Nodal signaling regulates asymmetric cellular behaviors, driving clockwise rotation of the heart tube in zebrafish.

Clockwise rotation of the primitive heart tube, a process regulated by restricted left-sided Nodal signaling, is the first morphological manifestation of left-right asymmetry. How Nodal regulates cell behaviors to drive asymmetric morphogenesis remains poorly understood. Here, using high-resolution live imaging of zebrafish embryos, we simultaneously visualized cellular dynamics underlying early heart morphogenesis and resulting changes in tissue shape, to identify two key cell behaviors: cell rearrangement and cell shape change, which convert initially flat heart primordia into a tube through convergent extension. Interestingly, left cells were more active in these behaviors than right cells, driving more rapid convergence of the left primordium, and thereby rotating the heart tube. Loss of Nodal signaling abolished the asymmetric cell behaviors as well as the asymmetric convergence of the left and right heart primordia. Collectively, our results demonstrate that Nodal signaling regulates the magnitude of morphological changes by acting on basic cellular behaviors underlying heart tube formation, driving asymmetric deformation and rotation of the heart tube.

Dorsoventral differences in cAMP levels and correlated changes in the subcellular distribution of the PKA catalytic domain, provide further evidence that PKA signaling coordinates dorsoventral patterning of the otocyst.

Dorsoventral (DV) patterning of the otocyst gives rise to formation of the morphologically and functionally complex membranous labyrinth composed of unique dorsal and ventral sensory organs. DV patterning results from extracellular signaling by secreted growth factors, which presumably form reciprocal concentration gradients across the DV axis of the otocyst. Previous work suggested a model in which two important growth factors, bone morphogenetic protein (BMP) and SHH, undergo crosstalk through an intersecting pathway to coordinate DV patterning. cAMP-dependent protein kinase A (PKA) lies at the heart of this pathway. Here, we provide further evidence that PKA signaling coordinates DV patterning, showing that both BMPs and SHH regulate cAMP levels, with BMPs increasing levels in the dorsal otocyst and SHH decreasing levels in the ventral otocyst. This, in turn, results in regional changes in the subcellular distribution of the catalytic domain of PKA, as well as DV regulation of PKA activity, increasing it dorsally and decreasing it ventrally. These new results fill an important gap in our previous understanding of how ligand signaling acts intracellularly during otocyst DV patterning and early morphogenesis, thereby initiating the series of events leading to formation of the inner ear sensory organs that function in balance and hearing.

Contributions of the chick embryo and experimental embryology to understanding the cellular mechanisms of neurulation.

The chick embryo has served as a workhorse for experimental embryological studies designed to elucidate mechanisms underlying neurulation, the process that forms the neural tube, the rudiment of the entire adult central nervous system. Early chick embryos developing in whole-embryo culture can be readily manipulated in cut-and-paste-type experiments, and this attribute makes this model system unparalleled for studying the morphogenesis of embryos and their organ rudiments. How the chick embryo and experimental embryology have contributed to our understanding of critical events of neurulation are summarized.

The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension.

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis.

Hearing crosstalk: the molecular conversation orchestrating inner ear dorsoventral patterning.

The inner ear is a structurally and functionally complex organ that functions in balance and hearing. It originates during neurulation as a localized thickened region of rostral ectoderm termed the otic placode, which lies adjacent to the developing caudal hindbrain. Shortly after the otic placode forms, it invaginates to delineate the otic cup, which quickly pinches off of the surface ectoderm to form a hollow spherical vesicle called the otocyst; the latter gives rise dorsally to inner ear vestibular components and ventrally to its auditory component. Morphogenesis of the otocyst is regulated by secreted proteins, such as WNTs, BMPs, and SHH, which determine its dorsoventral polarity to define vestibular and cochlear structures and sensory and nonsensory cell fates. In this review, we focus on the crosstalk that occurs among three families of secreted molecules to progressively polarize and pattern the developing otocyst. WIREs Dev Biol 2018, 7:e302. doi: 10.1002/wdev.302 This article is categorized under: Establishment of Spatial and Temporal Patterns > Gradients Signaling Pathways > Cell Fate Signaling Vertebrate Organogenesis > From a Tubular Primordium: Non-Branched.

Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.

Fgf3 and Fgf16 expression patterns define spatial and temporal domains in the developing chick inner ear.

