Abstract
Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions, namely the dorsal morula, yolk syncytial layer, and distal hypoblast/anterior visceral endoderm (in amphibians, teleosts and mammals, respectively), require the localised stabilisation of nuclear Beta-catenin (Ctnnb1), implying that localised Wnt/Beta-catenin signalling activity is critical in their establishment. However, it is becoming increasingly apparent that the stabilisation of Beta-catenin in this context may be initiated independently of secreted Wnt growth factor activity. In Xenopus, dorsal Beta-catenin stabilisation is initiated by a requisite microtubule-mediated symmetry-breaking event in the fertilised egg: “cortical rotation”. Vegetally-localised wnt11b mRNA has been implicated upstream of Beta-catenin in this context, as has the dorsal enrichment of Wnt ligand-independent activators of Beta-catenin, but the extent that each of these processes contribute to axis formation in this paradigm remains unclear. Here we describe a maternal effect mutation in Xenopus laevis wnt11b.L, generated by CRISPR mutagenesis. We demonstrate a maternal requirement for timely and complete gastrulation morphogenesis and a zygotic requirement for proper left-right asymmetry. We also show that a subset of maternal wnt11b mutants have axis and dorsal gene expression defects, but that Wnt11b likely does not act through the Wnt coreceptor Lrp6 or through Dishevelled, which we additionally show (using exogenous constructs) do not exhibit patterns of activity consistent with roles in early Beta-catenin stabilisation. Instead, we find that microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs, leading to less organised and directed vegetal microtubule arrays. In conclusion, we propose that Wnt11b signals to the cytoskeleton in the egg or early zygote to enable robust cortical rotation, and thus acts in the distribution of putative dorsal determinants rather than as a component or effector of the determinants themselves.
Introduction
The formation of a single dorsal midline axis of bilateral symmetry in the embryo is a central event in the early development of vertebrate animals. In amphibians, this process depends on symmetry-breaking in the fertilised egg, which occurs through a process of microtubule-mediated “cortical rotation” of the egg cortex, resulting in corticocytoplasmic translocation of putative dorsal axial determinants (Gerhart, 2004; Houston, 2012; Houston, 2017; Weaver and Kimelman, 2004). A major outcome of cortical rotation is the stabilisation and nuclear localization of Ctnnb1/Beta-catenin (Beta-catenin hereafter) during the 16-to-32 cell stages (Kao and Elinson, 1988; Yang et al., 2002). This process sets up chromatin states and later gene regulatory interactions (Blythe et al., 2010) that lead to the specification of cells comprising the Spemann organiser (reviewed in Houston, 2017). The organiser ultimately elaborates axial patterning through the formation of extra-cellular gradients of BMP (and other) growth factor antagonists.
At least two non-mutually exclusive views of dorsal signalling subsequent to cortical rotation have been considered: 1) the intracellular localization of cytoplasmic activators of Wnt signalling in the absence of Wnt ligand, and 2) the dorsal enrichment of vegetally localised Wnt ligand (wnt11b) mRNA. The (first) intracellular localization model is supported by various studies, including: cytoplasmic ablation/transplantation experiments involving egg cytoplasm (Darras et al., 1997; Kageura, 1997; Marikawa and Elinson, 1999; Marikawa et al., 1997), the visualisation of translocating particles containing exogenous Dishevelled (Dvl) and/or Frat1/GBP fusion proteins (Miller et al., 1999; Weaver et al., 2003), and the failure of overexpressed extracellular Wnt antagonists to inhibit endogenous beta-catenin activity or axis determination (Hoppler et al., 1996; Leyns et al., 1997; Wang et al., 1997).
Also, recent work in fish and frogs has shown that deficiency in maternal huluwa (hwa; (Yan et al., 2018), which encodes a localised RNA in both species, results in ventralization. These data also included dominant-negative results suggesting that Hwa acts independently of secreted Wnt signalling, supporting the prior results in frog embryos. Hwa accumulates dorsally and likely promotes the dorsal degradation of Axin1, a key scaffold protein that facilitates beta-catenin degradation (Yan et al., 2018). More broadly, Beta-catenin is required for the establishment of the Anterior Visceral Endoderm (AVE) in mouse embryos, the requisite axis-regulating region, but evidence is accumulating that the AVE can form in the absence of secreted Wnt ligand signalling (reviewed in Houston, 2017).
The (second) extracellular signalling model was suggested by experiments showing that depletion of maternal wnt11b mRNA using antisense oligonucleotides (oligos) results in axial defects, with corresponding loss of early Beta-catenin target gene expression (Tao et al., 2005). These studies also indicated that Wnt11b is required extracellularly, in a complex with Exostosin glycosyltransferase 1 (Ext1) and Tdgf1 (alias Cripto/FRL-1) homologues, and possibly with other Wnt ligands (Cha et al., 2008; Tao et al., 2005), implying an extracellular signalling event. TALEN-mediated mutagenesis in zebrafish has ruled out a maternal role for wnt8a in early development (Hino et al., 2018), although the roles of other Wnts have yet to be tested in this organism.
These studies on maternal Wnt signalling need to be reconciled with more general data on Wnt/Beta-catenin signalling showing the importance of activated Wnt receptor-coreceptor complexes (Lrp6 “signalosomes”) in association with Dvl (Bilic et al., 2007). A hybrid model of maternal Beta-catenin regulation has been proposed wherein cortical rotation might enrich active phospho-Lrp6 signalosomes on the dorsal side (Dobrowolski and Robertis, 2012). However, recent data suggest that endocytosis may not be required for Wnt/Beta-catenin activation (Rim et al., 2020). And, the extent that phospho-Lrp6 signalosomes become dorsally enriched is unknown.
To re-evaluate the role of maternal Wnt11b signalling in axis formation, we used targeted genome editing to generate Xenopus laevis deficient in wnt11b, obtaining embryos lacking Wnt11b function maternally, zygotically, or both. Our data show that maternal Wnt11b signalling is required for proper initiation of dorsal gene expression, axis formation, and gastrulation morphogenesis, but that axial tissues eventually form in many cases. Additionally, consistent with prior reports, we find that loss of Wnt11b activity is associated with left-right asymmetry defects. Regulation of Lrp6 phosphorylation and Dishevelled puncta were unchanged in wnt11b mutant oocytes and eggs. Furthermore, and in contrast to the existing models for Wnt11b in Beta-catenin activation, live imaging experiments show that vegetal microtubule dynamics and cortical rotation are disrupted in the absence of Wnt11b function, implicating Wnt11b in the initiation or maintenance of, rather than (or in addition to) the outcome of, cortical rotation.
