The AP-2 Transcription Factor APTF-2 Is Required for Neuroblast and Epidermal Morphogenesis in Caenorhabditis elegans Embryogenesis

The evolutionarily conserved family of AP-2 transcription factors (TF) regulates proliferation, differentiation, and apoptosis. Mutations in human AP-2 TF have been linked with bronchio-occular-facial syndrome and Char Syndrome, congenital birth defects characterized by craniofacial deformities and patent ductus arteriosus, respectively. How mutations in AP-2 TF cause the disease phenotypes is not well understood. Here, we characterize the aptf-2(qm27) allele in Caenorhabditis elegans, which carries a point mutation in the conserved DNA binding region of AP-2 TF. We show that compromised APTF-2 activity leads to defects in dorsal intercalation, aberrant ventral enclosure and elongation defects, ultimately culminating in the formation of morphologically deformed larvae or complete arrest during epidermal morphogenesis. Using cell lineaging, we demonstrate that APTF-2 regulates the timing of cell division, primarily in ABarp, D and C cell lineages to control the number of neuroblasts, muscle and epidermal cells. Live imaging revealed nuclear enrichment of APTF-2 in lineages affected by the qm27 mutation preceding the relevant morphogenetic events. Finally, we found that another AP-2 TF, APTF-4, is also essential for epidermal morphogenesis, in a similar yet independent manner. Thus, our study provides novel insight on the cellular-level functions of an AP-2 transcription factor in development.

All AP-2 transcription factors have a central basic region followed by a highly conserved helixspan-helix (HSH) motif at the carboxyl terminus [9]. The HSH is essential for dimerization and together with the adjacent basic region achieves a sequence-specific DNA binding function [10]. The less conserved proline-and glutamine-rich region at the amino terminus is required for transcription activation [11]. AP-2 transcription factors bind primarily to the palindromic core sequence 5'-GCCN 3 GGC-3' and serve a dual role as transcriptional activators or repressors [1].
AP-2 knockout mice display a wide spectrum of anomalies in early development such as craniofacial, neural tube and body wall defects, and polycystic kidney disease associated with uncontrolled apoptosis [12][13][14]. The phenotypic defects correspond to the diverse and overlapping expression patterns of murine AP-2 family genes in the neural crest cells, forebrain, facial and limb mesenchyme, and various types of epithelial cells [4,5,15,16]. In humans, mutations in TF AP-2-alpha (TFAP2A) have been associated with branchio-oculo-facial syndrome (BOFS), a congenital birth defect characterized by craniofacial abnormalities, skin and eye defects as well as hearing problems [17]. Char Syndrome, a congenital disease characterized by patent ductus arteriosus and facial and hand anomalies, was linked to mutations in TF AP-2-beta (TFAP2B) [18]. Multiple point mutations and deletions in BOFS and Char Syndrome patients have been mapped to the conserved basic region of the DNA binding domain in AP-2α and AP-2β [17][18][19][20]. However, the molecular mechanisms by which these mutations manifest in the disease symptoms are not well understood.
In C. elegans, there are four AP-2 TF family members: APTF-1, APTF-2, APTF-3 and APTF-4. APTF-1 functions in the GABAergic neuron RIS to induce sleep-like quiescence in C. elegans [21]. Other APTF members have not yet been studied. Using whole genome sequencing, we identified a mutant allele which gave rise to a single amino acid change in the basic region of APTF-2. Here, we describe the role of APTF-2 during C. elegans embryonic development, specifically during epidermal morphogenesis that involves the formation of a single epithelial layer that envelops the animal. We found APTF-2 is important for epithelial dorsal intercalation and ventral enclosure and mutation of aptf-2 results in larva with body morphology defects as well as embryonic lethality. Cell lineaging revealed misregulation of cell division timing, possibly leading to the phenotypic defects. Thus, C. elegans could serve as a model to study molecular and cellular consequences of mutations in the family of AP-2 TF analogous to those mutations in human AP-2 TF underlying BOFS and Char syndrome diseases.

A missense mutation in aptf-2(qm27) causes embryonic lethality and morphological defects in larva
In a genetic screen for maternal-effect mutations that have an impact on C. elegans development Hekimi et. al. isolated mal-1(qm27) as a mutation that causes extensive embryonic and larval lethality, with surviving homozygous mutants displaying morphological defects characterized by dorsal protrusions on the head and/or shortened body length [22] (Figs 1A and S1A and Tables 1 and S1). Genetic mapping predicted the approximate location of mal-1(qm27) on chromosome IV [22], but the molecular identity of the mal-1 gene has remained unknown. Whole genome sequencing of a mal-1(qm27) strain identified a missense mutation in aptf-2, one of four AP-2-like transcription factors in C. elegans. This mutation changes a highly conserved glutamic acid residue within the basic region of the DNA binding domain into a lysine residue (Fig 1B and  1C). Previous findings have indicated the basic region as a mutation hotspot for BOFS and Char Syndrome [17][18][19][20]. We also analysed gk902, a deletion allele of aptf-2 generated by the International C. elegans Gene Knockout Consortium. Similar to qm27, gk902 worms also displayed maternal effect embryonic lethality with 99 ± 0.5% of embryos not hatching and the few hatching larva displaying head and/or tail morphological defects and arresting as larva (Table 1, S1 Table). The gk902 and qm27 alleles failed to complement each other, as the progeny of trans-heterozygote aptf-2(gk902)/aptf-2(qm27) had a level of embryonic lethality in between homozygote aptf-2 (qm27) and homozygote aptf-2(gk902), consistent with them being mutations in the same gene ( Fig 1D and S2 Table). Moreover, expression of APTF-2::GFP from an integrated array driven by the aptf-2 promoter, completely rescued the embryonic lethality in both aptf-2(gk902) and aptf-2 (qm27) strains ( Fig 1D, S2 Table, S1 Movie), confirming the embryonic lethality in these strains is due to the mutations in aptf-2. Consistent with Hekimi et. al. [22] we found that qm27 homozygous progeny of +/qm27 worms are phenotypically normal, indicating maternal rescue.

