged Kif7 construct, respectively. We thank Susan Mackem, Steven Vokes and Brian Harfe for helpful discussions and Diana Lim for creating the graphic art. We thank Peter Gallagher for technical assistance.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols with WCR IACUC protocol number 203-14.

Reviewing Editor

  1. Anna Akhmanova, Utrecht University, Netherlands

Publication history

  1. Received: April 2, 2015
  2. Accepted: March 5, 2016
  3. Version of Record published: April 5, 2016 (version 1)

Copyright

© 2016, Emechebe et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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