Results
Mutagenesis of Xenopus laevis wnt11b
CRISPR/Cas9 mutagenesis was used to create insertion-deletions (indels) in the wnt11b.L locus of the inbred Xenopus laevis J-strain (Session et al., 2016; TOCHINAI and KATAGIRI, 1975). These mutants were generated and raised at the National Xenopus Resource (NXR; (Pearl et al., 2012). We designed guide RNAs targeting the first two exons of the wnt11b.L gene on chromosome 8L (chr8L; hereafter just wnt11b)(Fig. S1A, Table S1). These guide RNAs lack homology to the paralogous wnt11.L/S genes (alias wnt11-r), which are located on chromosomes 2L/2S and are not expressed maternally, but share similar zygotic expression patterns to wnt11b (Garriock et al., 2005) (Fig. S1B). The homeolog (alias alloallele) for wnt11b.S, which would be present on chromosome 8S, is absent, a likely consequence of widespread gene loss on the S subgenome chromosomes (Session et al. 2016). X. laevis also lacks a duplicated wnt11b gene found in X. tropicalis (alias xetro.H00536; (Dichmann et al., 2015)). Thus, X. laevis is functionally diploid for the wnt11b gene, and Wnt11b represents the sole contribution to maternal Wnt11 protein family function.
Guide RNAs against wnt11b were complexed with Cas9 protein and injected into one-cell embryos obtained from matings/in vitro fertilizations of wildtype J-strain X. laevis to begin generating a mutant pedigree (Fig. 1A). A set of resulting embryos were grown to sexual maturity and females were tested for germline transmission of indels. An F0 female was identified that transmitted a 13 bp deletion (−13del; allele designation Xla.wnt11bemNXR), located near the three prime end of exon1 (sgT1 guide RNA site). This deletion is predicted to generate a frameshift mutation resulting in a premature stop codon just after the Wnt11b signal sequence cleavage site (Fig. 1B). Outcrossing of this ‘founder’ female to a wildtype J strain male resulted in heterozygous F1 progeny that were then intercrossed. Three sexually mature F2 females homozygous for the 13 bp deletion were initially identified, in addition to several homozygous mutant males (Fig. 1A). These F2 generation Xla.wnt11bemNXR/emNXR (wnt11b-/-hereafter) frogs all developed normally (outwardly), indicating that zygotic wnt11b is not uniquely required for developmental viability.
To assess the functionality (or lack thereof) of the -13del/emNXR allele, we amplified and cloned the wnt11b.L coding region cDNAs from wildtype and mutant oocyte RNA by RT-PCR. Sequencing confirmed that the 13bp deletion created the predicted frameshift mutation, resulting in premature stop codons in the expressed RNA, and that it did not disrupt normal exon1-exon2 splicing (Fig. 1B). Additionally, we synthesised transcripts from these cDNAs in vitro and assessed their activity through overexpression in wildtype Xenopus embryos. In all cases (n>=30 each; two experiments), wildtype wnt11b induced axis shortening, consistent with known phenotypic effects of wnt11b injection (Fig. 1C; (Du et al., 1995). By contrast, overexpression of mutant wnt11b-13del transcripts had no effect on development and morphogenesis (Fig. 1C), a result further in line with this mutant allele lacking activity.
wnt11b is maternally required for normal progression through gastrulation and axial morphogenesis
Homozygous wnt11b-/- F2 females produced fertile eggs, and homozygous mutant F2 males were similarly fertile, indicating that zygotic Wnt11b function is not required for overall germline development. To assess the role of maternal Wnt11b in development, we fertilised eggs from homozygous mutant females using sperm from either wildtype or mutant males, alongside eggs from wildtype females. Because we wished to unambiguously describe the strictly maternal effects of wnt11b loss-of-function, we designated the origin of the mutant allele in these crosses using a capital ‘M’ or ‘Z’ for whether the mutant allele was maternally/egg derived (‘M’) or zygotically/sperm derived (‘Z’). Thus maternal-zygotic mutants are referred to using ‘M/Z’ in this work, whereas heterozygous embryos are indicated as ‘M/+’ or ‘+/Z’, corresponding to mutant or wildtype eggs fertilised with wildtype or mutant sperm, respectively. For simplicity, most experiments focused on comparing M/Z (MZwnt11b-/-) embryos with +/Z (+Zwnt11b+/-) embryos.
The crosses described above (Fig. 1A) resulted in embryos that developed normally until gastrulation. In embryos derived from mutant females (M/Z and M/+), gastrulation was consistently delayed with complete penetrance, regardless of paternal genotype (see below). Maternal mutant embryos subsequently developed a range of defects in embryogenesis (variable expressivity), ranging from severe axial truncation/ventralization, with or without open blastopores and neural tubes (spina bifida-like) and small ectopic tailbuds, to largely normal development (Fig. 1D; Table S2). We characterised the delays in gastrulation using time-lapse imaging of heterozygous (+/Z) and maternal/zygotic homozygous (M/Z) embryos. For these experiments, embryos were fertilised at the same time and imaged in the same dish (Fig. 1E, Supplemental Video 1). Movies were started when the control +/Z embryos were at the mid-gastrula stage (Nieuwkoop and Faber stage 10.5), the time when the majority of M/Z embryos were unequivocally delayed (Fig. 1E, time 00hr:00min:00sec). M/Z embryos did not initiate ventral blastopore closure until the equivalent stage 12 and most did not fully close the blastopore during the nine hours of imaging. Neural plate formation and morphogenesis commenced roughly on schedule in these abnormal embryos, suggesting a primary defect in gastrulation and not in dorsal signalling per se (Fig. 1E, time 07hr:00min:00sec). Similar results were seen using antisense Morpholino oligos against all wnt11 family transcripts (Van Itallie et al., 2022), suggesting that normal expression of either maternal (of which Wnt11b is the sole contributor) or pan zygotic Wnt11 proteins is required for timely and complete gastrulation.
The formation of normal dorsal axial structures was first verified by assessing antigen tissue markers at the tailbud stage. Immunostaining against notochord and somite antigens using monoclonal antibodies (mAbs Tor70 and 12/101; (Bolce et al., 1992; Kintner and Brockes, 1984; Kushner, 1984)) showed reduced and disorganised expression in the population of mutant embryos that formed dorsal axes (Fig. S2). Interestingly, immunostaining against NCAM (mAb 6F11; (Lamb et al., 1993)) revealed ectopic neural differentiation both in ectopic tailbud structures and in epidermal regions throughout the embryo (Fig. S2). Embryos lacking visible axes lacked staining for these antigens (not shown). Thus, whereas a subset of embryos maternally deficient in wnt11b lack dorsal axes, many are able to form dorsal structures and even ectopic neural tissues.
