IRX3/5 regulate mitotic chromatid segregation and limb bud shape.

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.

Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud.

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5a-dependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease.

Characterizing Inner Pressure and Stiffness of Trophoblast and Inner Cell Mass of Blastocysts.

It has long been recognized that mechanical forces underlie mammalian embryonic shape changes. Before gastrulation, the blastocyst embryo undergoes significant shape changes, namely, the blastocyst cavity emerges and expands, and the inner cell mass (ICM) forms and changes in shape. The embryo's inner pressure has been hypothesized to be the driving mechanical input that causes the expansion of the blastocyst cavity and the shape changes of the ICM. However, how the inner pressure and the mechanics of the trophoblast and the ICM change during development is unknown because of the lack of a suitable tool for quantitative characterization. This work presents a laser-assisted magnetic tweezer technique for measuring the inner pressure and Young's modulus of the trophoblast and ICM of the blastocyst-stage mouse embryo. The results quantitatively showed that the inner pressure and Young's modulus of the trophoblast and ICM all increase during progression of mouse blastocysts, providing useful data for understanding how mechanical factors are physiologically integrated with other cues to direct embryo development.

Cell and Tissue Scale Forces Coregulate Fgfr2-Dependent Tetrads and Rosettes in the Mouse Embryo.

What motivates animal cells to intercalate is a longstanding question that is fundamental to morphogenesis. A basic mode of cell rearrangement involves dynamic multicellular structures called tetrads and rosettes. The contribution of cell-intrinsic and tissue-scale forces to the formation and resolution of these structures remains unclear, especially in vertebrates. Here, we show that Fgfr2 regulates both the formation and resolution of tetrads and rosettes in the mouse embryo, possibly in part by spatially restricting atypical protein kinase C, a negative regulator of non-muscle myosin IIB. We employ micropipette aspiration to show that anisotropic tension is sufficient to rescue the resolution, but not the formation, of tetrads and rosettes in Fgfr2 mutant limb-bud ectoderm. The findings underscore the importance of cell contractility and tissue stress to multicellular vertex formation and resolution, respectively.

Mutations in prickle orthologs cause seizures in flies, mice, and humans.

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.

Nuclear localization of Prickle2 is required to establish cell polarity during early mouse embryogenesis.

The establishment of trophectoderm (TE) manifests as the formation of epithelium, and is dependent on many structural and regulatory components that are commonly found and function in many epithelial tissues. However, the mechanism of TE formation is currently not well understood. Prickle1 (Pk1), a core component of the planar cell polarity (PCP) pathway, is essential for epiblast polarization before gastrulation, yet the roles of Pk family members in early mouse embryogenesis are obscure. Here we found that Pk2(-/-) embryos died at E3.0-3.5 without forming the blastocyst cavity and not maintained epithelial integrity of TE. These phenotypes were due to loss of the apical-basal (AB) polarity that underlies the asymmetric redistribution of microtubule networks and proper accumulation of AB polarity components on each membrane during compaction. In addition, we found GTP-bound active form of nuclear RhoA was decreased in Pk2(-/-) embryos during compaction. We further show that the first cell fate decision was disrupted in Pk2(-/-) embryos. Interestingly, Pk2 localized to the nucleus from the 2-cell to around the 16-cell stage despite its cytoplasmic function previously reported. Inhibiting farnesylation blocked Pk2's nuclear localization and disrupted AB cell polarity, suggesting that Pk2 farnesylation is essential for its nuclear localization and function. The cell polarity phenotype was efficiently rescued by nuclear but not cytoplasmic Pk2, demonstrating the nuclear localization of Pk2 is critical for its function.

Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity.

Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1(-/-) epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of DeltaPET/LIM (DeltaP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCzeta, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.

Patterning the embryonic pulmonary mesenchyme.

Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

Transgenic force sensors and software to measure force transmission across the mammalian nuclear envelope in vivo.

Nuclear mechanotransduction is a growing field with exciting implications for the regulation of gene expression and cellular function. Mechanical signals may be transduced to the nuclear interior biochemically or physically through connections between the cell surface and chromatin. To define mechanical stresses upon the nucleus in physiological settings, we generated transgenic mouse strains that harbour FRET-based tension sensors or control constructs in the outer and inner aspects of the nuclear envelope. We knocked-in a published esprin-2G sensor to measure tensions across the LINC complex and generated a new sensor that links the inner nuclear membrane to chromatin. To mitigate challenges inherent to fluorescence lifetime analysis in vivo, we developed software (FLIMvivo) that markedly improves the fitting of fluorescence decay curves. In the mouse embryo, the sensors responded to cytoskeletal relaxation and stretch applied by micro-aspiration. They reported organ-specific differences and a spatiotemporal tension gradient along the proximodistal axis of the limb bud, raising the possibility that mechanical mechanisms coregulate pattern formation. These mouse strains and software are potentially valuable tools for testing and refining mechanotransduction hypotheses in vivo.

Magnetic Micromanipulation for In Vivo Measurement of Stiffness Heterogeneity and Anisotropy in the Mouse Mandibular Arch.

The mechanical properties of tissues are pivotal for morphogenesis and disease progression. Recent approaches have enabled measurements of the spatial distributions of viscoelastic properties among embryonic and pathological model systems and facilitated the generation of important hypotheses such as durotaxis and tissue-scale phase transition. There likely are many unexpected aspects of embryo biomechanics we have yet to discover which will change our views of mechanisms that govern development and disease. One area in the blind spot of even the most recent approaches to measuring tissue stiffness is the potentially anisotropic nature of that parameter. Here, we report a magnetic micromanipulation device that generates a uniform magnetic field gradient within a large workspace and permits measurement of the variation of tissue stiffness along three orthogonal axes. By applying the device to the organ-stage mouse embryo, we identify spatially heterogenous and directionally anisotropic stiffness within the mandibular arch. Those properties correspond to the domain of expression and the angular distribution of fibronectin and have potential implications for mechanisms that orient collective cell movements and shape tissues during development. Assessment of anisotropic properties extends the repertoire of current methods and will enable the generation and testing of hypotheses.

Oscillatory cortical forces promote three dimensional cell intercalations that shape the murine mandibular arch.

Multiple vertebrate embryonic structures such as organ primordia are composed of confluent cells. Although mechanisms that shape tissue sheets are increasingly understood, those which shape a volume of cells remain obscure. Here we show that 3D mesenchymal cell intercalations are essential to shape the mandibular arch of the mouse embryo. Using a genetically encoded vinculin tension sensor that we knock-in to the mouse genome, we show that cortical force oscillations promote these intercalations. Genetic loss- and gain-of-function approaches show that Wnt5a functions as a spatial cue to coordinate cell polarity and cytoskeletal oscillation. These processes diminish tissue rigidity and help cells to overcome the energy barrier to intercalation. YAP/TAZ and PIEZO1 serve as downstream effectors of Wnt5a-mediated actomyosin polarity and cytosolic calcium transients that orient and drive mesenchymal cell intercalations. These findings advance our understanding of how developmental pathways regulate biophysical properties and forces to shape a solid organ primordium.

The two domain hypothesis of limb prepattern and its relevance to congenital limb anomalies.

Functional annotation of mutations that cause human limb anomalies is enabled by basic developmental studies. In this study, we focus on the prepatterning stage of limb development and discuss a recent model that proposes anterior and posterior domains of the early limb bud generate two halves of the future skeleton. By comparing phenotypes in humans with those in model organisms, we evaluate whether this prepatterning concept helps to annotate human disease alleles. WIREs Dev Biol 2017, 6:e270. doi: 10.1002/wdev.270 For further resources related to this article, please visit the WIREs website.

FGF10 is a mesenchymally derived stimulator for epidermal development in the chick embryonic skin.

The development of avian cutaneous appendages, feathers and scales, is known to arise from the epithelial-mesenchymal interaction. Here we show that FGF10 is associated with this developmental process as an early signal from mesenchymal cells underlying nascent cutaneous placodes. Expression of Fgf10 was detected in the mesenchymal cells underneath the developing placodes. Forced expression of Fgf10 in the femoral skin suppressed expression of Shh and a zinc finger gene snail-related (cSnR), while induced expression of Bmp2 in the interbud region, resulting in thickening of the epidermal layer. Furthermore, forced expression of Fgf10 in the foot skin caused marked ingrowings of the epidermis. The cells in the epidermal ingrowings expressed beta-catenin, proliferating cell nuclear antigen, and an epidermal stem cell marker p63. These results support the idea that FGF10 is a mesenchymally derived stimulator of epidermal development through crosstalk with bone morphogenetic protein (BMP), beta-catenin, and other signaling pathways.

Anisotropic stress orients remodelling of mammalian limb bud ectoderm.

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a ß-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by ß-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

PRICKLE1 interaction with SYNAPSIN I reveals a role in autism spectrum disorders.

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Exogenous FGF10 can rescue an eye-open at birth phenotype of Fgf10-null mice by activating activin and TGFalpha-EGFR signaling.

Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit an eye-open phenotype at birth. It has previously been shown that FGF10 has a dual role in proliferation and migration during the early and later stages of eyelid development, respectively. To verify the role of FGF10 during eyelid closure, explant culture of Fgf10-null eyelid anlagen was performed, by which it was examined whether or not exogenous FGF10 could rescue the expression of activin betaB and transforming growth factor alpha, known to be required for eyelid closure. We found that the expression of these genes was markedly induced while that of Shh or Ptch1, Ptch2 was not. We also observed the distribution of filamentous actin (F-actin) after FGF10 application in the mutant eyelid explant, finding that the FGF10 protein induced F-actin accumulation. We further examined filopodia of the eyelid leading edge cells, finding the length of the filopodia was significantly reduced in the mutant. These results verify that FGF10 promotes eyelid closure through activating activin and TGFalpha-EGFR signaling.

A dual role of FGF10 in proliferation and coordinated migration of epithelial leading edge cells during mouse eyelid development.

The development of the eyelid requires coordinated cellular processes of proliferation, cell shape changes, migration and cell death. Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit open-eyelids at birth. To elucidate the roles of FGF10 during eyelid formation, we examined the expression pattern of Fgf10 during eyelid formation and the phenotype of Fgf10-null eyelids in detail. Fgf10 is expressed by mesenchymal cells just beneath the protruding epidermal cells of the nascent eyelid. However, Fgf10-null epithelial cells running though the eyelid groove do not exhibit typical cuboid shape or sufficient proliferation. Furthermore, peridermal clumps are not maintained on the eyelid leading edge, and epithelial extension does not occur. At the cellular level, the accumulation of actin fibers is not observed in the mutant epithelial leading edge. The expression of activin/inhibin betaB (ActbetaB/Inhbb) and transforming growth factor alpha (Tgfa), previously reported to be crucial for eyelid development, is down-regulated in the mutant leading edge, while the onset of sonic hedgehog (Shh) expression is delayed on the mutant eyelid margin. Explant cultures of mouse eyelid primordia shows that the open-eyelid phenotype of the mutant is reduced by exogenous FGF10 protein, and that the expression of ActbetaB and Tgfa is ectopically induced in the thickened eyelid epithelium by the FGF10 protein. These results indicate a dual role of FGF10 in mouse eyelid development, for both proliferation and coordinated migration of eyelid epithelial cells by reorganization of the cytoskeleton, through the regulation of activin, TGFalpha and SHH signaling.

Fibroblast growth factor 10 is required for proper development of the mouse whiskers.

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse.