Development, Growth & Differentiation

Choanoflagellate genetics has undergone rapid and impactful developments in the last decade. Currently, the primary method for genetic modification of choanoflagellates relies on proprietary nucleofection reagents to deliver transgenes for ectopic expression or CRISPR-Cas9 ribonucleoprotein complexes for targeted genome editing. The acquisition of proprietary buffers required for nucleofection can hamper advances in choanoflagellate research due to costs, shipping limitations, and restrictions that prevent buffer components from being optimized for understudied organisms. Therefore, we test whether a low-cost in-house electroporation buffer developed for other systems can replace the proprietary buffer currently used for choanoflagellate transfection. Here, we present an in-house buffer with transfection efficiency comparable to that of the previously established proprietary buffer. This work increases the accessibility of choanoflagellate genetics and can broaden research participation in investigating animal origins.

Understanding the functions of maternal effect genes during oocyte growth is essential for elucidating the mechanisms of oogenesis and early embryonic development. However, conventional gene knockout and conditional knockout approaches require extensive breeding and are time-consuming. Here, we present a rapid in vitro gene functional analysis system that combines microinjection of mRNA, siRNA and plasmid DNA into mouse secondary follicles with a two-step oocyte growth culture system. Mouse secondary follicles were subjected to microinjection of mCherry mRNA and subsequently cultured for 15 days to produce fully grown oocytes. mCherry fluorescence persisted throughout the oocyte growth period but declined rapidly after fertilization. Despite minor cellular damage occasionally caused by microinjection, injected follicles developed normally and retained developmental competence. To evaluate the efficiency of gene suppression, we introduced siRNA targeting Dnmt3l, which is abundantly expressed during oocyte growth phase. Although Dnmt3l deficiency is known not to affect oocyte growth, we observed that oocyte growth was maintained normally despite a marked reduction in endogenous Dnmt3l mRNA levels in our knockdown model. These results demonstrate that this method enables efficient manipulation of gene expression specifically during oocyte growth while preserving developmental competence, providing a versatile platform for rapid functional screening of maternal effect genes in vitro.

Enhancer RNAs (eRNAs) are non-coding transcripts produced at enhancer regions, which appear to be involved in transcriptional regulation. Up to date, these have been primarily investigated using labor-and cost-intensive genomic techniques. However, the precise mechanisms by which eRNA transcription or the eRNA transcripts themselves mediate transcriptional regulation remain unclear. Here, we present a novel experimental approach that allows us to analyze the characteristics of eRNA transcription in fixed and live whole Drosophila melanogaster embryos. We employ the anterior-posterior patterning genes as a model system to investigate the dynamics of eRNA expression, utilizing an imaging-based approach. We combined high-sensitivity fluorescence in situ hybridization (FISH) chain reaction (HCR) with high-resolution confocal microscopy to detect eRNA and mRNA molecules. Through this experimental assay, we identified foci of elevated transcriptional activity that generate eRNA transcripts correlated with mRNA production at the same gene locus. We could show that this eRNA transcription is independent of promoter activity. Additionally, we demonstrate that insulators can influence eRNA transcription, resulting in loss of eRNA transcription. Moreover, we observe that eRNAs can originate both within classical enhancer regions and outside of them, including from foreign bacterial sequences when these are placed near enhancer sequences, underscoring the strong influence of local regulatory context on eRNA initiation. In live embryos using MS2-MCP live imaging, our analysis of insulators showed a modest reduction in mRNA burst intensity accompanied by a slight increase in burst frequency. Overall, our imaging-based approach offers a novel platform for dissecting enhancer-eRNA interactions and could be adapted for wider applications.

Recent technological advances have made many "non-model" organisms accessible for experimental studies. However, reference inbred strains were not necessarily available, especially in marine invertebrates, and genetic background of organisms used for experiments are often non-uniform. This situation potentially affects experimental reproducibility. Although ascidians, Ciona intestinalis (type A, or C. robusta), are a widely used marine animal for many areas of experimental biology including developmental studies, no reference strains have been obtained despite extensive efforts. As an alternative way to improve reproducibility, we have established and maintained ascidian colonies through intra-population breeding every year from 2016 to 2020, and monitored genomic variants of these colonies. This method does not reduce genetic variations but instead manages and monitors genetic variations in the colonies, providing an easy and cost-effective way of increasing experimental reproducibility. Furthermore, we recently upgraded these genetically isolated, closed colonies that were re-established every year, and have maintained them for more than three years only through intra-population breeding and occasional back-cross using cryopreserved sperm. Genetic variants that we revealed using 3.7 tera-bases of sequence data will help to design future experiments in this species. Our data also show that two wild-populations, which were used to establish the colonies, have maintained distinct genetic backgrounds, although their habitats are directly linked to the Pacific Ocean and only 170 km apart. More importantly, genetic information regarding these colonies will undoubtedly improve experimental reproducibility and traceability, and our method will provide a realistic solution for performing reproducible experiments using non-model organisms.