Wnt11 family transcript expression is normal in wnt11b mutant oocytes and embryos
We next sought to determine the extent that the early stop codon in the emNXR mutant allele of wnt11b may have indirectly affected activities unrelated to Wnt11b protein function, resulting from either the degradation or delocalization of wnt11b transcripts during oogenesis. We also examined whether compensatory expression of wnt11.L/S genes (on chr2) might have occurred. We isolated oocytes and embryos from wnt11b homozygous mutant females and from wild type siblings and analysed the expression of RNAs localised to the vegetal cortex. RNAs representative of the three main classes of localised RNAs were examined: germ plasm, late pathway and intermediate RNAs, exemplified by nanos1, vegt and wnt11b itself (Houston, 2013). All three RNAs were expressed similarly when comparing wildtype and mutant oocytes (Fig. 2A), indicating that general oocyte polarisation and RNA localization occur normally in wnt11b-/- mutant animals.
We also assessed wnt11b and wnt11 RNA expression in oocytes and embryos by RT-PCR. Transcripts for wnt11b were expressed equivalently in wildtype and wnt11b-/- mutant oocytes and embryos throughout development (Fig. 2B), suggesting a lack of nonsense-mediated decay maternally. wnt11.L/S paralogues were not ectopically expressed maternally in wnt11b-/- mutant oocytes or during later stages (Fig. 2C), although their expression was delayed around stage 11 (mid-gastrulation), likely as part of the general delay in gastrulation in these embryos (see below).
These data suggest that the variable defects seen in wnt11b maternal mutants are the result of loss of maternal Wnt11b protein function, and that variable up-or down-regulation of the paralogous wnt11.L/S genes or disruption of RNA localization does not generally occur and does not appear to contribute to the wnt11b-deficient phenotypes.
wnt11b is required zygotically for left-right asymmetry
In the course of these initial studies on the role of maternal wnt11b, we noted that a homozygous wnt11b-/- F2 male used as a testis donor had reversed organ situs. Because wnt11b had previously been implicated in laterality signalling using Morpholino-based knockdown (i.e., loss of left-sided pitx2c expression; (Walentek et al., 2013)), we performed heterozygous crosses and scored resulting embryos at the swimming tadpole stage, when organ laterality is easily discernible (Yost, 1992). In a pilot experiment (47 total tadpoles), we identified three tadpoles that exhibited reversed heart orientation (heterotaxy or situs inversus; abnormal gut coiling and/or heart orientation). Genotyping revealed that all three heterotaxic tadpoles were homozygous for wnt11b-/-, whereas the remaining 8 homozygous mutant tadpoles and the heterozygotes and wildtype tadpoles had normal situs (situs solitus; Table S3), a frequency of about 25% laterality defects in homozygous F2 mutants.
Subsequent observations of surviving F3 MZwnt11b-/- mutant tadpoles with normal phenotypes showed a similar frequency of heterotaxy (24-25%; n >= 50; Table S3, Fig. 3A-B). Examination of pitx2c expression at the tailbud stage showed normal expression in the left lateral plate mesoderm of heterozygous embryos (+/Z; Fig. 3C-D) and in about half the maternal-zygotic mutants (M/Z; Fig. 3E-E’). Left-sided pitx2c could be seen in some axis deficient embryos, suggesting left-right defects could be independent from axial induction defects (Fig. 3E). The remaining maternal-zygotic mutant embryos lacked or were severely reduced in left-sided pitx2c expression, or exhibited bilateral pitx2c, with weak expression on the right side (Table S3). These frequencies are comparable to the frequency seen in the null embryos from heterozygous matings (above), and in published studies on wnt11b Morpholino-injected embryos (in which zygotic, but not maternal Wnt11b, function is affected; Walentek et al. 2013). These congruences suggest that the action of Wnt11b on left-right signalling is mainly performed by zygotically expressed wnt11b. We did not pursue this zygotic aspect of Wnt11b’s function further.
Maternal Wnt11b is required for normal early dorsal-ventral gene expression
To characterise the maternal effects of wnt11b loss-of-function, we analysed gene expression in embryos derived from wnt11b-/- F2 females at the gastrula and stages, when visible phenotypes first became apparent. Consistent with visible delays in gastrulation, the expression of mesoderm and endoderm markers was reduced and delayed during gastrulation in maternally wnt11b deficient embryos (Fig. 4). Notably, mesodermal genes myod1 (stages 12-13), and wnt11b itself and wnt8a (stage 10.5) were expressed in patterns consistent with delayed blastopore formation in maternal mutant embryos. (Fig. 4A-L, Q). This delay was particularly evident in delayed ventral endoderm expression of sox17a (Fig. 4M, O), whereas a general ventral fate marker, sizzled (szl), was expressed in its normal spatial pattern (Fig. 4D, F, Q), although at elevated overall levels. The expression of dorsal and Wnt/Beta-catenin target genes were also reduced in maternal wnt11b mutant embryos (MZwnt11b-/- and M+wnt11b-/+). In situ hybridization analyses of dorsal nodal3.1 expression showed either reduced or absent expression in all cases, with about 50% lacking visible signal (Fig. 4N, P; Fig. S3). Similarly, in RT-PCR analyses, levels of the organiser marker siamois homeodomain 1 (sia1; Fig. 4R) and nodal3.1 (Fig. S3) were reduced to =< 20% of control levels (on average) in embryos derived from wnt11b mutant eggs (M/Z and M/+). Conversely, expression of the ventral marker szl was expressed at normal (low) levels at stage 9, but was elevated in maternal wnt11b mutant embryos at stage 10.5 (Fig. 4S).
Preliminary RNA sequencing analysis showed that other mesendodermal genes (e.g., mixer, foxc2) and ectodermal/prospective neuroectoderm markers (e.g., lhx5, sox2) were either unaffected or slightly changed in mutants (up or down; Fig. S4A-B). Levels of wnt5a.L/S were slightly elevated in MZwnt11b-/- embryos at stage 9 (FigS4C) and stage 10.5 (not shown). There was variability across replicate samples, consistent with the phenotypic variability (e.g., see M/Z replicate one versus replicate two in Fig. S4B). Additionally, analyses of differentially expressed genes showed dysregulation of apparently unrelated genes involved in metabolism, particularly at stage 9, which may be indicative of developmental delay (Fig. S4D). Thus, wnt11b is strictly, but variably, required maternally for normal dorsoventral patterning and for the normal progression through gastrulation.