aptf-2(qm27) embryos fail during epidermal morphogenesis
To characterize the developmental defects in aptf-2(qm27) embryos leading to their lethality we used 4D differential interference contrast (DIC) microscopy to follow isolated embryos positioned with either their dorsal or ventral side facing the microscope objective. We identified three major defects, all related to epidermal morphogenesis: failure in dorsal epidermal cell intercalation, failure of ventral epidermal cell enclosure, and arrest during elongation ( Table 2). A small percentage of embryos also exhibited leakage of cells out of the body of the embryo during elongation ( Table 2). The exact cause for elongation arrest is not easily discerned, but we noted that one third of the ventrally-oriented embryos that arrested during elongation had previous ventral enclosure defects and nearly all of the dorsally-oriented embryos that arrested in elongation displayed earlier defects in dorsal intercalation.
We confirmed the phenotypes observed in DIC microscopy by imaging aptf-2(qm27) embryos expressing fluorescently-tagged cell-cell junction markers E-cadherin/HMR-1 and alpha-catenin/HMP-1. As shown in Fig 2, S2 and S3 Movies, these markers confirmed the failure of epidermal cells to dorsally intercalate (Fig 2A), ventrally migrate (Fig 2B), and elongate the embryo (Fig 2C). Previous studies have shown that ventral enclosure defects are often preceded by failure of ventral neuroblasts to seal the cleft at the end of gastrulation. We imaged gastrulation cleft closure in wild-type and aptf-2(qm27) embryos by DIC and by expression of the neuroblast marker KAL-1::GFP and found that the ventral cleft in the mutant embryos was larger to begin with, took up to four times the amount of time to close and in some cases did not completely close before the onset of epidermal ventral enclosure (S1 Fig). A missense mutation in aptf-2(qm27) gives rise to embryonic lethality and morphologically defective larva. (A) Wild-type L1 larva (left panel) and aptf-2(qm27) (right panel) L1 larva exhibiting morphological defects in the head (indicated by the arrows). (B) Gene and protein organization of APTF-2. qm27 is an aptf-2 allele with a missense mutation at amino acid 182 (indicated by an asterisk) in the basic region of the DNA binding domain converting glutamic acid to lysine, whereas gk902 is a frame shift deletion allele of aptf-2 spanning from the middle of intron 1 to the end of exon 3. (C) Multiple alignment of AP-2 transcription factors in C. elegans and H. sapiens shows that the glutamic acid residue mutated in qm27 is conserved across species. (D) Loss of aptf-2 function in aptf-2(gk902), aptf-2(qm27) and in trans heterozygote aptf-2(gk902)/aptf-2(qm27) animals results in high embryonic lethality. Stable expression of APTF-2::GFP rescues embryonic lethality in aptf-2(qm27) and aptf-2(gk902) worms. Data are presented as mean ± s.e.m. n > 500 embryos from five independent experiments (*P<0.05, **P<0.01, two-tailed test). See S1 Movie for rescue experiment and S2 Table for the numerical values and statistical analysis. doi:10.1371/journal.pgen.1006048.g001 APTF-2 in C. elegans Development aptf-2(gk902) embryos display excessive apoptosis in the early embryo in addition to epidermal morphogenesis defects We next examined the embryonic phenotypes of the null mutant aptf-2(gk902) by DIC microscopy. We found 60% of the embryos died prior to epidermal morphogenesis, and approximately half of these early embryonic deaths were associated with the appearance of many ectopic apoptotic cells ( Fig 3A and Table 3). The remaining 40% of embryos that made it to epidermal morphogenesis all exhibited defects in dorsal intercalation, a quarter of them had ventral enclosure defects, and they all arrested during elongation ( Fig 3B, 3C and 3D and Table 3). The massive apoptosis phenotype was completely rescued by the expression of APTF-2::GFP (S2 Table), suggesting that it is a result of the complete loss of APTF-2 function. However, this phenotype was never observed in the partial loss of function allele qm27. Neither was it observed in aptf-2(RNAi) nor following injection of aptf-2 dsRNA into aptf-2(qm27).
die-1, a putative APTF-2 target gene is downregulated in aptf-2(qm27) embryos Using TargetOrtho [23], a phylogenetic footprinting tool to identify transcription factor targets, we identified within the C. elegans genome 1631 putative AP-2 TF binding sites in the 3KB upstream promoter region of 872 genes (S1 Text). Protein domain analysis of these genes revealed enrichment in F-box, Homeobox, EF-hand, SET and CUB domain proteins, as well as others, and gene onthology analysis of biological processes showed enrichment in genes associated with embryonic development, tissue morphogenesis, locomotion, regulation of growth rate, and reproduction, among others (see S1 Text for full list). Among the putative AP-2 TF regulated genes classified as associated with epithelium development our attention was caught by die-1. The zinc finger transcription regulator DIE-1 is autonomously required in the posterior dorsal hypodermis for intercalation, for morphogenesis in other embryonic tissues, and for normal postembryonic growth and vulval development [24,25]. Given the defects we observed in epidermal morphogenesis we tested whether the expression of die-1 is altered in aptf-2 mutants. Indeed, we found that two out of seven aptf-2(qm27) embryos showed aberrant localization of DIE-1::GFP. Furthermore, we measured a 22.5% reduction in mean intensity of

aptf-2(qm27) embryo development is retarded compared to wild-type
Analyzing the DIC movies of embryonic development we found that in addition to the various defects in epidermal morphogenesis the aptf-2(qm27) embryos developed more slowly than wild-type embryos at the same temperature. To quantify the delay and find out whether there is a particular stage in development that is slower or if all of embryogenesis is inherently slower we chose easy-to-recognize developmental milestones in dorsally or ventrally oriented embryos and measured the time it took for an embryo to progress from one milestone to the next (S3A and S3B Table). We also measured the same developmental times in aptf-2(qm27) embryos stably expressing wild-type APTF-2::GFP. The results, graphically presented in Fig 5, show that all stages of development are slower, to varying degrees, in aptf-2(qm27) embryos, and the developmental timing is mostly rescued in embryos ectopically expressing APTF-2::GFP. Specifically, ventral cleft closure is three times slower and elongation to 2 fold stage is one and a half times slower, while early development until Ea/Ep ingression is only slightly slower.
Cell lineaging uncovers aberrant cell divisions in ABarp, C and D lineages in aptf-2(qm27) embryos To better understand the developmental defects in aptf-2(qm27) embryos we performed cell lineage analysis by following a nuclear marker, HIS-72::GFP, using 4D fluorescence microscopy. The cell division patterns in wild-type and aptf-2(qm27) embryos were captured, then movement of the dorsal epidermal nuclei towards the opposite side of their starting position (top panels). Intercalation of dorsal epidermal cells is defective in aptf-2(gk902) embryo as indicated by abnormal or absence of movement of dorsal epidermal nuclei (bottom panels). Nuclei of dorsal epidermal cells are colored to better visualize their positions during the intercalation process. t' marks the positioning of the two rows of epidermal cells at the dorsal side, whereas t+60' in wild type marks the completion of the intercalation process. (C) The end of gastrulation is marked by the closure of the ventral cleft. In wild-type embryos, the size of the ventral cleft was 23.14 μm 2 ± 1.595 (mean ± S.E.M, N = 19) and it closed within 20 minutes (top panels), whereas in the aptf-2(gk902) embryos, the ventral cleft was four times as large at 99.74 μm 2 ± 8.858 (mean ± S.E.M, N = 21) and often stayed open until the time of ventral epidermal enclosure and thereby prevented proper epidermal enclosure from taking place (bottom panels). The ventral clefts are colored in blue. t' marks the start of the ventral cleft and t+20' marks its closure in wild type. (D) The ventral epidermal enclosure is followed by elongation. While the wild-type embryo progressively elongates from 2-to 3.5-fold in 2 hours (top panels), the aptf-2(gk902) embryo is arrested during elongation slightly beyond 2 fold stage (bottom panels). t' marks the beginning of two fold elongation, whereas t+120' marks 3.5 fold elongation in wild type.
doi:10.1371/journal.pgen.1006048.g003 Table 3. Phenotypic analysis of aptf-2(gk902) embryos analyzed by DIC. APTF-2 in C. elegans Development analysed and edited using StarryNite and AceTree, respectively (n = 2 for wild-type and n = 6 for aptf-2(qm27) embryos). Cell division defects were consistently detected in three lineages: ABarp, C and D (Fig 6). The color markings drawn on the wild-type lineage trees illustrate the frequency of defects that occurred in the six aptf-2(qm27) mutant embryos analysed. Strikingly, failure in aptf-2(qm27) cell division occurs mostly in three lineages: ABarp, C and D with the Caaaa division absent in all six aptf-2(qm27) embryos analysed. The missing divisions resulted in the absence of epidermal seam cells and neuroblasts in the AB lineage and the absence of epidermal cells from the main body syncytium (hyp7), body wall muscle cells in the C and most of the D lineage (Fig 6 and S4 Table). In other cell lineages cell divisions appeared to be normal, except for an occasional division absent in the ABala or MSa lineages (S2-S14 Figs). We used embryos co-expressing HIS::mCherry and the translational fusion of APTF-2::GFP driven by the aptf-2 promoter to follow the subcellular localization of APTF-2 in specific cells during embryogenesis (Fig 6A). We found that in most cells APTF-2 is found uniformly in the nucleus and the cytoplasm. However, in certain cells at specific times during development, APTF-2 was enriched within the nucleus. Based on the lineaging of two embryos for 210 minutes we found significant nuclear enrichment of the APTF-2::GFP signal in neuroblasts and epidermal cells in AB lineage during ventral cleft closure and in epidermal cells in C lineage preceding dorsal intercalation (Fig 7B and S15 Fig). However, there does not appear to be a strong correlation between nuclear enrichment of APTF-2 and defects in cell division. While a high degree of nuclear enrichment was found in the C and ABarp lineages, in which the absence of cell division in aptf-2(qm27) embryos occured in 6/6 embryos, a high degree of nuclear enrichment was also found in ABpra and ABpla lineages that did not experience any defects in cell division. Similarly, in the D lineage, which did not show much nuclear enrichment, the failure in cell division was frequently observed.
Aberrant nuclear localization does not explain the functional defects of the qm27 allele of APTF-2 In light of the specific nuclear enrichment of APTF-2 in the cell lineages where we observed defects in cell division timing in the aptf-2(qm27) hypomorph, we wondered whether the mutant protein has a defect in nuclear enrichment. To test this possibility we introduced into the APTF-2::GFP construct the same point mutation present in the qm27 allele. As shown in Fig 8A, the mutant protein had no problem in becoming enriched in neuroblast nuclei during ventral cleft closure. To the contrary, once the mutant APTF-2 entered the nucleus, it appeared to remain enriched in the nucleus for longer than the wild-type protein. This raised the question whether abnormal nuclear retention of APTF-2 could explain the defects in aptf-2(qm27).

APTF-4 cooperates with APTF-2 to regulate epidermal morphogenesis
The worm genome encodes for four AP2-like transcription factors (S16 Fig). APTF-1 is expressed in only five head interneurons and is required for a sleep-active neuron to induce lethargus in molting larvae [21]. To test whether APTF-3 and/or APTF-4 may play a role in embryonic development we depleted zygotic and maternal products of the genes by RNAi and tested for embryonic lethality in the progeny. Knockdown of aptf-3 did not result in any embryonic lethality. In contrast, knockdown of aptf-4 resulted in 26 ± 3% embryonic lethality. Moreover, hatched aptf-4(RNAi) larvae often exhibited body morphology defects reminiscent defective in the aptf-2(qm27) embryos and S2a- S2m Fig for the  APTF-2 in C. elegans Development of the defects observed in aptf-2 mutants (Fig 9A). The deletion allele aptf-4(gk582) resulted in 100% larval arrest of homozygous worms, precluding analysis of embryonic phenotypes. Closer examination of embryonic development by 4D DIC and fluorescence microscopy revealed defects in dorsal intercalation, ventral cleft closure, and elongation ( Fig 9B-9D, S4 Movie). To test whether APTF-2 and APTF-4 work independently or cooperatively in the regulation of epidermal morphogenesis we tested the combined effect of aptf-4 KD in the background of aptf-2(qm27). We found the embryonic lethality upon co-depletion of aptf-2 and aptf-4 to be higher than the sum of the lethality of single depletions, suggesting synergy between aptf-2 and aptf-4 ( Fig 8E and S6 Table). As AP-2 transcription factors are believed to function as heterodimers in some cases [26], one possibility is that aptf-2 and aptf-4 work cooperatively. 4D DIC movie analysis revealed that 100% of the dorsally oriented dual-depleted embryos had dorsal intercalation defects and arrested during elongation and 57% of the ventrally oriented dualdepleted embryos displayed ventral cleft closure defects and 100% of them arrested in elongation (S7 Table). We used expression data for APTF-4 from the EPIC dataset (http://epic.gs. washington.edu/) to compare the nuclear expression pattern between APTF-2::GFP and APTF-4::GFP (S17 Fig). Both APTF-2::GFP and APTF-4::GFP showed similar nuclear enrichment in the AB and C lineages, consistent with their cooperativity in embryogenesis.

Discussion
Vertebrates and C. elegans AP-2 TF genes share high sequence similarities in their functional domains, although the duplications leading to four family members appear to have occurred  In this study, we report that partial loss of aptf-2 or aptf-4 resulted in body morphological defects. Patients with BOFS suffer from skin defects while complications associated with Char Syndrome result from derangement of neural-crest-cell derivatives [17,18]. Our findings from the characterization of aptf-2(qm27) share similarity with the pathological manifestation of BOFS and Char Syndrome patients in epidermal and neuronal tissues. The mutation in the aptf-2(qm27) allele lies in the basic region of the DNA binding domain, a region that was defined as a mutation hotspot for BOFS and Char Syndorme in the human TFAP2A and TFAP2B genes [17][18][19][20]. At least 24 mutations in the basic region have been identified for BOFS and five for Char Syndrome [18,20,27]. It is challenging to determine the genotype-phenotype relationship in BOFS and Char Syndrome patients due to the small sample size and the large spectrum of mutations affecting TFAP2A and TFAP2B. With recent advances in site-targeted mutagenesis in the C. elegans genome, it is an exciting possibility to generate worm strains carrying mutations of conserved residues in BOFS and Char Syndrome. The aptf-2(gk902) allele results in a frame shift, generating a null allele. The massive apoptotic phenotype observed following a complete loss of APTF-2 in aptf-2(gk902) embryos is drastically different from the epidermal morphogenesis defects observed when APTF-2 activity is partially compromised as with the aptf-2(qm27) allele. This suggests different thresholds of AP-2 transcriptional activity are required for different cellular functions. Interestingly, in Char Syndrome patients, hypomorphic mutations in TFAP2B result in congenital heart defect, whereas a complete deletion of the mouse ortholog, AP-2β, leads to polycystic kidney disease due to excessive apoptosis of renal epithelial cells [14,18].
In murine models, depletion of AP-2γ resulted in defective epidermal development due to delayed expression of epidermal differentiation genes [28]. This is consistent with our observation that aptf-2 mutants showed epidermal morphogenesis defects. Neural crest defects in mouse, zebrafish and Xenopus embryos have been attributed to loss of AP-2 transcription factors [1,29,30], parallel to the neuroblast migration defect we observed in the C. elegans embryo. Earlier expression studies of AP-2 transcription factors were largely conducted in mice, Drosophila and Xenopus by observing in-situ hybridization and staining patterns [5,16,[31][32][33]. Our work in the live C. elegans embryo provided spatio-temporal information at a resolution not described previously. We observed APTF-2::GFP to be enriched in the nuclei of neuroblasts and epidermal cells during ventral enclosure and dorsal intercalation respectively, lack of which (in the case of the mutant) resulted in aberrant cell division in the epidermal and neuroblast lineages. Thus, our work identified lineage-specific regulation of cell division timing by APTF-2. Similar mechanisms could be at play in mammals. Interestingly, we observed that nuclear enrichment of APTF-2 does not always correlate with regulation of cell division, as in the case of D, suggesting that a lower level of nuclear APTF-2 may be required for the division in this lineage. In contrast, nuclear APTF-2 enrichment was observed in ABpra and ABpla and yet an absence of cell division was not been observed in these lineages in aptf-2(qm27) embryos, indicating that either a stronger APTF-2 depletion is required to see cell division defects or APTF-2 plays a different role in these two lineages.
Although various members of the vertebrate AP-2 transcription family have been shown to have overlapping expression patterns, knockout studies in mice revealed specific and localized phenotypic defects. For example, Moser et. al. showed that the AP-2α and AP-2β expression in mouse embryos overlap significantly, [16], but the single knockout models of each gene did not share any phenotypic defects, suggesting non-redundant roles of the two genes [14]. In contrast to the vertebrate system, our results showed both similar phenotypes and similar expression pattern, mostly in AB and C lineages of aptf-2 and aptf-4 in the worm. The fact that their effect is synergistic suggests they may partially function through the same pathway.
For wild-type APTF-2::GFP, expression in the majority of cells was evenly distributed between the nucleus and cytoplasm and was enriched in the nucleus of neuroblasts during ventral cleft closure and in epidermal cells preceding dorsal intercalation. It is possible that APTF-2 functions to regulate gene expression at a basal level, while enrichment in the nucleus of specified cells during epidermal morphogenesis upregulates genes required for proliferation of the neuroblasts and epidermal cells. This would be consistent with observations in Drosophila, where different levels of AP-2 have been shown to result in a variety of morphological defects [32].
AP-2 transcription factors are known to play a dual role as transcription activators and repressors [33]. Pfisterer et. al. identified multiple genes repressed by AP-2α known to induce apoptosis and retards proliferation [34]. There has also been evidence in Xenopus epidermal development regarding the importance of AP-2 TF in promoting the expression of epidermal specific genes [31]. We used TargetOrtho to identify putative APTF-2 targets. Among the candidates, we tested die-1, a well known regulator of epidermal dorsal intercalation, and observed the reduction of DIE-1 nuclear signal in aptf-2(qm27) embryos, suggesting that DIE-1 is likely a target of APTF-2. Future work must determine APTF-2 target genes in neuroblasts and epidermal cells in order to further elucidate its function during morphogenesis.
In conclusion, we have characterized a hypomorphic mutant of C. elegans APTF-2 and have shown it to share genetic and anatomical similarities with human Char Syndrome and Bronchio-occular-facial Syndrome. We propose mutations in C. elegans AP-2 TF genes can serve as disease models to study the cellular mechanisms and tissue dynamics that lead from mutant genotype to disease phenotype.

Strains and alleles
Strains were maintained at 20°C under standard conditions [35]. Wild-type Bristol strain N2 was used as a control. The aptf-2(qm27) IV line was retrieved in an EMS screen conducted by Hekimi et al. [22] and aptf-2(gk902) was generated by the C. elegans Reverse Genetics Core Facility at the University of British Columbia and was maintained as heterozygotes using the nT1[qIs51] (IV;V) balancer. For analysis using GFP reporters, F 2 progeny exhibiting aptf-2 phenotypes and carrying the markers were selected from crosses between aptf-2(qm27) and the following strains:

Whole genome sequencing and mutation validation
Genomic DNA was extracted from mal-1(qm27) mutant worms using standard method and subjected to whole genome sequencing using Illumina platform and annotated using MAQ-Gene [41]. The whole genome sequencing and its annotation were performed by Hobert lab (Columbia University). Candidate genes altered in mal-1(qm27) were narrowed down using genetic mapping results done by Hekimi et al. [22]. Point mutation in aptf-2 gene was confirmed by amplification of aptf-2 gene in aptf-2(qm27) mutant worms, subcloning into pJET vector (Thermo Scientific) and followed by conventional sequencing (First Base).

Complementation assay, brood size analysis and larva phenotype scoring
For complementation assay, aptf-2(gk902)/nT1[qIs51] males was crossed with aptf-2(qm27) hermaphrodites. Non-GFP F 1 animals were singled and incubated to lay embryos for 24 hours. The F 1 animals were genotyped for the gk902 deletion and only the cross progeny between qm27 and gk902 alleles was scored for embryonic lethality of their F 2 s.
For brood size analysis, ten L4 larvae of wild-type, aptf-2(qm27) and aptf-2(gk902) were singled and incubated for 24 hours. Each animal was shifted to a new plate every day for 5 consecutive days to the point that no more embryos were laid. The total number of embryos laid was determined as the brood size. The number of hatched animals was calculated and used to determine the percentage of embryonic lethality. Larvae that did not grow into adult in 48-92 hours after hatching were considered as being arrested. aptf-2(qm27) and aptf-2(gk902) larvae of any stage were subjected to phenotypic analysis to determine the presence and the position of the morphological defects.

Quantification of embryonic lethality
Besides wild-type, aptf-2(qm27) and aptf-2(gk902) animals whose embryonic lethality was determined as described above, the embryonic lethality of the remaining strains were determined as follows: ten to fifteen gravid hermaphrodites were placed on the plate and incubated at 20°C for several hours to lay more than 100 embryos. Hermaphrodites were then removed and the number of embryos laid was counted. Twenty-four hours later, the number of larvae hatched was determined. Each experiment was repeated at least five times.

4D microscopy
Larvae or embryos collected from gravid hermaphrodite were mounted onto 3% agarose-padded glass slide, closed with a coverslip and sealed with wax. DIC images shown in Figs 1A, 3A, 3B, 3C, 8A, 8C, 8E and S1A were captured using a Nikon Ti Eclipse widefield microscope equipped with DIC 1.40NA oil condenser and a charged-coupled device camera Cool Snap HQ 2 (Photometrics). All other images and movies were acquired using a spinning disk confocal system composed of a Nikon Ti Eclipse microscope with a CSU-X1 spinning disk confocal head (Yokogawa), DPSS-Laser (Roper Scientific) at 491 and 568 nm excitation wavelengths and an Evolve Rapid-Cal electron multiplying charged-coupled device camera (Photometrics). For both microscopes, Metamorph software (Molecular Devices) was used to control acquisition. Projected images were created using Fiji. All imaging was done at 20°C in an environmental chamber encompassing the microscope stage heated by a JCS temperature controller (Shinko Technos Co, Japan) within a microscope room kept at 18°C by a CITEC precision air conditioning unit.
Knockdown experiments aptf-4 dsRNA was synthesized as described [42] and injected into the gonad of twenty wildtype or aptf-2(qm27) L4 larvae. Each animal was singled into a separate plate and its embryonic lethality was examined 24 hours post injection.
Predicting AP2-TF target genes using TargetOrtho AP-2 has been shown to bind to the palindromic consensus sequence 5'-GCCN3GGC-3', as well as the binding motif 5'-GCCN3/4GGG-3' [2]. We used either the 9bp or 10bp motif as an input for TargetOrtho [23]. From the program output we selected only putative targets that are conserved in at least 4 Caenoharbditis species, and are located within the 3 kb region upstream of the start codon. Functional annotation was performed using DAVID Bioinformatics Resources 6.7 [45,46] and the threshold we used for enrichment was an EASE score equal or smaller than 0.05.

Cell lineaging
For cell lineaging, six aptf-2(qm27) embryos expressing nuclear signal of GFP::HIS-72 and two embryos co-expressing APTF-2::GFP and mCherry::HIS-72 were analysed for at least 270 minutes according to the protocol described in [47][48][49]. The lineage tree was built using AceTree [50] and compared to that of wild-type. To visualize the temporal enrichment of the nuclear APTF-2::GFP signal during embryogenesis, the minimum/ maximum threshold values were set to display the 75% highest signal. All movies used for lineaging in this paper can be downloaded from http://epic2.gs.washington.edu/Epic2.

Statistical analysis
Statistical analyses were done using Prism 6 (GraphPad Software, La Jolla, CA). Two-tailed Student's t-test was applied to compare the values.  Table. Expression of APTF-2::GFP in aptf-2(gk902) and aptf-2(qm27) animals rescues their embryonic lethality. (DOCX) S3 Table. APTF-2 is required for timely embryogenesis events (A dorsally-oriented embryos B ventrally-oriented embryos). (DOCX) S4 Table. Affected lineages in aptf-2(qm27) mutant embryos. (DOCX) S5 Table. Nuclear targeting of APTF-2 is sufficient to rescue aptf-2(gk902) and aptf-2 (qm27) embryonic lethality. (DOCX) S6 Table. APTF-2 and APTF-4 synergistically regulate C. elegans embryogenesis. (DOCX) S7 Table. Phenotypic analysis of aptf-2(qm27) embryos injected with aptf-4 dsRNA and analyzed by DIC. (DOCX) S1 Movie. Time lapse DIC and spinning disk microscopy showing that the expression of APTF-2::GFP rescues aptf-2(qm27) embryonic lethality. Movie starts from early embryogenesis and ends at the time of hatching. Only a single focal plane of the embryo epidermis is shown. Time is indicated in minutes. Embryo at the bottom expresses APTF-2::GFP, whereas embryo at the top does not. (AVI) S2 Movie. Dorsal epidermal cell intercalation in wild-type and aptf-2(qm27) embryos expressing HMR-1::GFP. Movie starts with two rows of dorsal epidermal cells ready to intercalate and ends with the wild-type embryo hatching and the aptf-2(qm27) embryo arrested during elongation. Time is indicated in minutes. (AVI) S3 Movie. Ventral enclosure in wild-type and aptf-2(qm27) embryos expressing HMP-1:: GFP. Movie starts when ventral epidermal cells initiate embryo enclosure and ends with the wild-type embryo hatching, whereas the mutant embryo is not properly enclosed, resulting in the internal tissues leaking out from the gap on the ventral side. Time is indicated in minutes. (AVI) S4 Movie. Time lapse spinning disk microscopy showing dorsal epidermal cell intercalation in aptf-4 dsRNA embryo expressing HMR-1::GFP. Movie starts with two rows of dorsal epidermal cells ready to intercalate. The intercalation is abnormal and ends with the embryo being arrested during elongation. Time is indicated in minutes. (AVI) S1 Text. List of putative target genes of AP-2 transcription factors in the C. elegans genome generated by TargetOrtho (see materials and methods for details). (XLSX) S2 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #1 produced by Starry-Nite [47] in a format readable by AceTree. (ZIP) S3 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #2 produced by Starry-Nite [47] in a format readable by AceTree. (ZIP) S4 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #3 produced by Starry-Nite [47] in a format readable by AceTree. (ZIP) S5 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #4 produced by Starry-Nite [47] in a format readable by AceTree. (ZIP) S6 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #5 produced by Starry-Nite [47] in a format readable by AceTree. (ZIP) S7 Text. zip files containing the nuclei files of aptf-2(qm27) mutant #6 produced by Starry-Nite [47] in a format readable by AceTree.

Supporting Information
(ZIP)