To get insights into the conservation of itpka gene location and Itpka protein homology of X. laevis in comparison to Homo sapiens, in silico analysis was performed. Comparative synteny analysis revealed that the location of itpka and the surrounding genes on the chromosome are highly conserved across species (Figure 1A). A schematic overview of the ITPKA protein illustrates the region required for cytoskeleton location, the inositol polyphosphate kinase (IPK) region as well as calmodulin binding region (Figures 1B,C). The full-length protein of human ITPKA demonstrated comparable amino acid lengths and a high evolutionary conservation of the protein sequences across different species (Figure 1D).

The knowledge of the spatio-temporal expression pattern of itpka during the early development of vertebrates such as X. laevis remains unknown so far. To provide a detailed expression pattern analysis of itpka during the development of X. laevis, we used the whole mount in situ hybridization (WMISH) technique. First, we have designed a sense and an antisense probe to validate if the itpka digoxigenin-labeled antisense probe binds specifically to the X. laevis itpka mRNA in comparison to the digoxigenin-labeled sense probe. Expression analysis revealed a specific expression pattern of the itpka antisense probe and thus we used it for the detailed analysis (Supplementary Figure S1). At early cleavage stages, itpka transcripts were detected in the neuroectoderm next to the blastopore of X. laevis embryos (Figure 2A; arrowhead). At stage 13, itpka-positive cells were found in the anterior neural tissue (Figure 2B). At stage 15, itpka is expressed in a distinct region in the anterior neural plate and the notochord (Figures 2C,D). From stage 20 onward, we found itpka transcripts predominantly in the developing eye, brain, epidermis and migrating neural crest cells (NCCs) such as the mandibular (ma), hyoid (ha), and branchial arches (ba) (Figures 2E–J). The RT-PCR analyses using isolated tissues of different stages (Figure 2K) revealed an expression of itpka in the anterior (head) region and the eye (Figure 2L).

Our data indicated that during early X. laevis development, itpka transcripts were enriched in developing anterior neural tissue (Figure 2). We thus introduced an antisense-based morpholino oligonucleotide (MO) KD approach to investigate the possible effects of Itpka depletion in anterior neural development of X. laevis. First of all, we investigated whether the Itpka MO efficiently binds to the X. laevis itpka MO binding site. Therefore, the respective MO binding site of X. laevis (Xitpka MObs) and the corresponding Δ 5′UTR X. laevis itpka MO binding site (Δ 5′UTR XitpkaMObs) were cloned in front of and in frame with green fluorescent protein (GFP). RNA of these constructs was co-injected together with Itpka MO and control morpholino oligonucleotide (CoMO) into two-cell stage embryos. GFP expression was then monitored in stage 20. Itpka MO blocked GFP expression upon Xitpka MObs-GFP RNA co-injection, whereas CoMO did not, indicating the interference of Itpka MO with Xitpka MObs-GFP translation. In addition, Itpka MO did not block translation of Δ 5′UTR XitpkaMObs-GFP demonstrating that RNA coding for Δ 5′UTR itpka RNA is suitable for rescue experiments (Supplementary Figure S2).

To investigate the Itpka MO KD, we injected Itpka MO unilaterally into one animal dorsal blastomere of X. laevis embryos at the eight-cell stage that gives rise to the anterior neural tissue (Moody and Kline, 1990). To monitor the targeted region, we co-injected 0.4–0.5 ng GFP RNA and sorted the embryos according to its specific expression in the anterior neural tissue. CoMO that cannot bind to any X. laevis mRNA was used as injection control (Eisen and Smith, 2008). Itpka MO-injected embryos exhibited a spectrum of anomalies ranging from significantly smaller and/or deformed heads or eyes on the injected side to a lack of anterior head structures in an Itpka MO-dose dependent manner (Figures 3A,B).

To analyze the head phenotype upon Itpka MO KD in more detail, we first quantified the embryos revealing significantly more embryos with smaller and/or deformed head compared to wildtype (WT) and CoMO-injected embryos (Figures 4A,B). The measurement of head width and area of X. laevis embryos showed significantly smaller heads (Figures 4A,C). Co-injection of X. laevis itpka (Xitpka) RNA that is not targeted by Itpka MO significantly rescued the Itpka MO-induced head width and area indicating the specificity of Itpka MO-induced phenotypes (Figures 4D–F). The smaller heads of Itpka morphants prompted us to focus more on the cartilage. Alcian blue staining at late tadpole stages showed a reduction of branchial arch and Meckel’s cartilage in Itpka MO-injected embryos (Figure 4G). In addition, 3A10 antibody staining revealed a clear shortening and/or disorganization of the cranial nerves upon Itpka KD (Figures 5A,B). For instance, the Nervus opticus (N. opticus) and Nervus glossopharyngeus (N. glossop.) were shortened in length (Figures 5A,C). Furthermore, confocal images revealed a more diffuse organization of the N. opticus, Nervus trigeminus (N. trigeminus) and Nervus mandibularis (N. mandibularis) (Figures 5D,E).

After observing smaller head structures in Itpka MO-induced embryos, we thought that a possible reason of smaller heads could be fewer proliferative cells or more apoptotic cells upon Itpka KD, but neither pH H3 nor TUNEL staining was altered in the anterior neural region of the head compared to the control embryos at stage 23 (Supplementary Figure S3). These results indicate that the observed changes are not due to altered or abnormal proliferation or apoptosis in the respective area and at this early stage in development.

Since itpka is expressed in NCCs (Figures 2F–H,J), and Itpka KD led to deformed NCC derivates such as cranial cartilage and nerves, we investigated the NCC-specific genes twist1 (twist family bHLH transcription factor 1), snai2 (snail family transcriptional repressor 2), foxd3 (forkhead box D3) and egr2 (early growth response 2) by WMISH. At stage 15, the neurula stage, the induction of the neural crest cells is initiated. At this stage expression of twist1 and snai2 was strongly reduced upon Itpka KD (black arrowheads) (Figures 6A,B). Detailed analysis revealed that the expression area and intensity were significantly diminished (black arrowheads) (Figures 6C–E). At stage 20, the expression of twist1 and snai2 in the migrating NCC was also reduced upon Itpka KD (black arrowheads) (Figures 7A,B), which was confirmed by measuring the expression area (black dotted lines) (Figures 7C,D). The expression intensity was not altered (Figure 7E). At stage 23, the expression of the marker genes twist1, foxd3 and egr2 was decreased upon Itpka MO-injection (black arrowheads) (Figures 8A,B) as evidenced by a reduction in the expression area, intensity (black dotted lines) and length of the three branchial arches (black lines) (Figures 8C–H). In conclusion, Itpka depletion impairs NCC induction and migration, which may contribute to craniofacial malformations in X. laevis embryos.

Previous studies have shown that Itpka loss of function negatively affects brain morphogenesis in adult mice (Köster et al., 2016). To analyze Itpka deficiency in X. laevis brain development, we isolated the brains of Itpka morphants (stage 42) and observed a significantly smaller brain area on the injected side (Figures 9A,B). Afterwards, we performed WMISHs with several brain-specific genes in different developmental stages of X. laevis embryos. At stage 13, where the induction of brain-specific cells starts, the expression of egr2 (rhombomeres 3 and 4) and pax6 (paired box 6; posterior neural tube) was significantly reduced upon Itpka MO KD (black arrowheads) (Figures 9C,D). Additionally, egr2 showed a reduced expression at stage 15 (black arrowhead) (Figures 9E,F). At a later stage (stage 23) brain-specific genes such as pax6 (forebrain and posterior neural tube), otx2 (orthodenticle homeobox 2; forebrain and midbrain) and egr2 (hindbrain) showed a reduced expression in Itpka MO-manipulated embryos in the fore-, mid- and hindbrain (black arrowheads) (Figures 9E,F). Taken together, these data indicate that Itpka depletion in very early stages affects brain cell induction and differentiation presumably contributing to brain defects in X. laevis.

As we investigated the head and brain phenotype, an eye phenotype was also noticeable. Itpka MO-injected embryos showed significantly smaller and/or deformed eyes on the injected side compared to WT and CoMO-injected embryos (Figures 10A,B). A detailed analysis of eye area (red dotted line) revealed a smaller eye size upon Itpka KD (Figures 10A,C). Itpka MO-mediated KD was partially rescued by co-injection of Xitpka RNA (Figures 10D,E). The detailed analysis of the eye area revealed that Xitpka significantly rescued the Itpka MO-induced eye phenotype (Figures 10D,F).