To confirm the specificity of these effects, we conducted rescue experiments by injecting oocytes obtained from a wnt11b-/- F2 female with wnt11b mRNA and then fertilising these oocytes using host-transfer methods. We injected a low dose (20 pg) of transcript to avoid overstimulation of Wnt signalling. Injected and uninjected mutant oocytes were cultured for 24 hours, stimulated to mature, and then transferred to the body cavity of a wild type female. After subsequent ovulation, host-transferred eggs were fertilised with sperm from a homozygous mutant male. Embryos derived from mutant oocytes injected with wildtype wnt11b exhibited somewhat restored levels of organiser genes and underwent normal gastrulation (Fig. S5; Table S2).
Phospho-Lrp6 and Dvl regulation are normal in wnt11b mutant eggs
Lrp6 activation has been hypothesised to initiate Beta-catenin stabilisation during axis formation, either in oocytes, or as part of signalling endosomes translocated during cortical rotation. We therefore sought to determine the patterns of Lrp6 phosphorylation during early development and to identify the extent to which these patterns were dependent on maternal Wnt11b. We assessed the state of Lrp6 activation in oocytes, eggs and early embryos by immunoblotting against a phospho-epitope of Lrp6, S1490, that is elevated by Wnt ligand binding and essential for the response to Wnt signals (Tamai et al., 2004; Zeng et al., 2005). To generate a more robust and reproducible signal, we injected oocytes with ‘trace’ amounts of exogenous mouse Lrp6 mRNA (∼20-50 pg).
In wildtype samples, S1490 Lrp6 phosphorylation was absent in stage VI oocytes but stimulated in progesterone-treated/mature oocytes (Fig. 5A). Endogenous phospho-Lrp6 was weakly detected in mature oocytes but not in untreated stage VI oocytes. Furthermore, this phosphorylation was transient and was downregulated following either prick activation or normal fertilisation of host-transferred eggs. Notably, phospho-Lrp6 was substantially reduced by 60 minutes post-fertilization, when cortical rotation would be occurring, and remained absent in cleavage stage embryos (Fig. 5B), when Beta-catenin stabilisation would be occurring. Phenotypic analysis of sibling embryos confirmed that the low doses of Lrp6 did not induce axial duplication/dorsalization when expressed in oocytes, whereas higher doses would do so (Fig. S6), indicating that the trace amounts of mRNA used are below the threshold for strongly activating Beta-catenin under these conditions.
Analysis of Lrp6 phosphorylation in wnt11b mutant oocytes and eggs showed that the patterns of phospho-S1490 stimulation in eggs and subsequent downregulation were identical to wildtype samples. These data suggest that Lrp6 is phosphorylated during oocyte maturation in a Wnt11b-independent manner, and that phospho-Lrp6 disappears before cortical rotation is complete and before the critical period for Beta-catenin stabilisation (i.e., the 16-to-32-cell stages).
We also tested the working hypothesis that Wnt11b might regulate the formation or activity of Dvl puncta, which are thought to act as dorsal determinants in Xenopus (Dobrowolski and Robertis, 2012; Miller et al., 1999), potentially in association with activated Wnt co-receptor Lrp6 (“signalosomes”; (Dobrowolski and Robertis, 2012; Miller et al., 1999)). We injected transcripts encoding Dvl2-GFP into both wildtype and wnt11b-/- mutant oocytes. In both cases we observed numerous Dvl2-GFP puncta by live epifluorescence microscopy (Fig. 6). We also had hoped to examine the behaviour of these puncta during cortical rotation. However, we instead found that Dvl2-GFP puncta disappeared following oocyte maturation and did not reappear after prick-activation in both wildtype and wnt11b-/- mutant oocytes (Fig. 6A-F). Immunoblotting against GFP showed comparable levels of fusion protein expression, indicating that the loss of puncta is not the result of Dvl protein degradation (Fig. 6G). Additionally, we tested the propensity of Dvl2 puncta associate with endosomal structures in Xenopus oocytes but we failed to observe co-localization of Dvl2-mCherry with exogenous endosome markers Rab5- and Rab11-GFP in coinjected wildtype oocytes (Fig. 6H-I).
These data show that Lrp6 is phosphorylated (activated) during oocyte maturation independently of Wnt11b function. This phosphorylation is downregulated after egg activation/fertilisation, and although reduced and present during cortical rotation, becomes undetectable by the cleavage stages, when Beta-catenin stabilisation is required to occur. In contrast, Beta-catenin levels tend to mirror phospho-Lrp6 levels in cultured cell experiments using exogenous Wnt stimulation (Kim et al., 2013; Li et al., 2012). Also, these data show that large aggregates/puncta of Dvl are found in a complementary pattern to Lrp6 phosphorylation and do not localise with endosomal markers in oocytes. Thus, these results generally fail to support models invoking dorsally localised signalosomes in axis specification, although it is possible that a subset of dorsally localised complexes with smaller Dvl oligomers (Ma et al., 2020) are present, but below the limit of detection of methods used here.
Maternal Wnt11b regulates cortical microtubule assembly and cortical rotation
Because wnt11b mutant embryos showed considerable variability with regard to dorsal gene expression and axis formation despite the presence of Lrp6 phosphorylation, we considered whether Wnt11b might be required for the proper distribution of so-called dorsal determinants rather than their generation. To test this idea, we first assessed general microtubule organisation in eggs derived from wnt11b-/- homozygous mutant females. Eggs from wildtype and mutant females were fertilised (with sperm from wildtype or mutant males) and fixed in methanol during cortical rotation, around 60 and 80 minutes after fertilisation. Immunostaining against Beta-tubulin revealed that zygotes lacking maternal wnt11b (regardless of sperm genotype) exhibited disorganised microtubules (Fig. 7B-B’, Table S4) that remained in a loose/sparse network and did not form parallel arrays. Control eggs invariably developed well-formed parallel microtubule arrays at both time points (Fig. 7A-A’, Table S4). Similar results were seen in prick-activated eggs derived from in vitro-matured oocytes from wildtype and mutant females (Table S4).
To more accurately measure and quantify the effect of maternal Wnt11b deficiency on microtubule dynamics and on cortical rotation, we performed live imaging of microtubule plus end dynamics. We isolated wildtype and wnt11b-/- mutant oocytes and injected eb3-gfp mRNA as a microtubule plus-end marker. These oocytes were treated with progesterone in vitro to induce maturation and then prick activated. Live imaging and analysis was performed as described (Olson et al. 2015) using oocytes, eggs, and prick-activated eggs undergoing cortical rotation (at 60 minutes post-fertilization).