The inner ear is a morphologically complex sensory structure with auditory and vestibular functions. The developing otic epithelium gives rise to neurosensory and non-sensory elements of the adult membranous labyrinth. Extrinsic and intrinsic signals manage the patterning and cell specification of the developing otic epithelium by establishing lineage-restricted compartments defined in turn by differential expression of regulatory genes. FGF3 and FGF16 are excellent candidates to govern these developmental events. Using the chick inner ear, we show that Fgf3 expression is present in the borders of all developing cristae. Strong Fgf16 expression was detected in a portion of the developing vertical and horizontal pouches, whereas the cristae show weaker or undetected Fgf16 expression at different developmental stages. Concerning the rest of the vestibular sensory elements, both the utricular and saccular maculae were Fgf3 positive. Interestingly, strong Fgf16 expression delimited these Fgf16-negative sensory patches. The Fgf3-negative macula neglecta and the Fgf3-positive macula lagena were included within weakly Fgf16-expressing areas. Therefore, different FGF-mediated mechanisms might regulate the specification of the anterior (utricular and saccular) and posterior (neglecta and lagena) maculae. In the developing cochlear duct, dynamic Fgf3 and Fgf16 expression suggests their cooperation in the early specification and later cell differentiation in the hearing system. The requirement of Fgf3 and Fgf16 genes in endolymphatic apparatus development and neurogenesis are discussed. Based on these observations, FGF3 and FGF16 seem to be key signaling pathways that control the inner ear plan by defining epithelial identities within the developing otic epithelium.

SHH ventralizes the otocyst by maintaining basal PKA activity and regulating GLI3 signaling.

During development of the inner ear, secreted morphogens act coordinately to establish otocyst dorsoventral polarity. Among these, Sonic hedgehog (SHH) plays a critical role in determining ventral polarity. However, how this extracellular signal is transduced intracellularly to establish ventral polarity is unknown. In this study, we show that cAMP dependent protein kinase A (PKA) is a key intracellular factor mediating SHH signaling through regulation of GLI3 processing. Gain-of-function experiments using targeted gene transfection by sonoporation or electroporation revealed that SHH signaling inactivates PKA, maintaining a basal level of PKA activity in the ventral otocyst. This, in turn, suppresses partial proteolytic processing of GLI3FL, resulting in a low GLI3R/GLI3FL ratio in the ventral otocyst and the expression of ventral-specific genes required for ventral otocyst morphogenesis. Thus, we identify a molecular mechanism that links extracellular and intracellular signaling, determines early ventral polarity of the inner ear, and has implications for understanding the integration of polarity signals in multiple organ rudiments regulated by gradients of signaling molecules.

BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways.

The inner ear consists of two otocyst-derived, structurally and functionally distinct components: the dorsal vestibular and ventral auditory compartments. BMP signaling is required to form the vestibular compartment, but how it complements other required signaling molecules and acts intracellularly is unknown. Using spatially and temporally controlled delivery of signaling pathway regulators to developing chick otocysts, we show that BMP signaling regulates the expression of Dlx5 and Hmx3, both of which encode transcription factors essential for vestibular formation. However, although BMP regulates Dlx5 through the canonical SMAD pathway, surprisingly, it regulates Hmx3 through a non-canonical pathway involving both an increase in cAMP-dependent protein kinase A activity and the GLI3R to GLI3A ratio. Thus, both canonical and non-canonical BMP signaling establish the precise spatiotemporal expression of Dlx5 and Hmx3 during dorsal vestibular development. The identification of the non-canonical pathway suggests an intersection point between BMP and SHH signaling, which is required for ventral auditory development.

A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development.

Fibroblast growth factor (FGF) signaling is a critical regulator of skeletal development. Fgf9 and Fgf18 are the only FGF ligands with identified functions in embryonic bone growth. Mice lacking Fgf9 or Fgf18 have distinct skeletal phenotypes; however, the extent of overlapping or redundant functions for these ligands and the stage-specific contributions of FGF signaling to chondrogenesis and osteogenesis are not known. To identify separate versus shared roles for FGF9 and FGF18, we generated a combined series of Fgf9 and Fgf18 null alleles. Analysis of embryos lacking alleles of Fgf9 and Fgf18 shows that both encoded ligands function redundantly to control all stages of skeletogenesis; however, they have variable potencies along the proximodistal limb axis, suggesting gradients of activity during formation of the appendicular skeleton. Congenital absence of both Fgf9 and Fgf18 results in a striking osteochondrodysplasia and revealed functions for FGF signaling in early proximal limb chondrogenesis. Additional defects were also noted in craniofacial bones, vertebrae, and ribs. Loss of alleles of Fgf9 and Fgf18 also affect the expression of genes encoding other key intrinsic skeletal regulators, including IHH, PTHLH (PTHrP), and RUNX2, revealing potential direct, indirect, and compensatory mechanisms to coordinate chondrogenesis and osteogenesis.

Getting published well requires fulfilling editors' and reviewers' needs and desires.

Publication in international scientific journals provides an unparalleled opportunity for authors to showcase their work. Where authors publish affects how the community values the work. This value directly determines the impact of the work on the field-papers must be read and cited to advance the field, and because the scientific literature is vast, only a subset of the literature is widely read and cited. Moreover, the value placed on the work also affects the authors' scientific reputation and career advancement. Consequently, it is essential that manuscripts receive the recognition they deserve by being published in one of the "best" journals that the scientific findings allow. Several factors determine where a paper is published: how well the topic of the paper fits the scope of the journal, the quality of the study and the manuscript describing it, the advance the paper makes in its field, the importance of the advance, and the extent to which the paper impacts the broader community of science. As scientists, we assume that our papers will be assessed objectively using only well defined scientific standards, but editors and reviewers also view papers subjectively, having biases of what defines a high-quality publication based on Western standards. Therefore, scientists trained in other parts of the world can be significantly disadvantaged in getting their papers published in the best journals. Here, I present concrete suggestions for improving the perception of a paper in the reader's minds, increasing the likelihood that it will get published well.

Detection of isoform-specific fibroblast growth factor receptors by whole-mount in situ hybridization in early chick embryos.

We have developed "b" and "c" isoform-specific chicken fibroblast growth factor (FGF) receptor 1-3 probes for in situ hybridization. We rigorously demonstrate the specificity of these probes by using both dot blot hybridization and whole-mount in situ hybridization during neurulation and early postneurulation stages, and we compare expression patterns of each of the three isoform-specific probes to one another and to generic probes to each of the three (non-isoform-specific) FGF receptors. We show that the expression pattern of each receptor is represented by the collective expression of each of its two isoforms, with the expression of each FGF receptor being most similar to that of its "c" isoform at two of the three stages studied, and that tissue and stage differences exist in the patterns of expression of the six isoforms. We demonstrate the usefulness of these probes for defining the differential tissue expression of FGF receptor 1-3 isoforms.

BMP/SMAD signaling regulates the cell behaviors that drive the initial dorsal-specific regional morphogenesis of the otocyst.

During development of the otocyst, regional morphogenesis establishes a dorsal vestibular chamber and a ventral auditory chamber, which collectively constitute the membranous labyrinth of the inner ear. We identified the earliest morphogenetic event heralding the formation of the vestibular chamber, a rapid thinning and expansion of the dorsolateral wall of the otocyst, and showed that this process is generated by changes in otocyst cell shape from columnar to squamous, as opposed to changes in other cell behaviors, such as localized changes in cell proliferation or cell death. Moreover, we showed that thinning and expansion of the dorsolateral otocyst is regulated by BMP/SMAD signaling, which is both sufficient and necessary for localized thinning and expansion. Finally, we showed that BMP/SMAD signaling causes fragmentation of E-cadherin in the dorsolateral otocyst, occurring concomitantly with cell shape change, suggesting that BMP/SMAD signaling regulates cell-cell adhesion during the initial morphogenesis of the otocyst epithelium. Collectively, our results show that BMP signaling via SMADs regulates the cell behaviors that drive the initial dorsal-specific morphogenesis of the otocyst, providing new information about how regional morphogenesis of a complex organ rudiment, the developing membranous labyrinth, is initiated.

The cessation of gastrulation: BMP signaling and EMT during and at the end of gastrulation.

An integral component of gastrulation in all organisms is epithelial to mesenchymal transition (EMT), a fundamental morphogenetic event through which epithelial cells transform into mesenchymal cells. The mesenchymal cells that arise from epithelial cells during gastrulation contribute to various tissue rudiments during subsequent development, including the notochord, somites, heart, gut, kidney, body wall and lining of the coelom. The process of gastrulation has been the subject of several hundred scientific papers. Despite all that has been written, it is likely that what we currently know about gastrulation is still considerably less than what remains to be learned. One critical remaining question that we consider here is how does gastrulation cease at the right place along the body axis, and at the right time? In this commentary, we focus on the molecular mechanism for the cessation of gastrulation, using the chick embryo as a model system.