Bone morphogenetic proteins (BMPs) play an important role in dorsal spinal cord patterning. Their presence in the roof plate of the midbrain indicates a role in its development. We examined whether the BMP signaling contributes to dorsal midbrain size expansion in chick embryos by missexpressing pathway activators and inhibitors. Overactivation of BMP4 did not affect midbrain development, whereas GDF7 reduced midbrain growth. In contrast, expression of a truncated dominant-negative BMP receptor type 1b or the extracellular inhibitor Chordin had no detectable effect. Ectopic expression of SMAD6, the intracellular inhibitor of the BMP/ TGF-{beta} pathway, significantly reduced midbrain size, which correlated with decreased proliferation rates of SMAD6-overexpressing cells. In some cases, SMAD6 also disrupted MTN axon trajectory. These results indicate an important role for SMAD-dependent signaling pathways in early dorsal midbrain growth.

Stem cell technologies have become a vital component of conservation efforts around the globe. Biobanks and pluripotent stem cell lines help to ensure species and their genetic diversity are preserved. These efforts have however, focussed mostly on mammals and birds, and the cryopreservation protocols for embryos and cells were developed decades ago laying the basis for artificial reproductive techniques for species conservation. With over 20% of non-avian reptile species facing extinction, it is imperative to establish protocols for reptiles to ensure species preservation and also to facilitate the establishment of new reptile model organisms to match the standard of mammals. Here, we have generated a cryopreservation method for preserving early gastrulating veiled chameleon embryos as a representative squamate species. To this end, we first developed a tissue culture method for maintaining cells extracted from peri-gastrulation chameleon embryos and then tested different cryopreservation methods altering the concentration of the penetrating cryoprotectant DMSO and assessing the effect of the addition of non-penetrating cryoprotectants Trehalose and Sucrose. We then optimised a protocol for whole embryo vitrification in 20% DMSO with added Trehalose or Sucrose that can easily be adapted for fieldwork. Taken together, our method not only provides a protocol for conservation efforts but also lays the basis for mechanistic studies of early squamate embryo development by enabling cryopreservation of whole embryos in a fieldwork setting, which facilitates their live transport back to a laboratory for functional experiments or molecular analyses.

Neural tube closure is a critical developmental process, essential to the proper formation of the vertebrate nervous system. This process starts with the invagination of neural plate cells. Its borders then converge, leading to the closure of the neural tube, propagating like a zipper. Afterwards, cell intercalation and proliferation allow the tube to elongate. Neural tube closure involves thousands of cells in vertebrates. However, the closest invertebrates to vertebrates, the tunicates, such as Ciona, close a hollow dorsal neural tube with fewer than 20 neural cells. This minimal model makes it easier to study the mechanisms of this intricated process. In Ciona, the transcription factor Lmx1 is expressed in the most dorsal cells of the developing neural tube, like its vertebrate orthologs. In vertebrates, Lmx1 paralogs are involved in neural tube patterning. However, no function related to morphogenesis has been uncovered. Here, we explore Ciona Lmx1 roles during neural tube closure. Lmx1 Knockdown leads to slight but significant defects in neural tube closure. The overexpression of a repressive Lmx1 variant prevents the proper intercalation of the dorsal neural tube cells, impeding the anterior progression of the zipper. Furthermore, studies of Lmx1 regulatory sequences indicate that Pax3/7, ZicL, and Nodal signaling may directly regulate its transcription. These transcription factors are present at the vertebrate neural plate border, suggesting that Lmx1 regulation is conserved across chordates. It raises the possibility of an unrecognized role for Lmx1 during vertebrate neural tube morphogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/709676v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f409b1org.highwire.dtl.DTLVardef@1a88180org.highwire.dtl.DTLVardef@1ce2a89org.highwire.dtl.DTLVardef@4aba89_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cultured meat technology, with its significant advantages of shortening meat production cycles, reducing natural resource consumption, minimizing the risk of zoonotic disease transmission, and enabling precise control over nutritional composition and texture, offers a novel alternative source for human meat consumption. One of the major challenges to produce cultured meat in large scale is how to establish high.quality seed cells, which should have long term proliferative capacities and are able to differentiate into muscles efficienuy with simple procedures. Here, we first established an engineered porcine expanded potential stem cells (Tet-On-PAX7 EPSCs) containing Tet-On regulated PAX7 gene. Then the Tet-On-PAX7 EPSCs were induced to somite-liKe mesodermal cells. These somite-liKe mesodermal cells can be expanded over 1025-fold even after 40 passages in-vitro culture while retaining strong myogenic potential. The somite-like mesodermal cells treated with DOX for one day would differentiate into muscle stem cells (Muses), and the later were able to differentiate into muscles with an efficiency of up to 90% within just 7 days in 11-FSDeDa without Dox. Moreover, when somite-liKe mesodermal cells were seeded on patterned scaffolds, microcarrier scaffolds, or cultured in anchorage-independent suspension, they maintained high efficiency in muscle differentiation, confirming their potential to be used as seed cells for scaled cultured meat production.

Antisense vivo-morpholino oligonucleotides (vivo-MOs) allow transient gene knockdown in adult organisms with high specificity and low toxicity. Vivo-MOs are used in cell culture and in many established model organisms, but a method for their use has not been described in threepsine stickleback (Gasterosteus aculeatus (Linnaeus, 1758)). Stickleback are an emerging model system used in evolutionary and ecological genetic studies. While genomic techniques are commonly used in stickleback research, there are few studies and tools available to assess gene function in-vivo, especially for genes that may be difficult to knock out by CRISPR (e.g., lethal knock-outs). Here, we test the use of splice-blocking vivo-MOs for gene knockdown in stickleback using intraperitoneal injection of vivo-MOs targeting three candidate genes. Gene expression was assessed in the liver, spleen, and intestine. Successful knockdown of Spi1b was observed in the spleen, however, we observed no other significant knockdown at either timepoint tested. Injection of a fluorescently labeled control vivo-MO confirmed delivery to each target organ, validating this approach, but delivery was variable which may explain inconsistent effects. These results indicate that vivo-MOs have potential as a tool for in-vivo gene knockdown in stickleback. Optimizing delivery methods could improve reproducibility and knockdown efficiency in future studies.

Somatic cell nuclear transfer (SCNT) holds great promise for regenerative medicine and agriculture, but its application is severely hampered by low efficiency, primarily attributable to aberrant epigenetic reprogramming. Although embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have been successfully derived from cloned embryos, an in vitro counterpart of the primitive endoderm (PrE) lineage has remained unavailable. To address this gap, this study reports the first successful establishment of extra-embryonic endoderm stem cell lines (XENs) from mouse SCNT-derived blastocysts (NT-XENs). Under conventional culture conditions, NT-XENs were generated from hybrid B6D2F1 blastocysts at a high efficiency of 55%, comparable to that of fertilization-derived XEN lines (FD-XENs, 50%), whereas derivation from inbred C57BL/6J SCNT-derived blastocysts was markedly lower (12.5%). Immunofluorescence and NanoString multiplex gene expression profiling confirmed that NT-XENs robustly expressed specific marker genes for PrE/XENs (e.g., Gata4, Gata6, Sox17), while exhibiting negligible or absent expression of pluripotency and trophoblast markers. Based on NanoString assay data, NT-XENs and FD-XENs shared highly similar global gene expression patterns, yet also exhibited some nonnegligible differences, exemplified by the differentially expressed genes (DEGs) Pecam1, Gtl2, Thbd and Xlr3b, which may suggest that the NT-XENs resided in a more differentiated state (potentially biased toward parietal endoderm (PE)) and retained SCNT-specific epigenetic imprinting errors, including aberrant X-chromosome inactivation and dysregulation of imprinted domains. In summary, this study successfully establishes NT-XEN cell lines, providing a valuable in vitro model for investigating the reprogramming scenarios of PrE lineage in SCNT and offering novel insights into the mechanisms underlying developmental failure of cloned embryos.

Mechanical regulation plays an essential role in the organization of early embryogenesis. In ascidian embryos, cells in the animal hemisphere exhibit periodic stiffening-softening cycles, and their cell rheological properties follow a common master curve, indicating a form of universality. In contrast, cells in the vegetal hemisphere show cell-to-cell differences in stiffness even within the same endodermal lineage, but their rheological behavior has not been characterized. Here, using atomic force microscopy (AFM), we investigated the spatiotemporal dynamics of single-cell power-law rheological states in the vegetal hemisphere during early cleavage. We found that both the elastic modulus (stiffness) and the fluidity (power-law exponent) differ among endodermal cells, and that these differences change in a stage-dependent manner. This result indicates that vegetal cells do not exhibit a single common rheological behavior, in contrast to animal hemisphere cells, suggesting that mechanical properties in the vegetal hemisphere are not uniformly regulated but are patterned in space and time during cleavage. Our findings indicate that this mechanical diversification is linked to the progression of early morphogenesis and may contribute to the emergence of distinct cell behaviors during development.

Kinesin-3 motor proteins are increasingly recognized for their important roles in cilia. The mammalian kinesin-3 motor KIF13B moves bidirectionally in primary cilia and regulates ciliary content, but its relationship to the intraflagellar transport (IFT) machinery is unclear. Here, we combine quantitative live-cell imaging with a new kymograph analysis based on dynamic mode decomposition (DMD) to separate mobile from immobile protein populations in primary cilia. This approach simplifies extraction of molecular velocities from kymographs and reveals that a KIF13B deletion mutant retaining only the motor domain and part of the forkhead-associated domain does not alter steady-state IFT velocity or frequency. However, when retrograde dynein-2 function is inhibited by Ciliobrevin D, both anterograde and retrograde IFT velocities decrease in parental cells, as expected, but remain unchanged in KIF13B mutant cells. Structured illumination, confocal, and STED microscopy further show that KIF13B localizes to the ciliary membrane and concentrates at the periciliary membrane region and the centriolar subdistal appendages, below the distal appendage marker FBF1. Our improved kymograph approach provides new insight into KIF13B ciliary function and simplifies the quantitative analysis of ciliary protein transport.

Primarily inhabiting the harsh, high-altitude environment of the Qiangtang National Nature Reserve exceeding 5,000 meters above the sea (m.a.s.l.), the golden wild yak is critically endangered, with fewer than 300 individuals remaining in the world, a situation exacerbated by the significant challenges of conducting research and conservation of their genetic resources. Somatic cell nuclear transfer (SCNT) can be an effective method for their preservation, but facing several obstacles in this context, including the hypoxic stress at high altitude that impairs embryonic development due to in vitro manipulation, and constraints of long-distance embryo transport. In the present study, the ear tissue was collected from a childhood male golden wild yak at Xizang Geye Wildlife Rescue Station (4800 m.a.s.l.) and send to Institute of Animal Science at Beijing to derive fibroblast cells. Using fibroblast cells of the golden wild yak as nuclear donors, and bovine oocytes from a local slaughterhouse at Beijing as recipients, the interspecific SCNT (iSCNT) embryos were generated and in vitro developed to blastocysts. To maintain the embryonic viability after long-distance transportation from Beijing to Xizang, iSCNT blastocysts were subjected to cryopreservation by vitrification method. Thawing of vitrified iSCNT blastocysts were completed at Xizang Dangxiong Yak Breeding Innovation Base (4200 m.a.s.l.), and transferred into the uterine horn of domestic yaks. 257 days after blastocyst transfer, a cloned golden wild yak was successfully harvested on January 10, 2026. This work demonstrates, for the first time, that interspecies somatic cell nuclear transfer can successfully produce a cloned offspring under extreme conditions, spanning 4800 m.a.s.l. donor origin, long-distance vitrified embryo transportation, and high-altitude blastocyst transfer at 4200 m.a.s.l., establishing a viable strategy for conserving critically endangered high-altitude species.

MicroRNAs (miRNAs) play essential roles in the posttranscriptional regulation of gene expression in organisms. In the process of synthesizing mature miRNAs from miRNA precursors, the miRNA precursors are cleaved via Dicer at their loop structure, after which the miRNA precursors become mature and regulate transcription. However, the consequences of altering the loop sequence are not fully understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. We identified a mutant of the miRNA miR-3260 whose the part of the loop structure was lacking in a silkworm strain with translucent larval skin. Here, we aimed to analyze the role of wild-type miR-3260 and the influence of the mutation of the loop structure in B. mori. First, we identified the genomic region responsible for the translucent larval skin phenotype and determined that the mutated miR-3260 nucleotide sequences. Then, we predicted the binding partners of wild-type miR-3260 using the RNA hybrid tool and found two juvenile hormone (JH)-related genes as targets of wild-type miR-3260. Next, we assessed the relationships between miR-3260 and JH and found that miR-3260 was highly expressed in the Corpora allata and its expression responded to JH treatment. Meanwhile, miR-3260 mimic and inhibitor did not induce the typical phenotypes associated with JH in B. mori. Then, we compared the dicing products from wild-type and mutant miR-3260 precursors and observed that neither form underwent Dicer-mediated cleavage when the loop structure was altered. These results suggest that loop mutations in the miR-3260 precursor may not influence dicing activity, consistent with the lack of observable phenotypic effects.

Low take rates and inter-tumor variability in growth rates can limit the effectiveness of mouse xenograft models when comparing between groups. To address this problem we developed a simple method to compare multiple cell types within a single mixed xenograft. Individual cell lines or clones were transduced with a lentiviral vector that includes a unique PCR tag, allowing the use of qPCR to determine the proportion of each tagged cell type within a mixed xenograft tumor. We generated vectors with six distinct PCR tags, and two different selectable markers, and have optimized the approach for determining their relative proportions within a mix. An initial pre-amplification step is used to increase the amount of material for subsequent qPCR reactions. This also removes the bulk of the genomic DNA, increasing the specificity of the qPCR step. Samples are then used for qPCR with specific pairs of primers that distinguish between each of the individual PCR tags, and the relative proportion of each tag is determined relative to that in the starting mix. We have tested this approach for in vitro growth of mixed cell cultures and in an orthotopic cecal xenograft model using a human colon cancer cell line. Since each individual tumor is initiated with a mix of cells, multiple tumors within a single animal can be analyzed separately, and overall tumor size is not important. Similarly, multiple metastatic lesions from the same animal can be analyzed individually. Thus, each tumor provides a direct comparison between individually tagged cell lines or clones. This low throughput "bar-coding" approach is simple and cost effective and has the potential to reduce the number of animals needed for xenograft experiments.

Genetic mosaicism can arise from in vivo CRISPR-Cas9 gene editing, especially in the embryos. This study evaluates the extent of genetic mosaicism resulted from CRISPR-Cas9-mediated knockout for 11 genes in the freshwater microcrustacean Daphnia magna, D. pulex, and D. sinensis. Based on extensive genotyping data of the asexually produced progenies of successfully edited females, we find strong evidence of mosaicism in 9 of these genes. The genotyping data also suggest the gene editing activity can take place as early as the one-cell embryo stage and extends into the 32-cell and later stages. This study establishes genetic mosaicism as an important feature of Cas9-mediated gene editing in Daphnia.

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

Mammalian spermatogenesis occurs in the seminiferous tubules, which exhibit unique spatiotemporal differentiation patterns known as cellular association patterns. In mice, these patterns can be regarded as one-dimensional wavetrains that consistently propagate inward from both ends, resulting in one or more "sites of reversal." Segmented wavetrain pattern, in which the wave propagation direction spatially switches, was observed in our previous three-species reaction-diffusion model for interspecific species difference in spermatogenic waves (Kawamura et al., 2021). However, the biological mechanisms of the formation of sites of reversal and of this directional bias, as well as the principle of pattern formation, remain unknown. Here, we refined our previous model to match the actual biological spatiotemporal scale and examined its dynamics through extensive numerical simulations. The modified model frequently generated segmented wavetrain patterns, corresponding to the sites of reversal, but without directional bias. We systematically examined possible biological mechanisms for the bias and found that tubule elongation, especially near the rete testis, most effectively accounts for the bias among the tested. Extensive simulations revealed that the segmented pattern is numerically stable, emerges more frequently in longer domains, and shows an exponential segment size distribution with a lower limit for the stably existing segment length. These explorations imply that locally emerged unidirectional wavetrains serve as building blocks to generate the stable segmented wavetrains through their interactions. HighlightsO_LISegmented wavetrains reflect sites of reversal in seminiferous tubules. C_LIO_LISegmented patterns frequently emerge but show no inherent directional bias. C_LIO_LITubule elongation may contribute to inward propagation near the rete testis. C_LIO_LISegmented wavetrains are numerically stable and more frequent in longer domains. C_LIO_LIInteractions of local unidirectional wavetrains generate stable segmented structures. C_LI

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull, a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus-oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.

BackgroundDuring development, axons are organized into bundles, a process known as axonal fasciculation. The zebrafish lateral line nerve has been used as a model to study axonal fasciculation; however, the underlying mechanisms are not yet fully understood. Although Fgf3 and Fgf10a are well known to regulate the migration of the lateral line primordium along which the lateral line nerve projects, their roles in the organization of the lateral line nerve itself have not been clarified. Resultsfgf3,10a double mutants exhibited lateral line axonal defasciculation accompanied by an increased number of Schwann cells. Live imaging revealed a marked increase in Schwann cell proliferation and demonstrated that newly divided Schwann cells migrate along axons and infiltrate interaxonal spaces, thereby expanding these spaces and disrupting axonal fasciculation. Pharmacological manipulations further implicated a contribution of Nrg1-ErbB signaling to this phenotype. ConclusionsOur findings suggest that Fgf3 and Fgf10a are required to restrict Schwann cell proliferation and infiltration, thereby ensuring axonal fasciculation during lateral line development.