Plus end dynamics in both oocytes and progesterone-treated oocytes (eggs; n=8 each) were comparable to previous reports (Olson et al. 2015) in both wildtype and mutants. Although wnt11b-/- mutant samples tended to have more plus end growths in oocytes with fewer plus end growths in eggs, and more rapid growth speed (Table S5) compared to wildtype cases, none of the differences between groups at these stages were sufficient to reject statistical non-significance. Movies of activated eggs in wildtype control samples during cortical rotation (n=12) revealed numerous clearly aligned plus-end growths, indicative of robust cortical rotation (Fig. 7C-D). Plus-end tracking analyses identified significant concentration around a mean angle in each case, using the Rayleigh test for circular uniformity (Batschelet, 1981; Berens, 2009). Additionally, we previously established a mean resultant vector length of r = 0.3 as an empirical measure of directionality associated with the establishment of cortical rotation (Olson et al., 2015). This value was chosen because the 95% confidence intervals around the mean angle become asymptotically minimal as r approaches this magnitude. Wildtype activated eggs exhibited an average mean resultant vector length of 0.3, with over half the r values above this number (Fig. 7G). Only one sample registered below 0.1, a mean vector length indicative of poor directional organisation (where r=0 would be completely undirected, and r=1 would be uniformly directed on one angle).
Movies of wnt11b mutant eggs during cortical rotation (n=14) revealed considerable variability in plus-end growth (Fig. 7E-F), in line with the variability seen in anti-tubulin immunostaining and in axial development. Some eggs exhibited apparently normal directional growth, whereas others showed randomly directed, disorganised growth. Curiously, plus-end tracking analyses showed none of the mutant eggs exhibited a mean resultant vector length > 0.3, despite clearly directional growth direction in some cases. Several cases reached a magnitude of r∼0.25, but many were much lower, resulting in a lower average mean vector length (0.16 compared to 0.29 in controls; Fig. 7G, Table S5).
A comparison of microtubule growth parameters during cortical rotation showed that all dynamic parameters were more variable (i.e., higher variances) in the mutant eggs (Fig. 7G). Mean growth speed was similar in both wildtype and mutant eggs at 60 minutes post-activation, and activated wnt11b eggs exhibited fewer plus-end growth events in the cortex, as well as shorter growth lifetime and lengths, although only the latter difference was significant enough reject a null statistical hypothesis (Table S6). Plus end growth speed was also generally correlated with more robust directionality. However in one mutant sample with higher than average growth speed, directionality was compromised (9.5 µm/min, and r=0.1), suggesting a possible limit to this relationship (Fig. 7G).
Additional analyses of growth parameters showed significant correlation of the mean resultant vector length with plus end growth length in both wildtype samples and mutant samples (ρ=0.74 and 0.79, respectively; Fig. 7G). Other correlations between mean vector and growth numbers, speed and lifetime were also similar between mutant and controls, but weaker (ρ =< 0.6, p > 0.01), as were correlations of the parameters with each other (data not shown). However, correlation is a relatively weak measure of association, detecting only linear dependency independent of magnitude, and non-linear or other complex relationships might exist.
Together, these data show that the earliest detectable consequence of maternal wnt11b deficiency is an overall reduction in the overall organisation and alignment of vegetal cortical microtubules during cortical rotation, leading to less ‘robust’ directional orientation. This disruption is coincident with shorter growth length of microtubule plus-ends in the fertilised/activated egg. The data do not address the extent that this effect is owing to direct regulation of microtubule dynamics at the time of cortical rotation or to a more general effect on the cytoskeleton or other cellular structures or organelles following oocyte maturation.
Discussion
We have used CRISPR/Cas9 technology to generate a loss-of-function mutation in Xenopus laevis wnt11b.L, the only maternally expressed member of the wnt11 family in this species. Our analyses of these mutant embryos revealed a novel role for maternal Wnt11b as a permissive factor (at least) enabling robust microtubule-mediated cortical rotation and axis induction. In addition, we find that maternal Wnt11b is necessary for normal gastrulation morphogenesis, whereas zygotic Wnt11b is necessary for normal left-right asymmetry. Both of these latter findings are in line with the more traditional views of Wnt11 function obtained through antisense-mediated knockdown and dominant-negative experiments. The extent that Wnt11b might act through a similar molecular activity in all these different contexts is unknown. Wnt11b had been previously suggested to represent the main dorsalizing determinant upstream of Beta-catenin activation in Xenopus, whereas our results suggest that its role in this process is likely indirect.
Using targeted CRISPR/Cas9 mutagenesis of wnt11b.L, we generated a 13 base pair deletion near the end of exon1, which resulted in an early in-frame stop codon that was inherited through the germline. This deletion disrupts the normal signal peptide cleavage site and creates a premature stop codon two amino acids after a new (suboptimal) cleavage site. Thus, the mutant wnt11b.L locus would generate at most a two amino acid peptide (Serine-Proline). Several other in-frame stop codons are also generated downstream of the initial stop codon, and none of the potential peptides generated match known proteins. Based on these sequence predictions and the observation that mRNA encoding the mutant transcript lacks activity in over-expression assays, we interpret our results under the premise that this 13 base pair deletion nonsense mutation represents a null allele. Further genetic analysis, such as failure to complement deletion alleles would more conclusively demonstrate nullness, but this would require additional mutant lines.
In some contexts, premature stop codons can cause transcript degradation through nonsense-mediated mRNA decay. This mechanism does not seem to occur in the case of the -13bp deletion, which is consistent with the position of the premature stop codons at the 5’end of the transcript (Dyle et al., 2020). We examined the persistence of wnt11b RNA using both RT-PCR and in situ hybridization, and both assays showed normal (or slightly lowered) mRNA levels. Studies in zebrafish and mouse embryos have identified a genetic compensation response of related genes that is triggered by nonsense-mediated mRNA decay (El-Brolosy et al., 2019; Ma et al., 2019). In the absence of nonsense-mediated decay, we would also predict an absence of genetic compensation. Although our data do not directly address this phenomenon, we do note that the related wnt11.L/S genes show reduced, rather than elevated expression, a result inconsistent with compensation.
Nonetheless, also not addressed in this study, a structural or regulatory role may exist for wnt11b transcript, based on oligo-mediated transcript degradation experiments in host-transferred embryos (Tao et al., 2005), which exhibit a more severe ventralization phenotype. Other localised RNAs have non-coding roles in cytoskeletal organisation in Xenopus (Kloc, 2009), including plin2 (alias fatvg), which is required for microtubule organisation and cortical rotation, hence axis formation (Chan et al., 2007).
