Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest.

The histone methyltransferase Polycomb repressive complex 2 (PRC2) is required for specification of the neural crest, and mis-regulation of the neural crest can cause severe congenital malformations. PRC2 is required for induction of the neural crest, but the embryonic, cellular, and molecular consequences of PRC2 activity after neural crest induction are incompletely understood. Here we show that Eed, a core subunit of PRC2, is required for craniofacial osteoblast differentiation and mesenchymal proliferation after induction of the neural crest. Integrating mouse genetics with single-cell RNA sequencing, our results reveal that conditional knockout of Eed after neural crest cell induction causes severe craniofacial hypoplasia, impaired craniofacial osteogenesis, and attenuated craniofacial mesenchymal cell proliferation that is first evident in post-migratory neural crest cell populations. We show that Eed drives mesenchymal differentiation and proliferation in vivo and in primary craniofacial cell cultures by regulating diverse transcription factor programs that are required for specification of post-migratory neural crest cells. These data enhance understanding of epigenetic mechanisms that underlie craniofacial development, and shed light on the embryonic, cellular, and molecular drivers of rare congenital syndromes in humans.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.

A Suite of Mouse Reagents for Studying Amelogenesis.

Amelogenesis, the formation of dental enamel, is driven by specialized epithelial cells called ameloblasts, which undergo successive stages of differentiation. Ameloblasts secrete enamel matrix proteins (EMPs), proteases, calcium, and phosphate ions in a stage-specific manner to form mature tooth enamel. Developmental defects in tooth enamel are common in humans, and they can greatly impact the well-being of affected individuals. Our understanding of amelogenesis and developmental pathologies is rooted in past studies using epithelial Cre driver and knockout alleles. However, the available mouse models are limited, as most do not allow targeting different ameloblast sub-populations, and constitutive loss of EMPs often results in severe phenotype in the mineral, making it difficult to interpret defect mechanisms. Herein, we report on the design and verification of a toolkit of twelve mouse alleles that include ameloblast-stage specific Cre recombinases, fluorescent reporter alleles, and conditional flox alleles for the major EMPs. We show how these models may be used for applications such as sorting of live stage specific ameloblasts, whole mount imaging, and experiments with incisor explants. The full list of new alleles is available at https://dev.facebase.org/enamelatlas/mouse-models/ .

Exome sequencing efficacy and phenotypic expansions involving esophageal atresia/tracheoesophageal fistula plus.

Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a life-threatening birth defect that often occurs with other major birth defects (EA/TEF+). Despite advances in genetic testing, a molecular diagnosis can only be made in a minority of EA/TEF+ cases. Here, we analyzed clinical exome sequencing data and data from the DECIPHER database to determine the efficacy of exome sequencing in cases of EA/TEF+ and to identify phenotypic expansions involving EA/TEF. Among 67 individuals with EA/TEF+ referred for clinical exome sequencing, a definitive or probable diagnosis was made in 11 cases for an efficacy rate of 16% (11/67). This efficacy rate is significantly lower than that reported for other major birth defects, suggesting that polygenic, multifactorial, epigenetic, and/or environmental factors may play a particularly important role in EA/TEF pathogenesis. Our cohort included individuals with pathogenic or likely pathogenic variants that affect TCF4 and its downstream target NRXN1, and FANCA, FANCB, and FANCC, which are associated with Fanconi anemia. These cases, previously published case reports, and comparisons to other EA/TEF genes made using a machine learning algorithm, provide evidence in support of a potential pathogenic role for these genes in the development of EA/TEF.

Cellular and molecular mechanisms of EPH/EPHRIN signaling in evolution and development.

The EPH receptor tyrosine kinases and their signaling partners, the EPHRINS, comprise a large class of cell signaling molecules that plays diverse roles in development. As cell membrane-anchored signaling molecules, they regulate cellular organization by modulating the strength of cellular contacts, usually by impacting the actin cytoskeleton or cell adhesion programs. Through these cellular functions, EPH/EPHRIN signaling often regulates tissue shape. Indeed, recent evidence indicates that this signaling family is ancient and associated with the origin of multicellularity. Though extensively studied, our understanding of the signaling mechanisms employed by this large family of signaling proteins remains patchwork, and a truly "canonical" EPH/EPHRIN signal transduction pathway is not known and may not exist. Instead, several foundational evolutionarily conserved mechanisms are overlaid by a myriad of tissue -specific functions, though common themes emerge from these as well. Here, I review recent advances and the related contexts that have provided new understanding of the conserved and varied molecular and cellular mechanisms employed by EPH/EPHRIN signaling during development.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

Tracheal separation is driven by NKX2-1-mediated repression of Efnb2 and regulation of endodermal cell sorting.

The mechanisms coupling fate specification of distinct tissues to their physical separation remain to be understood. The trachea and esophagus differentiate from a single tube of definitive endoderm, requiring the transcription factors SOX2 and NKX2-1, but how the dorsoventral site of tissue separation is defined to allocate tracheal and esophageal cell types is unknown. Here, we show that the EPH/EPHRIN signaling gene Efnb2 regulates tracheoesophageal separation by controlling the dorsoventral allocation of tracheal-fated cells. Ventral loss of NKX2-1 results in disruption of separation and expansion of Efnb2 expression in the trachea independent of SOX2. Through chromatin immunoprecipitation and reporter assays, we find that NKX2-1 likely represses Efnb2 directly. Lineage tracing shows that loss of NKX2-1 results in misallocation of ventral foregut cells into the esophagus, while mosaicism for Nkx2-1 generates ectopic NKX2-1/EPHRIN-B2 boundaries that organize ectopic tracheal separation. Together, these data demonstrate that NKX2-1 coordinates tracheal specification with tissue separation through the regulation of EPHRIN-B2 and tracheoesophageal cell sorting.

Investigating the effects of compound paralogous EPHB receptor mutations on mouse facial development.

BACKGROUND: Variation in facial shape may arise from the combinatorial or overlapping actions of paralogous genes. Given its many members, and overlapping expression and functions, the EPH receptor family is a compelling candidate source of craniofacial morphological variation. We performed a detailed morphometric analysis of an allelic series of E14.5 Ephb1-3 receptor mutants to determine the effect of each paralogous receptor gene on craniofacial morphology. RESULTS: We found that Ephb1, Ephb2, and Ephb3 genotypes significantly influenced facial shape, but Ephb1 effects were weaker than Ephb2 and Ephb3 effects. Ephb2-/- and Ephb3-/- mutations affected similar aspects of facial morphology, but Ephb3-/- mutants had additional facial shape effects. Craniofacial differences across the allelic series were largely consistent with predicted additive genetic effects. However, we identified a potentially important nonadditive effect where Ephb1 mutants displayed different morphologies depending on the combination of other Ephb paralogs present, where Ephb1+/- , Ephb1-/- , and Ephb1-/- ; Ephb3-/- mutants exhibited a consistent deviation from their predicted facial shapes. CONCLUSIONS: This study provides a detailed assessment of the effects of Ephb receptor gene paralogs on E14.5 mouse facial morphology and demonstrates how the loss of specific receptors contributes to facial dysmorphology.

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts.

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.

NMDA receptors control development of somatosensory callosal axonal projections.

Callosal projections from primary somatosensory cortex (S1) are key for processing somatosensory inputs and integrating sensory-motor information. How the callosal innervation pattern in S1 is formed during early postnatal development is not clear. We found that the normal termination pattern of these callosal projections is disrupted in cortex specific NMDAR mutants. Rather than projecting selectively to the primary/secondary somatosensory cortex (S1/S2) border, axons were uniformly distributed throughout S1. In addition, the density of this projection increased over postnatal life until the mice died by P30. By combining genetic and antibody-mediated loss of function, we demonstrated that it is GluN2B-containing NMDA receptors in target S1 that mediate this guidance phenotype, thus playing a central role in interhemispheric connectivity. Furthermore, we found that this function of NMDA receptors in callosal circuit formation is independent of ion channel function and works with the EPHRIN-B/EPHB system. Thus, NMDAR in target S1 cortex regulates the formation callosal circuits perhaps by modulating EPH-dependent repulsion.

Delineating the early transcriptional specification of the mammalian trachea and esophagus.

