ABSTRACT An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10 , Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo , respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.

ABSTRACT Advanced 3D imaging modalities, such as micro-computed tomography (micro-CT), have been incorporated into the high-throughput embryo pipeline of the International Mouse Phenotyping Consortium (IMPC). This project generates large volumes of raw data that cannot be immediately exploited without significant resources of personnel and expertise. Thus, rapid automated annotation is crucial to ensure that 3D imaging data can be integrated with other multi-dimensional phenotyping data. We present an automated computational mouse embryo phenotyping pipeline that harnesses the large amount of wild-type control data available in the IMPC embryo pipeline in order to address issues of low mutant sample number as well as incomplete penetrance and variable expressivity. We also investigate the effect of developmental substage on automated phenotyping results. Designed primarily for developmental biologists, our software performs image pre-processing, registration, statistical analysis and segmentation of embryo images. We also present a novel anatomical E14.5 embryo atlas average and, using it with LAMA, show that we can uncover known and novel dysmorphology from two IMPC knockout lines.

ABSTRACT To gain a deeper understanding of pancreatic β-cell development, we used iterative weighted gene correlation network analysis to calculate a gene co-expression network (GCN) from 11 temporally and genetically defined murine cell populations. The GCN, which contained 91 distinct modules, was then used to gain three new biological insights. First, we found that the clustered protocadherin genes are differentially expressed during pancreas development. Pcdhγ genes are preferentially expressed in pancreatic endoderm, Pcdhβ genes in nascent islets, and Pcdhα genes in mature β-cells. Second, after extracting sub-networks of transcriptional regulators for each developmental stage, we identified 81 zinc finger protein (ZFP) genes that are preferentially expressed during endocrine specification and β-cell maturation. Third, we used the GCN to select three ZFPs for further analysis by CRISPR mutagenesis of mice. Zfp800 null mice exhibited early postnatal lethality, and at E18.5 their pancreata exhibited a reduced number of pancreatic endocrine cells, alterations in exocrine cell morphology, and marked changes in expression of genes involved in protein translation, hormone secretion and developmental pathways in the pancreas. Together, our results suggest that developmentally oriented GCNs have utility for gaining new insights into gene regulation during organogenesis.

ABSTRACT Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila , we also find that single short tags can be used for POI manipulation in vivo .

ABSTRACT Characterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes.

ABSTRACT CRISPR-stop converts protein-coding sequences into stop codons, which, in the appropriate location, results in a null allele. CRISPR-stop induction in one-cell-stage zygotes generates Founder 0 (F0) mice that are homozygous mutants; this avoids mouse breeding and serves as a rapid screening approach for nonlethal genes. However, loss of function of 25% of mammalian genes causes early lethality. Here, we induced CRISPR-stop in one of the two blastomeres of the zygote, a method we name mosaic CRISPR-stop, to produce mosaic Atoh1 and Sox10 F0 mice; these mice not only survived longer than regular Atoh1 / Sox10 knockout mice but also displayed their recognized cochlear phenotypes. Moreover, by using mosaic CRISPR-stop, we uncovered a previously unknown role of another lethal gene, Rbm24 , in the survival of cochlear outer hair cells (OHCs), and we further validated the importance of Rbm24 in OHCs by using our Rbm24 conditional knockout model. Together, our results demonstrated that mosaic CRISPR-stop is reliable and rapid, and we believe this method will facilitate rapid genetic screening of developmentally lethal genes in the mouse inner ear and also in other organs.

ABSTRACT Efficient genetic manipulation in the developing central nervous system is crucial for investigating mechanisms of neurodevelopmental disorders and the development of promising therapeutics. Common approaches including transgenic mice and in utero electroporation, although powerful in many aspects, have their own limitations. In this study, we delivered vectors based on the AAV9.PHP.eB pseudo-type to the fetal mouse brain, and achieved widespread and extensive transduction of neural cells. When AAV9.PHP.eB-coding gRNA targeting PogZ or Depdc5 was delivered to Cas9 transgenic mice, widespread gene knockout was also achieved at the whole brain level. Our studies provide a useful platform for studying brain development and devising genetic intervention for severe developmental diseases.

ABSTRACT Craniofacial development is regulated through dynamic and complex mechanisms that involve various signaling cascades and gene regulations. Disruption of such regulations can result in craniofacial birth defects. Here, we propose the first developmental stage-specific network approach by integrating two crucial regulators, transcription factors (TFs) and microRNAs (miRNAs), to study their co-regulation during craniofacial development. Specifically, we used TFs, miRNAs and non-TF genes to form feed-forward loops (FFLs) using genomic data covering mouse embryonic days E10.5 to E14.5. We identified key novel regulators (TFs Foxm1, Hif1a, Zbtb16, Myog, Myod1 and Tcf7, and miRNAs miR-340-5p and miR-129-5p) and target genes ( Col1a1 , Sgms2 and Slc8a3 ) expression of which changed in a developmental stage-dependent manner. We found that the Wnt-FoxO-Hippo pathway (from E10.5 to E11.5), tissue remodeling (from E12.5 to E13.5) and miR-129-5p-mediated Col1a1 regulation (from E10.5 to E14.5) might play crucial roles in craniofacial development. Enrichment analyses further suggested their functions. Our experiments validated the regulatory roles of miR-340-5p and Foxm1 in the Wnt-FoxO-Hippo subnetwork, as well as the role of miR-129-5p in the miR-129-5p– Col1a1 subnetwork. Thus, our study helps understand the comprehensive regulatory mechanisms for craniofacial development.

ABSTRACT Epithelia are dynamic tissues that self-remodel during their development. During morphogenesis, the tissue-scale organization of epithelia is obtained through a sum of individual contributions of the cells constituting the tissue. Therefore, understanding any morphogenetic event first requires a thorough segmentation of its constituent cells. This task, however, usually involves extensive manual correction, even with semi-automated tools. Here, we present EPySeg, an open-source, coding-free software that uses deep learning to segment membrane-stained epithelial tissues automatically and very efficiently. EPySeg, which comes with a straightforward graphical user interface, can be used as a Python package on a local computer, or on the cloud via Google Colab for users not equipped with deep-learning compatible hardware. By substantially reducing human input in image segmentation, EPySeg accelerates and improves the characterization of epithelial tissues for all developmental biologists.

ABSTRACT Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5β-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.

ABSTRACT The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a ‘big data’ resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

ABSTRACT Identifying cell states during development from their mRNA profiles provides insight into their gene regulatory network. Here, we leverage the sea urchin embryo for its well-established gene regulatory network to interrogate the embryo using single cell RNA sequencing. We tested eight developmental stages in Strongylocentrotus purpuratus , from the eight-cell stage to late in gastrulation. We used these datasets to parse out 22 major cell states of the embryo, focusing on key transition stages for cell type specification of each germ layer. Subclustering of these major embryonic domains revealed over 50 cell states with distinct transcript profiles. Furthermore, we identified the transcript profile of two cell states expressing germ cell factors, one we conclude represents the primordial germ cells and the other state is transiently present during gastrulation. We hypothesize that these cells of the Veg2 tier of the early embryo represent a lineage that converts to the germ line when the primordial germ cells are deleted. This broad resource will hopefully enable the community to identify other cell states and genes of interest to expose the underpinning of developmental mechanisms.

ABSTRACT To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, we generated an sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila . The homozygous fly appears similar to wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, we quantified an average of 1100, 6200 and 2500 receptors per follicle cell (FC) at stages 8/9, 10 and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGFα-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, we followed the endosomal localization of EGFR in the FCs. The EGFR colocalized with the late endosome, but no significant colocalization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource for studying cellular localization and regulation of EGFR in tissues.

ABSTRACT Kidney formation requires the coordinated growth of multiple cell types including the collecting ducts, nephrons, vasculature and interstitium. There is a long-held belief that interactions between progenitors of the collecting ducts and nephrons are primarily responsible for kidney development. However, over the last several years, it has become increasingly clear that multiple aspects of kidney development require signaling from the interstitium. How the interstitium orchestrates these various roles is poorly understood. Here, we show that during development the interstitium is a highly heterogeneous patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. Our analysis indicates that the heterogeneity is not a mere reflection of different stages in a linear developmental trajectory but instead represents several novel differentiated cell states. Further, we find that β-catenin has a cell autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent epithelia. These findings suggest the intriguing possibility that the different interstitial subtypes may create microenvironments that play unique roles in development of the adjacent epithelia and endothelia.

ABSTRACT Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

ABSTRACT Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an otic placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9 P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other pan-otic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9 P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses.

ABSTRACT The ways in which placental defects affect embryonic development are largely overlooked because of the lack of a trophoblast-specific approach for conditional gene ablation. To tackle this, we have established a simple, fast and efficient method for trophectodermal Tat-Cre/loxP recombination. We used the natural permeability barrier in mouse blastocysts in combination with off-the-shelf Tat-Cre recombinase to achieve editing of conditional alleles in the trophoblast lineage. This direct approach enables gene function analysis during implantation and placentation in mice, thereby crucially helping to broaden our understanding of human reproduction and development.

ABSTRACT Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis- regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.

ABSTRACT Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila .

ABSTRACT For decades, clearing and staining with Alcian Blue and Alizarin Red has been the gold standard to image vertebrate skeletal development. Here, we present an alternate approach to visualise bone and cartilage based on X-ray microCT imaging, which allows the collection of genuine 3D data of the entire developing skeleton at micron resolution. Our novel protocol is based on ethanol fixation and staining with Ruthenium Red, and efficiently contrasts cartilage matrix, as demonstrated in whole E16.5 mouse foetuses and limbs of E14 chicken embryos. Bone mineral is well preserved during staining, thus the entire embryonic skeleton can be imaged at high contrast. Differences in X-ray attenuation of ruthenium and calcium enable the spectral separation of cartilage matrix and bone by dual energy microCT (microDECT). Clearing of specimens is not required. The protocol is simple and reproducible. We demonstrate that cartilage contrast in E16.5 mouse foetuses is adequate for fast visual phenotyping. Morphometric skeletal parameters are easily extracted. We consider the presented workflow to be a powerful and versatile extension to the toolkit currently available for qualitative and quantitative phenotyping of vertebrate skeletal development.