It has long been problematic to reconcile and incorporate various embryological, cell biological and biochemical studies on Wnt signalling and early development into a coherent understanding of the initiating steps in axis specification in Xenopus (or indeed in any vertebrate embryo). The data presented here allow several refinements to current models. First, our data suggest that wnt11b mRNA is unlikely to be the major axis-inducing agent enriched dorsally by cortical rotation because axis signalling can occur in its absence. High-resolution transcriptomic analyses have also generally failed to identify significant dorsal enrichment or polyadenylation of wnt11b (Domenico et al., 2015; Flachsova et al., 2013). Second, we find that Wnt11b signalling is not required for Lrp6 phosphorylation, which is activated during oocyte maturation and then attenuated during egg activation/fertilisation. The presence of phospho-Lrp6 in the egg and its diminution following activation had been noted by Davidson et al. (2009); our observations support this idea, but also show that phosphorylation is stimulated during oocyte maturation. Third, Dvl protein forms visible puncta in oocytes but not in eggs, a pattern complementary to phospho-Lrp6 (and unchanged in wnt11b mutants), suggesting that these puncta (as traditionally viewed) are likely unrelated to Lrp6 activation or signalosomes. Furthermore, the disappearance of visible Dvl puncta during oocyte maturation indicates that these (large) aggregates are unlikely to be Beta-catenin-stabilising agents transported by cortical rotation either.
Our data do not rule out a small domain of persistent phospho-Lrp6 and/or small Dvl oligomers at the cell surface (à la Ma et al., 2020), which might be below the limits of detection in our assays. It is not clear to what extent small Dvl oligomers might behave differently than the larger ‘puncta’ in this context however. Other wnts are not highly expressed maternally, and with our present work, in conjunction with recent evidence for a cytoplasmic role for Huluwa in fish and frogs (Yan et al., 2018), the preponderance of evidence is accumulating that direct Beta-catenin stabilisation in the early morula is Wnt ligand-independent. Intriguingly, a growing body of evidence shows that regulation of Beta-catenin in the mammalian AVE occurs independently of secreted Wnt ligand signalling, but does involve Tdgf1, as is the case in frogs as well (reviewed in Houston, 2017).
One implication of these findings is that Wnt11b would be signalling in para/autocrine fashion, acting on the single-cell egg to modulate microtubule dynamics (directly or indirectly). Recent proteomic data support the idea that Wnt11b protein expression is elevated during oocyte maturation (Van Itallie et al., 2022; Peshkin et al., 2019 - visualised on Xenbase) and other studies indicate active translation in the egg (Michael D. Sheets, personal communication). This pattern is mirrored by Fzd7, a Wnt11 family receptor, suggesting that this signalling pathway becomes active in the egg.
A large body of work has demonstrated the importance of Wnt/PCP signalling mediated by Wnt11 and other ligands in the regulation of gastrulation and axial morphogenesis through the control of convergent extension and other cellular behaviours (Solnica-Krezel, 2005). In this regard, Wnt11-Frizzled7 (Fzd7) signalling is thought to regulate cortical actin/actomyosin cytoskeletal dynamics (Huebner and Wallingford, 2018), resulting in either increased or decreased cell adhesion/cohesion depending on context (Dzamba et al., 2009; Heisenberg et al., 2000; Kraft et al., 2012; Ulrich et al., 2003; Ulrich et al., 2005; Winklbauer et al., 2001; Witzel et al., 2006). Our microtubule data show that Wnt11b signalling can have an effect on the cytoskeleton independently of controlling cell motility or cell adhesion (the egg is a single cell). Wnt11b-/- mutant eggs might thus serve as a useful system in which to study the Wnt/PCP or Wnt/Calcium regulation of cytoskeletal dynamics without confounding feedback effects resulting from changes in cell adhesion and or polarity.
In addition to signalling in the egg, our data also show that maternal Wnt11b is essential for blastopore closure and other cell behaviours during gastrulation, in this context potentially through regulation of actomyosin and convergent extension (see above). Maternal wnt11b compensates for zygotic loss of wnt11b (in F2 homozygous mutants derived from wildtype females), and maternal mRNA injection of wnt11b is sufficient to rescue the M/Z phenotype. MZwnt11b-/- mutants exhibit a delay in gastrulation, suggesting that other ligands (e.g., wnt5 homologues) or signals may eventually partially compensate. We note that gastrulation in MZwnt11b-/- embryos does seem to progress once zygotic expression of wnt11 (wnt11r) reaches normal levels (∼ stage 13) even though wnt5 homologues are expressed before this time. However, double Morpholino-based knockdown of Wnt11b/Wnt11 results in similarly delayed gastrulation, with abnormal extension of the archenteron (Van Itallie, et al., 2022). Thus, low levels of Wnt11 family function (whether through delayed wnt11 expression in M/Z mutants or through residual protein in knockdowns), with or without Wnt5 function, might be sufficient for ultimate closure of the blastopore. Alternatively, Wnt5 proteins could be sufficient for gastrulation to progress in the absence of Wnt11 family function, but with different (slower) kinetics. Future work, enabled by the wnt11b-/- mutants described here, will help distinguish these possibilities.
Maternal Wnt/PCP components (dvl2/3, fzd7, ptk7, gpc4 and vangl2) are similarly required for full convergent extension in zebrafish embryos and suggest a conserved role for Wnt signals near the onset of gastrulation (Jussila and Ciruna, 2017; Xing et al., 2018). Maternal Wnt11b could regulate general cell adhesion or cell behaviours prior to gastrulation, such as those occurring in the vegetal endoderm cells (e.g., vegetal rotation, separation behaviour; (Wen and Winklbauer, 2017; Winklbauer and Schürfeld, 1999; Winklbauer et al., 2001), and thus could potentially affect convergent extension or other morphogenetic movements indirectly. Loss of Wnt11b could also alter convergent extension indirectly by altering (elevating) BMP signalling and/or expression, which is inversely correlated with convergent extension (Myers et al., 2002) or through regulation of Nodal3.1-FGFR1 signalling (Yokota et al., 2003). A more extensive analysis of signalling interactions and perigastrular cell behaviours in wnt11b mutants would help distinguish these possibilities.
The genetic studies presented here also support the idea of a strictly zygotic role for Wnt11b in the establishment of left-right asymmetry in Xenopus (Walentek et al., 2013). F2 homozygous mutants for wnt11b derived from heterozygous crosses undergo normal axis formation and gastrulation and yet a subset undergo altered left-right asymmetry (∼25%). A similar proportion is seen in MZwnt11b-/- homozygotes. Taken together with the Morpholino-based observations of Walentek et al. (2013), which would affect only zygotic Wnt11b, these data suggest that left-right signalling is a function of zygotic Wnt11b. However, this must be only a biassing signal because roughly half the maternal/zygotic mutants develop reduced or bilateral pitx2c expression in the left lateral plate mesoderm – a condition which itself randomises asymmetry, thus potentially accounting for the 25% incidence of abnormal asymmetry.