To investigate retinal lamination, we performed WMISHs of well-characterized retina-cell-type genes followed by transversal vibratome sectioning (Cizelsky et al., 2013). Most of the specific genes for retina cell layers such as the photoreceptor (rho; rhodopsin), horizontal (prox1; prospero homeobox 1), bipolar (vsx1; visual system homeobox 1), amacrine/ganglion (pax6) and ganglion (pou4f1; pou class 4 homeobox 1) cells displayed a disorganized localization and/or a smaller area in the retina upon Itpka MO KD (Figure 10G). Probes specific for the lens, celf1 (CUGBP elav-like family member 1; mature lens fiber cells) and cryba1 (crystallin beta A1; stem cells), show a diffuse expression in Itpka morphants (Figure 10H).

To determine the molecular basis of the eye phenotype, we then investigated key steps of eye development. During eye field induction at stage 13, the anterior expression of the eye-specific genes rax (retina and anterior neural fold homeobox) and pax6 was significantly reduced (black arrowheads) in Itpka morphants at the injected side, while the expression of the pan-neural gene sox3 (sex-determining region Y-box 3) remained unaffected (Figures 11A,B). At a later stage (stage 23) during eye cell differentiation, Itpka deficiency caused a reduction in the expression of all investigated eye cell-specific genes rax, pax6, and otx2 (black arrowheads) (Figures 11C,D). Taken together, we have shown that Itpka is essential for proper eye development as well as eye cell differentiation and that its depletion already affects early eye field induction in X. laevis.

In our study, in silico analysis of ITPKA identified strong evolutionary conservation across species. Consistent with previous findings that demonstrated a high degree of similarity of ITPKA and ITPKB in their C-terminal amino acid sequences and less conservation in the N-terminal region (Erneux et al., 1992), our data additionally show that there is a high homology of ITPKA protein alignment across species. Notably, this conservation is particularly strong within the C-terminal region, which includes the IPK and the calmodulin-binding regions.

To date, there is only one study in vertebrates that describes the expression kinetics of itpka during X. tropicalis development (Owens et al., 2016), showing that itpka is increased expressed from stage 10 on. Northern blot analysis revealed ubiquitous and strong expression of Itpka in the brain (especially in the hippocampus, neocortex and cerebellum) and testis of adult mice, but only a very low expression during early mouse embryogenesis. This expression increased with postnatal development to adulthood (Mailleux et al., 1993; Vanweyenberg et al., 1995). In our study, we observed an enrichment of transcripts in neuroectoderm-derived structures such as the cells of neural crest, brain and retina starting at stage 10. At the transcriptional level, itpka has been detected in tissues of the anterior neural head region and the eye during early embryonic development of X. laevis. These findings confirm a mild expression in the developing anterior neural structures during embryogenesis and indicate a potential function of Itpka in the development of the central nervous system of X. laevis. Since we only investigated embryonic stages, we could only speculate about the expression levels in the metamorphosis of X. laevis and the adult frog.

Our functional analysis using Itpka MO-mediated KD in X. laevis revealed severe developmental defects in anterior structures, particularly affecting the head, brain and retina. These Itpka morphants displayed microcephaly, cranial cartilage and nerve defects, as well as microphthalmia, accompanied by disrupted retinal lamination and altered lens morphology. These severe phenotypes were successfully rescued by co-injection of X. laevis itpka RNA, confirming the validity of our model for studying the functional role of Itpka in vertebrate development.

Itpka has a bifunctional activity, being involved in actin bundling and calcium signaling (Schell et al., 2001; Windhorst et al., 2012) – both molecular processes are crucial for neuronal development and function. Regarding actin, expression of Itpka in hippocampal neurons leads to an elongation of dendritic spines (Köster et al., 2016) while the overexpression of the full protein in primary neurons results in an increase of dendritic spine number (Windhorst et al., 2012). To our knowledge the severe developmental defects in anterior neural structures that we described were not investigated or shown in mice or rats (Jun et al., 1998). It is also reported that retardation of spine development in young mice could recover with age (Kim et al., 2009), therefore it would be interesting to investigate adult Itpka KO frogs. Moreover, the lack of information about Itpka KO mouse embryos gives rise to further investigations in early mouse embryogenesis.

Calcium signaling regulates various cellular processes including metabolism, cell proliferation, and brain development (Berridge, 2016). Additionally, phospholipase C, which initiates the InsP₃/Ca²⁺ signaling pathway that is regulated by Itpka, plays a crucial role in brain development (Kang et al., 2016). In line with the aforementioned involvement of Itpka in crucial processes regulating neuronal development and morphology, our study shows that Itpka MO KD leads to a significant impairment of brain development. This aligns with previous findings in adult Itpka knockout mice and rats, which show both neuromorphological (Köster et al., 2016) and neurobehavioral abnormalities (Blechner et al., 2020). Interestingly, such findings are commonly observed in neurodevelopmental disorders like schizophrenia (Takahashi et al., 2011) or autism spectrum disorder (ASD).

To explore the reason for smaller head structures and eyes, we investigated cell proliferation and apoptosis, but neither pH H3 nor TUNEL staining was altered in the anterior neural region of the head compared to the control embryos at stage 23. These results indicate that the observed changes are not caused by altered or abnormal cell proliferation or apoptosis measured with these methods at this stage.

This prompted us to further investigate the molecular basis of the developmental phenotypes observed and analyzed the expression of several key neurodevelopmental genes upon Itpka KD. Given that embryonic development involves coordinated processes such as cell differentiation and migration, we focused on genes associated with neural crest cell (NCC), eye, and brain development.

NCCs contribute to a wide range of cell types and structures, including craniofacial skeleton, peripheral nervous system, and ocular tissues (Le Douarin and Dupin, 2018). Previous studies have shown that abnormalities in NCC development can result in craniofacial defects and ocular anomalies (Siismets and Hatch, 2020; Williams and Bohnsack, 2015). In line, aberrant NCC development and differentiation give rise to a group of severe disorders known as neurocristopathies. Patients with these conditions present a phenotype comparable to those observed in Itpka KD X. laevis embryos, including craniofacial abnormalities such as malformed skull bones, cleft palate and visual impairments (Vega-Lopez et al., 2018). Our data demonstrate that Itpka KD led to a significant reduction in the expression of key NCC genes during cell induction, differentiation and migration, including snai2, egr2, twist1, and foxd3. This downregulation and altered migration likely contribute to the craniofacial and cranial nerve defects observed. Furthermore, transcriptional regulators such as snai2 or foxd3 are known to play a crucial role in neural differentiation, with mutations leading to impaired neurogenesis (Méndez-Maldonado et al., 2020). In summary, the decreased expression of NCC genes upon Itpka KD may represent a fundamental impact on the molecular basis underlying the developmental defects we observed.

We further examined genes essential for eye development, such as rax and pax6, and observed reduced expression during early eye field induction and cell differentiation upon Itpka KD. Specifically, the downregulation of rax, a gene required for retinal progenitor cell proliferation and cell fate specification, contributes to retinal malformations (Rodgers et al., 2018). Decreased expression of PAX6, OTX2, and EGR2, which are all associated with human brain and eye defects (Deml et al., 2016; Gonzalez-Rodriguez et al., 2010; Nakayama et al., 2015; Sevilla et al., 2015), further supports the relevance of our findings. Interestingly, expression of the pan-neural gene sox3 remained unchanged at stage 13, indicating that early neural induction is not broadly disrupted by Itpka KD.

Taken together, our results show that Itpka KD leads to a reduced expression of key developmental genes, resulting in severe head, brain, and eye abnormalities. Based on our findings, we propose that Itpka plays an essential role in early development by possibly regulating calcium signaling and/or actin dynamics, both crucial for cellular processes that shape the anterior nervous system. Further investigation into the precise mechanisms of Itpka function during central nervous system development is essential.

Synteny analysis of itpka was performed by comparing the gene location between the species: human, mouse, frog, and fish using the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/). To determine the protein regions within Itpka such as the regions required for cytoskeleton location, IPK and calmodulin binding in the different species, the NCBI GenBank was used. Afterwards, multiple sequence alignments and calculations of the homology from Itpka were done using the ClustalW and Clustal Omega multiple sequence alignment tools from the EMBL-EBI homepage. The following sequences from the NCBI GenBank were used: Homo sapiens: NP_002211.1; Mus musculus: NP_666237.1; Xenopus laevis S: XP_018087711.1; Xenopus laevis L: XP_018086619.1; Xenopus tropicalis: XP_002935396.3; and Danio rerio: NP_001313405.1.

X.laevis embryos were generated, cultured, and staged according to the standard protocols (Nieuwkoop and Faber, 1994; Sive et al., 2010). All experimental procedures were performed in agreement with the German animal use and care law. Furthermore, in vivo experiments were approved by the administration of the state of Baden-Württemberg (Regierungspräsidium Tübingen). Embryos were cultivated in 0.1× modified Barth’s saline with HEPES buffer (MBSH) at 12.5°C–20°C and fixed with 1× MEMFA(T) [0.1 M MOPS, pH 7.4; 2 mM EGTA, 1 mM MgSO4 (H2O)7, 4% formaldehyde, 0.1% Tween] at the desired stage.

To study the spatio-temporal expression profile during X. laevis embryogenesis, whole mount in situ hybridization (WMISH) analysis was performed according to the established protocols (Hemmati-Brivanlou et al., 1990; Lufkin, 2007). Digoxigenin (DIG)-labeled antisense RNA probes were generated by in vitro transcription using T7, SP6, or T3 RNA polymerase (Roche, Basel, Switzerland). Subsequently, the embryos were stained with BM Purple (Roche, Basel, Switzerland) for up to 14 days for exterior view or NBT/BCIP (Roche, Basel, Switzerland) for up to 14 days for sectioning. BM Purple-stained embryos were bleached with 30% H₂O₂. For more detailed tissue analysis, NBT/BCIP-stained or wildtype X. laevis embryos were equilibrated in 1 mL gelatin/albumin solution (2.2 g gelatin, 135 g BSA, 90 g sucrose, and 500 mL 1× PBS) overnight at 4°C and embedded in 1 mL gelatin/albumin solution with 75 μL glutaraldehyde (Fluka, Switzerland). Using a vibratome (Vibratome 1500 Classic, The Vibratome Company), we made sections with a thickness of 25 μm according to (Guo et al., 2011). For investigating the spatio-temporal expression pattern of itpka during X. laevis development, we cloned cDNA fragments of 1151 bp (itpka.L, part of 5′UTR and coding sequence) with the cloning primers itpka_l 5′-GAG AAA GGA GGA AGT GAG-3′ and itpka_r 5′-AAG GTT GAG GTT GAA CTG-3′, into the pSC-B vector (Stratagene, La Jolla, California, United States). For all PCR amplifications, we used cDNA from stage 28 of X. laevis embryos and the proof reading PfuUltraTM II fusion HS DNA polymerase (Agilent Tech., Santa Clara, California, United States). We used the following RNA antisense probes as described previously: celf1 (CUGBP elav-like family member 1) (Day and Beck, 2011; Rothe et al., 2017), cryba1 (crystallin beta A1) (Day and Beck, 2011), egr2 (early growth response 2) (Cizelsky et al., 2013), foxd3 (forkhead box D3) (Gessert et al., 2007; Li et al., 2009), itpka (inositol-) (this publication), otx2 (orthodenticle homeobox 2) (Lamb and Harland, 1995), pax6 (paired box 6) (Hitchcock et al., 1996; Hollemann et al., 1998), pou4f1 (pou class 4 homeobox 1) (Liu et al., 2000), prox1 (prospero homeobox 1) (Dyer et al., 2003), rax (retina and anterior neural fold homeobox) (Furukawa et al., 1997), rho (rhodopsin) (Chang and Harris, 1998), snai2 (snail family zinc finger 2) (clone ID: pMX363), sox3 (sex-determining region Y-box 3) (Maurus et al., 2005), twist1 (twist family bHLH transcription factor 1) (Gessert et al., 2007) and vsx1 (visual system homeobox 1).

To analyze the temporal expression of itpka, X. laevis embryos were collected, tissues were isolated as indicated and fixed by freezing at −80°C at different developmental stages (13, 23, 32, 42). The total RNAs were isolated from X. laevis embryos using the peqGOLD RNAPure Kit (Peqlab, Erlangen, Germany) by following the manufacturer’s protocol. cDNA was generated using random primers and the SuperScript II reverse transcriptase (Invitrogen, Carlsbad, California, United States). RT-PCRs were performed with the same set of cDNA using the Phire Hot Start II DNA polymerase (Thermo Scientific, Waltham, Massachusetts, United States).

To perform knockdown (KD) experiments, the Itpka morpholino oligonucleotide (MO) was designed to the sequence of itpka L and S homologue: 5′-CAT CCA AAA CAC AAA GCT GCG GG-3′. Itpka MO and the standard control morpholino oligonucleotide (CoMO) (5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′) were purchased from Gene Tools (Philomath, OR, United States). The MOs were diluted in autoclaved diethyl-pyro-carbonate (DEPC)-treated water. To target anterior neural tissue, 15 ng of Itpka MO was unilaterally injected (inj.) into one animal-dorsal blastomere of eight-cell-stage X. laevis embryos (Moody and Kline, 1990). The uninjected (uninj.) side served as an internal control. As injection control, CoMO was injected (15 ng). Successful and correct injections were controlled by the co-injection of 0.4–0.5 ng RNA coding for GFP. For rescue experiments, X. laevis Δ-5′UTR-itpka-l (Xitpka) RNA was cloned into the pCS2+ vector (Rupp and Weintraub) using StuI for restriction and the following primers: Δ5′UTRitpka-l 5′-AGG CCT ATG ATT CCT ACG GAA GTT GCA-3′ and Δ5′UTRitpka-r 5′-AGG CCT TCC TAG TGT AGT GGT CAG TAA-3’. Generation of injection mRNA was performed by in vitro transcription using SP6 and T7 polymerase. Rescue experiments of Itpka MO were accomplished by injection of 10 ng Itpka MO with 0.25 ng Xitpka RNA co-injection into one animal-dorsal blastomere of an 8-cell-stage embryo. Xitpka cannot target Itpka MO because of the altered sequence as shown in Supplementary Figure S2A. To adjust the amount of RNA per injection, GFP RNA were used for all experiments.

To show the specificity of the Itpka MO to its corresponding Itpka MO binding site a MO binding efficiency test was performed. We cloned the MO binding site of Itpka MO and the binding site of the rescue Xitpka in front and in frame with the green fluorescent protein (GFP) gene. Following cloning primers were used: itpka-MO-bs_l 5′-GA TCC CCC CGC AGC TTT GTG TTT TGG ATG GGG-3′, itpka-MO-bs_r 5′-AA TTC CCC ATC CAA AAC ACA AAG CTG CGG GGG-3′, Δ-itpka-MO-bs_l 5′-GA TCC ATC GAT TCG AAT TCA AGG CCT ATG GGG-3′ and Δ-itpka-MO-bs_r 5′-AA TTC CCC ATA GGC CTT GAA TTC GAA TCG ATG-3’. To test the Itpka MO binding efficiency, either 10 ng of CoMO or Itpka MO was co-injected with 1 ng of the respective fusion construct RNA into two-cell stage embryos, and the successful or blocked translation of the green fluorescent protein (GFP) was monitored at stage 20–25.

In order to investigate the craniofacial cartilage and cranial nerves, embryos injected with 15 ng Itpka MO and CoMO were fixed at late tadpole stages. Alcian blue staining: embryos were stained with Alcian blue as previously described (Gessert et al., 2007), and afterwards, the cranial cartilage was dissected and imaged. 3A10 antibody staining: embryos were treated with the monoclonal 3A10 antibody (DSHB, Iowa City) to visualize the cranial nerves (Schuff et al., 2007).

Proliferative cells of stage 23 X. laevis embryos were stained for phospho-histone H3 (pH H3) and apoptotic cells of stage 23 X. laevis embryos were stained with TUNEL according to the established protocols (Cizelsky et al., 2013; Gessert et al., 2007).

Representative embryos/experiments were imaged. Whole X. laevis embryos from the exterior view were imaged by using an Olympus MVX10 (fluorescence) and an Olympus UC75 camera. Vibratome sections were imaged with an Olympus RX60 microscope and an Olympus DP28 camera. Images were processed with ImageJ2 version 2.9.0 (Rueden et al., 2017) and Affinity Designer 1.10.8.

For all quantitative measurements, one/two representative experiments of unilaterally injected control MO (CoMO) and Itpka MO embryos were used. The area and width of the head as well as the eye area were measured using the software ImageJ2 version 2.9.0 (Rueden et al., 2017). For brain area measurements, the brains of stage 42 embryos were dissected and imaged. ImageJ2 (Rueden et al., 2017) was used to measure the area of the brain as well as the expression area and intensity of the different marker genes. The values (injected/uninjected side) were then calculated and analyzed using GraphPad Prism 10.4.0 for macOS (Boston, Massachusetts, United States) as well as the expression area and intensity of the different marker genes.

Data were analyzed with GraphPad Prism 10.4.2 for macOS (Boston, Massachusetts, United States, www.graphpad.com). Only experiments with a higher survival rate than 50% were considered for statistical evaluation. Statistical evaluation was performed only with more than three independent experiments. To determine statistical differences, the non-parametric Mann-Whitney rank-sum test was used, and the error bars represent the standard errors of the mean (SEM). Statistical significances are indicated as ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001.