FGF signaling regulates otic placode induction and refinement by controlling both ectodermal target genes and hindbrain Wnt8a.

The inner ear epithelium, with its complex array of sensory, non-sensory, and neuronal cell types necessary for hearing and balance, is derived from a thickened patch of head ectoderm called the otic placode. Mouse embryos lacking both Fgf3 and Fgf10 fail to initiate inner ear development because appropriate patterns of gene expression fail to be specified within the pre-otic field. To understand the transcriptional "blueprint" initiating inner ear development, we used microarray analysis to identify prospective placode genes that were differentially expressed in control and Fgf3(-)(/)(-);Fgf10(-)(/)(-) embryos. Several genes in the down-regulated class, including Hmx3, Hmx2, Foxg1, Sox9, Has2, and Slc26a9 were validated by in situ hybridization. We also assayed candidate target genes suggested by other studies of otic induction. Two placode markers, Fgf4 and Foxi3, were down-regulated in Fgf3(-)(/)(-);Fgf10(-)(/)(-) embryos, whereas Foxi2, a cranial epidermis marker, was expanded in double mutants, similar to its behavior when WNT responses are blocked in the otic placode. Assays of hindbrain Wnt genes revealed that only Wnt8a was reduced or absent in FGF-deficient embryos, and that even some Fgf3(-)(/)(-);Fgf10(-)(/+) and Fgf3(-)(/)(-) embryos failed to express Wnt8a, suggesting a key role for Fgf3, and a secondary role for Fgf10, in Wnt8a expression. Chick explant assays showed that FGF3 or FGF4, but not FGF10, were sufficient to induce Wnt8a. Collectively, our results suggest that Wnt8a provides the link between FGF-induced formation of the pre-otic field and restriction of the otic placode to ectoderm adjacent to the hindbrain.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms.

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.

Identification of differentially expressed genes in early inner ear development.

To understand the etiology of congenital hearing loss, a comprehensive understanding of the molecular genetic mechanisms underlying normal ear development is required. We are identifying genes involved in otogenesis, with the longer term goal of studying their mechanisms of action, leading to inner ear induction and patterning. Using Agilent microarrays, we compared the differential expression of a test domain (which consisted of the pre-otic placodal ectoderm with the adjacent hindbrain ectoderm and the underlying mesendodermal tissues) with a rostral control domain (which included tissue that is competent, but not specified, to express inner ear markers in explant assays). We identified 1261 transcripts differentially expressed between the two domains at a 2-fold or greater change: 463 were upregulated and 798 were downregulated in the test domain. We validated the differential expression of several signaling molecules and transcription factors identified in this array using in situ hybridization. Furthermore, the expression patterns of the validated group of genes from the test domain were explored in detail to determine how the timing of their expression relates to specific events of otic induction and development. In conclusion, we identified a number of novel candidate genes for otic placode induction.

Brachymesomelic dysplasia with Peters anomaly of the eye results from disruptions of the X chromosome near the SHOX and SOX3 genes.

We report on a mother and son affected with an unusual skeletal dysplasia and anterior segment eye abnormalities. Their skeletal phenotype overlaps with the SHOX-related skeletal dysplasias and is intermediate between Leri-Weill dyschondrosteosis (LWD) and Langer Mesomelic dysplasia (LMD). The mother has bilateral Peters anomaly of the eye and was reported as having a new syndrome; the son had severe bilateral sclerocornea. Chromosome analysis showed that the mother has a pericentric inversion of the X chromosome [46,X,inv(X)(p22.3q27)] and the son, a resultant recombinant X chromosome [46,Y,rec(X)dup(Xq)inv(X)(p22.3q27)]. The observed skeletal and ophthalmologic abnormalities in both patients were similar in severity. The additional features of developmental delay, growth retardation, agenesis of the corpus callosum, cryptorchidism and hypoplastic scrotum in the son are consistent with Xq28 duplication. Analysis of the son's recombinant X chromosome showed that the Xp22.33 breakpoint lies 30-68 kb 5' of the SHOX gene. This finding suggests that the skeletal dysplasia in both mother and son is allelic with LWD and LMD and results from a novel misexpression of SHOX. Analysis of the Xq27.1 breakpoint localized it to a 90 kb interval 3' of the SOX3 gene, supporting a novel role of SOX3 misexpression in the development of Peters anomaly of the eye.