In the context of left-right asymmetry, prior evidence suggests that Wnt11b controls cilia polarisation and cell shape in the ciliated cells of the posterior notochord/gastrocoel roof plate to establish leftward fluid flow (Schweickert et al., 2007; Walentek et al., 2013). Inhibition of “nodal flow” results in randomised left-right asymmetry in frogs and mice (Nonaka et al., 1998; Schweickert et al., 2010); we would therefore expect abnormal cilia and reduced fluid flow in wnt11b mutants. Wnt/Calcium signalling was implicated in this aspect of Wnt11b signalling, based on comparative inhibitor studies (Walentek et al., 2013) and this branch of the Wnt network was implicated in tissue separation behaviour as well (Winklbauer et al., 2001). It will thus be interesting to determine the extent that Wnt11b-mediated Wnt/Calcium signalling underlies the multiple roles of Wnt11b during development.
Other proposed functions of Wnt11b during development were not indicated by the genetic data presented here, including roles in pronephros, heart and neural crest development. It is possible that wnt11 or other functionally related wnts can compensate in these contexts, or that these defects might be secondary to alterations in gastrulation caused by the various loss-of-function reagents.
Overall, it is hoped the development of maternal-effect wnt11b mutants (the first such engineered maternal mutation in Xenopus) will be valuable for studying Wnt signalling activities within individual cells, as well as in conserved gastrulation movements and cell polarity signalling at the genetic level. Wnt11b mutants could be combined with other tools commonly used in Xenopus embryology including Morpholio/oligo-or F0 CRISPR-mediated gene knockdowns to further our understanding of diverse processes in early vertebrate development.
Materials and methods
Xenopus embryos and oocytes
Adult Xenopus laevis wildtype J-strain females (RRID:NXR_0024) were induced to ovulate using human chorionic gonadotropin (hCG, MP Biomed.). Eggs were collected and fertilised in 0.3x Marc’s Modified Ringer’s (MMR)[1x MMR: 1 M NaCl, 18 mM KCl, 20 mM CaCl2,10mM MgCl2, and 150 mM HEPES (pH 7.6)], using a sperm suspension (J-strain wildtype or wnt11b-/- mutant). Embryos were dejellied at the 2-8 cell stage in 2% cysteine in 0.1x MMR (pH7.8) for 4 min before washing the embryos with 0.1x MMR. Embryos were cultured to the desired stage at 18°-24°C in 0.1x MMR. For microinjection, fertilised eggs were dejellied one hour after fertilisation as above and transferred to 2% Ficoll (Pharmacia)/0.3x MMR and injected with 2-10 nl of solution as desired using an air-driven injector (Harvard Apparatus). Injected embryos were washed into 0.1x MMR for several hours-to-overnight after injection.
Ovary was isolated from anaesthetised wild type and mutant females and divided into one centimetre segments prior to storage at 18°C. Oocytes were manually defolliculated in a modified oocyte culture medium (OCM; 67% L-15, 0.05% PVA, 1x Pen-Strep, pH 7.6-7.8; (Houston, 2018; Houston, 2019)) using watchmakers forceps (Dumont #4 or #5) and cultured at 18°C. Alternatively, for in situ hybridization, oocytes were enzymatically defolliculated by treatment with 0.02% Liberase™ (Roche Applied Science) for 1-1.5 hours in 0.5x Delbecco’s PBS with rocking, followed by extensive washing in OCM.
CRISPR/Cas9 Mutagenesis
Mutations in wnt11b.L were generated in F0 embryos using CRISPR/Cas9 RNP injection. Guide RNAs were designed against exons 1-2 of X. laevis (J-strain) wnt11b.L (Supplemental Table 1). Wnt11b.L is the only homeolog of Wnt11b, and is the only member of the family to be expressed maternally (Session et al. 2016). In vitro transcribed guide RNAs were made from PCR-generated templates (Bhattacharya et al. 2015) and were complexed with Cas9 protein (CP01; PNA Bio) at 37°C before injection into the animal pole of fertilised eggs at the 1-2 cell stage. Mutagenesis was verified by sequencing of PCR products using DNA obtained through genotyping of whole embryo or tail samples, or through biopsy of the foot webbing of post-metamorphic animals. DNA isolation was performed as in (Bhattacharya et al., 2015) and sequencing was performed at MBL or at the Carver Center for Genomics (University of Iowa). A founder female was identified that transmitted a 13 base pair deletion in exon1 at the sgT1 site (chr8L:21220561..21220583) and was used to generate heterozygous male and female F1 offspring. These were interbred by in vitro fertilizations to generate homozygous F2 wnt11b.L mutant animals (RRID:NXR_2112). Breeding and colony maintenance were done by the NXR at MBL.
Plasmids
Full-length cDNAs for wnt11b.L were amplified from wildtype or wnt11b-/- mutant oocyte total RNA using a high-fidelity polymerase (Q5, New England Biolabs). PCR products were cloned into pCR8/GW/TOPO (Invitrogen) and individual clones were verified by sequencing. Primer sequences are presented in Supplemental Table 1. Desired clones in the (correct) 5’L1-3’L2 orientation were inserted via recombination into a pCS2+ Gateway-converted vector (Custom vector conversion kit; Invitrogen). Details of the Gateway plasmid are available upon request. Template DNAs for sense transcripts were prepared from wnt11b/pcs2+ plasmids by NotI digestion. Capped messenger RNA was synthesised using SP6 mMessage mMachine kits (Ambion). Eb3-gfp/pcs2+ RNA was similarly prepared as described (Olson et al., 2015). Mouse Lrp6 in pβ/RN3P was used as described (Kofron et al., 2007), prepared by SfiI/T3 digestion and transcription. RNAs for zebrafish dvl2-mcherry, rab5-gfp and rab11-gfp were gifts from D. Slusarski).
Analysis of gene expression using real-time RT-PCR
Total RNA was prepared from oocytes, embryos and explants using proteinase K and then treated with RNase-free DNase as described (Oh and Houston, 2017). Real-time RT-PCR was done using the LightCycler™ 480 system (Roche Applied Science). Samples were normalised to ornithine decarboxylase (odc) or to the geometric mean of odc and fgfr1, and relative expression values were calculated against a standard curve of control cDNA dilutions. Samples lacking reverse transcriptase in the cDNA synthesis reaction failed to give specific products. Primer sequences are listed in Supplemental Table 1. Charts were generated directly from the text file output of the Roche LightCycler software using a custom Python script.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed essentially as described (Kerr et al., 2008; Sive et al., 2000). Template DNAs for in vitro transcription were prepared by digestion, followed by transcription, with appropriate restriction enzymes and polymerases: wnt11b/pcs2+ (SalI/T7), nodal3.1 (from R. Harland; EcoRI/T7), eomes and myod1 (from J. Gurdon; EcoRI/T3 and BamHI/SP6 respectively), sox17a (from A. Zorn; Asp718/T3), pitx2c/pbluescript (a gift from M. Blum; NotI/T7) and sizzled/pcs2+ (from M. Kirschner, Addgene plasmid 16688, BamHI/T3), wnt8a/pcs2+ (XE10, from R. Moon; Addgene plasmid 16865; BamHI/T3). Antisense RNA probes labelled with digoxygenin-11-UTP (Roche) were synthesised using polymerases and reaction buffers from Promega. Processing of in situ hybridization was performed manually or using a robotic system (Biolane, Intavis AG).
Immunoblotting
Samples were lysed in cell lysis buffer (150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 1% Triton X-100, with protease and/or phosphatase inhibitors) and clarified by centrifugation (10 minutes at 10,000 x g). Lysates were heated in sample buffer (LAEMMLI, 1970) and the equivalent of 0.5 - 3 oocytes/embryos were electrophoresed on SDS-PAGE TGX AnyKD Ready Gels (BioRad) or large homemade gels and transferred to nitrocellulose (Power Blotter, Thermo-Pierce). For anti-phospho-LRP6 analysis, samples were lysed fresh (without freezing), and anti-phospho-LRP6 was used first, before stripping and reblotting.
Membranes were blocked in 5% BSA in TBS, 0.1% Tween 20 (for anti-phospho-LRP6) or 5% nonfat dry milk (HyVee) in PBS, 0.1% Tween 20, and incubated in primary antibody overnight at 4°C. Detection was performed using mouse or rabbit secondary antibodies conjugated to peroxidase (1:10000, Jackson Immunoresearch), and Licor reagents and equipment (C-Digit Scanner). Antibodies and dilutions used were: rabbit anti-phospho-LRP6 polyclonal antibodies (S1490; 1:500; Cell Signaling Technology (CST); RRID:AB_2139327), anti-LRP6 rabbit mAbs (C5C7 and C47E12; 1:500 each, mixed together; CST; RRID:AB_2139329 and RRID:AB_1950408), anti-Beta-tubulin (mAb E7; 1:1000; DSHB; RRID:AB_528499), and diphospho-ERK-1 and ERK-2 (1:4000; clone MAPK-YT; Sigma; RRID:AB_477245). In our hands, anti-phospho-LRP6 recognises endogenous Xenopus and injected mouse phospho-S1490 Lrp6, whereas the non-phospho mAbs recognise mouse but not frog Lrp6.
Immunostaining
Whole-mount immunostaining was performed on embryos fixed in MEMFA and stored in 100% methanol (embryo stages) or fixed directly in methanol (eggs), using modifications of previously described methods (Cuykendall and Houston, 2009; Elinson and Rowning, 1988). Fixed samples were rehydrated gradually to 1x PBS, then to PBT(PBS/0.5% Triton X-100/0.2% BSA (fraction V)) and then blocked for two hours at room temperature in PBT/2.5% BSA. Samples were washed for one hour the next day in PBT and incubated with primary antibodies diluted in PBT overnight at 4°C with rocking, followed by five one hour washes in PBT. Incubation with secondary antibodies diluted in PBT was done for 2 hrs followed by washing as above.
Antibodies were anti-Beta-tubulin mAb E7 (1:200 dilution of monoclonal antibody supernatant concentrate; DSHB Hybridoma Product E7, deposited with the DSHB by M. Klymkowsky; (Chu and Klymkowsky, 1989); RRID:AB_528499). Secondary antibodies were goat anti-mouse Alexa-488, diluted in PBT (1:500; Invitrogen/Molecular Probes). Fluorescence was visualised on a Leica DMI4000B inverted microscope using 20X - 63X dry objectives (Leica Microsystems). For confocal analysis, samples were imaged on an SP8 confocal imaging system (Leica Microsystems) using a 20X objective with or without tile-scanning to visualise the entire vegetal surface.
Time-lapse image analysis of microtubule plus end dynamics
Samples were imaged at room temperature on an inverted, wide-field epi fluorescence microscope (DMI4000B, Leica Microsystems) using an oil-immersion Leica 100x /1.30 N.A. PLANAPO objective. Image acquisition was done using a Leica DFC3000G monochrome camera at a frame rate of two seconds per frame and using Leica LAS software. The pixel size was 0.0536 × 0.0536 × 0.200 mm and the image size was 1296 × 966 pixels. Leica files (.lif) were imported directly into MATLAB_2021a using u-track (v2.3; https://github.com/DanuserLab/u-track; (Applegate et al., 2011; Jaqaman et al., 2008; Matov et al., 2010)) and processed using similar parameters as described (Olson et al., 2015). Custom code was inserted to incorporate code from the CircStat circular statistics toolbox (Berens, 2009) into the u-track analysis (available upon request). Statistical analyses of these data were performed using MATLAB; the fdr_bh package was used for adjusted p-values (Groppe, 2022; (Benjamini, 2010; Benjamini and Hochberg, 1995)). Consecutive frame averaging was performed using the Time-lapse Series Painter for FIJI (Vaart et al., 2011). Images for publication were prepared in Adobe Illustrator and Photoshop using only level and contrast adjustments applied over the entire image. Other modifications included resizing, changing stroke/fill weights and colours and annotated overlays.
Competing interests
D.W.H is interim director of the Developmental Studies Hybridoma Bank and serves on the Xenbase advisory board; M.E.H. is director of the National Xenopus Resource.
Funding
See Acknowledgements
Data availability
GEO accession GSE195806.
Acknowledgements
The authors would like to thank Cindy Toll for technical assistance in library preparation. We also thank Profs. R. Harland, J. Gurdon, A. Zorn, M. Kirschner, M. Blum, R. Lang, D. Slusarski, and R. Moon for contributing reagents (either directly or through Addgene and DSHB). We acknowledge Xenbase.org, the National Xenopus Resource, Addgene, and the Developmental Studies Hybridoma Bank (DSHB) for critical community resources. This work was funded by the University of Iowa (D.W.H) and by grants from the NIH, R01GM083999 (D.W.H) and R24OD030008, P40OD010997 (M.E.H.).