The genome-scale transcriptional programs that specify the mammalian trachea and esophagus are unknown. Though NKX2-1 and SOX2 are hypothesized to be co-repressive master regulators of tracheoesophageal fates, this is untested at a whole transcriptomic scale and their downstream networks remain unidentified. By combining single-cell RNA-sequencing with bulk RNA-sequencing of Nkx2-1 mutants and NKX2-1 ChIP-sequencing in mouse embryos, we delineate the NKX2-1 transcriptional program in tracheoesophageal specification, and discover that the majority of the tracheal and esophageal transcriptome is NKX2-1 independent. To decouple the NKX2-1 transcriptional program from regulation by SOX2, we interrogate the expression of newly-identified tracheal and esophageal markers in Sox2/Nkx2-1 compound mutants. Finally, we discover that NKX2-1 binds directly to Shh and Wnt7b and regulates their expression to control mesenchymal specification to cartilage and smooth muscle, coupling epithelial identity with mesenchymal specification. These findings create a new framework for understanding early tracheoesophageal fate specification at the genome-wide level.

The trachea or windpipe is a tube that connects the throat to the lungs, while the esophagus connects the throat to the stomach. The trachea has cartilage rings that help to ensure clear airflow to the lungs, while the esophagus walls are lined with muscles that help to move food to the stomach. Although there are many differences between them, both the trachea and esophagus form from the same group of cells during development. Proteins called transcription factors help to control the formation of different body parts by switching different groups of genes on and off in different subsets of cells. Existing research has suggested that a transcription factor called NKX2.1 drives trachea formation, while another, called SOX2, is important in esophagus formation. An absence of either of these two proteins is thought to be associated with serious birth defects including loss of the trachea or esophagus, or failure of the two to separate fully. How these two transcription factors interact and drive the development of the trachea and esophagus, however, is currently unclear. Kuwahara et al. used mice to study the role of NKX2.1 and SOX2 in the formation of the trachea and esophagus. The findings identify many new genes that are active in the trachea and esophagus and reveal that NKX2.1 is not a master regulator that controls all of the genes involved in trachea formation. However, NKX2.1 does control several key genes, particularly those involved in forming cartilage in the trachea instead of muscle in the esophagus. The investigation also revealed many genes that are not controlled by NKX2.1 suggesting that other, currently unknown, systems play a major role in trachea formation. More work is required to understand the wider genetic regulators involved in differentiating the trachea from the esophagus. The findings in this study will help researchers to understand birth defects in the trachea and esophagus that result from genetic errors. They also reveal information about gene regulation processes that are relevant to the formation of other body parts and in the context of other diseases. In the long term, they could support regenerative medicine to regrow or replace lost or damaged body parts using lab-grown stem cells.

Forced to communicate: Integration of mechanical and biochemical signaling in morphogenesis.

Morphogenesis is a physical process that requires the generation of mechanical forces to achieve dynamic changes in cell position, tissue shape, and size as well as biochemical signals to coordinate these events. Mechanical forces are also used by the embryo to transmit detailed information across space and detected by target cells, leading to downstream changes in cellular properties and behaviors. Indeed, forces provide signaling information of complementary quality that can both synergize and diversify the functional outputs of biochemical signaling. Here, we discuss recent findings that reveal how mechanical signaling and biochemical signaling are integrated during morphogenesis and the possible context-specific advantages conferred by the interactions between these signaling mechanisms.

The UCSF Mouse Inventory Database Application, an Open Source Web App for Sharing Mutant Mice Within a Research Community.

The UCSF Mouse Inventory Database Application is an open-source Web App that provides information about the mutant alleles, transgenes, and inbred strains maintained by investigators at the university and facilitates sharing of these resources within the university community. The Application is designed to promote collaboration, decrease the costs associated with obtaining genetically-modified mice, and increase access to mouse lines that are difficult to obtain. An inventory of the genetically-modified mice on campus and the investigators who maintain them is compiled from records of purchases from external sources, transfers from researchers within and outside the university, and from data provided by users. These data are verified and augmented with relevant information harvested from public databases, and stored in a succinct, searchable database secured on the university network. Here we describe this resource and provide information about how to implement and maintain such a mouse inventory database application at other institutions.

Aberrant cell segregation in the craniofacial primordium and the emergence of facial dysmorphology in craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder characterized by craniofacial, skeletal, and neurological anomalies and is caused by mutations in EFNB1. Heterozygous females are more severely affected by CFNS than hemizygous males, a phenomenon called cellular interference that results from EPHRIN-B1 mosaicism. In Efnb1 heterozygous mice, mosaicism for EPHRIN-B1 results in cell sorting and more severe phenotypes than Efnb1 hemizygous males, but how craniofacial dysmorphology arises from cell segregation is unknown and CFNS etiology therefore remains poorly understood. Here, we couple geometric morphometric techniques with temporal and spatial interrogation of embryonic cell segregation in mouse mutant models to elucidate mechanisms underlying CFNS pathogenesis. By generating EPHRIN-B1 mosaicism at different developmental timepoints and in specific cell populations, we find that EPHRIN-B1 regulates cell segregation independently in early neural development and later in craniofacial development, correlating with the emergence of quantitative differences in face shape. Whereas specific craniofacial shape changes are qualitatively similar in Efnb1 heterozygous and hemizygous mutant embryos, heterozygous embryos are quantitatively more severely affected, indicating that Efnb1 mosaicism exacerbates loss of function phenotypes rather than having a neomorphic effect. Notably, neural tissue-specific disruption of Efnb1 does not appear to contribute to CFNS craniofacial dysmorphology, but its disruption within neural crest cell-derived mesenchyme results in phenotypes very similar to widespread loss. EPHRIN-B1 can bind and signal with EPHB1, EPHB2, and EPHB3 receptor tyrosine kinases, but the signaling partner(s) relevant to CFNS are unknown. Geometric morphometric analysis of an allelic series of Ephb1; Ephb2; Ephb3 mutant embryos indicates that EPHB2 and EPHB3 are key receptors mediating Efnb1 hemizygous-like phenotypes, but the complete loss of EPHB1-3 does not fully recapitulate the severity of CFNS-like Efnb1 heterozygosity. Finally, by generating Efnb1+/Δ; Ephb1; Ephb2; Ephb3 quadruple knockout mice, we determine how modulating cumulative receptor activity influences cell segregation in craniofacial development and find that while EPHB2 and EPHB3 play an important role in craniofacial cell segregation, EPHB1 is more important for cell segregation in the brain; surprisingly, complete loss of EPHB1-EPHB3 does not completely abrogate cell segregation. Together, these data advance our understanding of the etiology and signaling interactions underlying CFNS dysmorphology.

Getting direction(s): The Eph/ephrin signaling system in cell positioning.

In vertebrates, the Eph/ephrin family of signaling molecules is a large group of membrane-bound proteins that signal through a myriad of mechanisms and effectors to play diverse roles in almost every tissue and organ system. Though Eph/ephrin signaling has functions in diverse biological processes, one core developmental function is in the regulation of cell position and tissue morphology by regulating cell migration and guidance, cell segregation, and boundary formation. Often, the role of Eph/ephrin signaling is to translate patterning information into physical movement of cells and changes in morphology that define tissue and organ systems. In this review, we focus on recent advances in the regulation of these processes, and our evolving understanding of the in vivo signaling mechanisms utilized in distinct developmental contexts.

Cellular organization and boundary formation in craniofacial development.

Craniofacial morphogenesis is a highly dynamic process that requires changes in the behaviors and physical properties of cells in order to achieve the proper organization of different craniofacial structures. Boundary formation is a critical process in cellular organization, patterning, and ultimately tissue separation. There are several recurring cellular mechanisms through which boundary formation and cellular organization occur including, transcriptional patterning, cell segregation, cell adhesion and migratory guidance. Disruption of normal boundary formation has dramatic morphological consequences, and can result in human craniofacial congenital anomalies. In this review we discuss boundary formation during craniofacial development, specifically focusing on the cellular behaviors and mechanisms underlying the self-organizing properties that are critical for craniofacial morphogenesis.

Live Imaging of Mouse Secondary Palate Fusion.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

EPHRIN-B1 Mosaicism Drives Cell Segregation in Craniofrontonasal Syndrome hiPSC-Derived Neuroepithelial Cells.

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types, they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial, skeletal, and neurological anomalies. Heterozygous females are more severely affected than hemizygous males, a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation, no direct evidence for this has been demonstrated. Here, by generating hiPSCs from CFNS patients, we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells, thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients.