ABSTRACT Male-female differences in many developmental mechanisms lead to the formation of two morphologically and physiologically distinct sexes. Although this is expected for traits with prominent differences between the sexes, such as the gonads, sex-specific processes also contribute to traits without obvious male-female differences, such as the intestine. Here, we review sex differences in developmental mechanisms that operate at several levels of biological complexity – molecular, cellular, organ and organismal – and discuss how these differences influence organ formation, function and whole-body physiology. Together, the examples we highlight show that one simple way to gain a more accurate and comprehensive understanding of animal development is to include both sexes.

ABSTRACT The phytohormone cytokinin regulates diverse aspects of plant growth and development. Our understanding of the metabolism and perception of cytokinin has made great strides in recent years, mostly from studies of the model dicot Arabidopsis . Here, we employed a CRISPR/Cas9-based approach to disrupt a subset of cytokinin histidine kinase (HK) receptors in rice ( Oryza sativa ) in order to explore the role of cytokinin in a monocot species. In hk5 and hk6 single mutants, the root growth, leaf width, inflorescence architecture and/or floral development were affected. The double hk5 hk6 mutant showed more substantial defects, including severely reduced root and shoot growth, a smaller shoot apical meristem, and an enlarged root cap. Flowering was delayed in the hk5 hk6 mutant and the panicle was significantly reduced in size and infertile due to multiple defects in floral development. The hk5 hk6 mutant also exhibited a severely reduced cytokinin response, consistent with the developmental phenotypes arising from a defect in cytokinin signaling. These results indicate that HK5 and HK6 act as cytokinin receptors, with overlapping functions to regulate diverse aspects of rice growth and development.

ABSTRACT Notch signaling plays a pleiotropic role in a variety of cellular processes, including cell fate determination, differentiation, proliferation and apoptosis. The increasingly complex regulatory mechanisms of Notch signaling account for the many functions of Notch during development. Using a yeast two-hybrid screen, we identified the Drosophila DNA-binding protein Hat-trick (Htk) to be an interacting partner of Notch-intracellular domain (Notch-ICD); their physical interaction was further validated by co-immunoprecipitation experiments. htk genetically interacts with Notch pathway components in trans-heterozygous combinations. Loss of htk function in htk mutant somatic clones resulted in the downregulation of Notch targets, whereas its overexpression caused ectopic expression of Notch targets, without affecting the level of the Notch protein. In the present study, immunocytochemical analyses demonstrate that Htk and overexpressed Notch-ICD colocalize in the same nuclear compartment. Here, we also show that Htk cooperates with Notch-ICD and Suppressor of Hairless to form an activation complex and binds to the regulatory sequences of Notch downstream targets such as Enhancer of Split complex genes, to direct their expression. Together, our results suggest a novel mode of regulation of Notch signaling by the chromatin-modeling protein Htk.

ABSTRACT Canonical Wnts promote myoblast differentiation; however, the role of β-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated β-catenin-null primary adult mouse myoblasts, we found that β-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a β-catenin-TCF/LEF interaction is not required for differentiation, and that β-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by β-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of β-catenin with α-catenin was important for efficient differentiation. Overall the study suggests dual roles for β-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an α-catenin-dependent membrane function that helps control cell-cell interactions. β-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of β-catenin and thus prevent precocious/excessive myoblast fusion.

ABSTRACT The phytohormone cytokinin plays diverse roles in plant development, influencing many agriculturally important processes, including growth, nutrient responses and the response to biotic and abiotic stresses. Cytokinin levels in plants are regulated by biosynthesis and inactivation pathways. Cytokinins are perceived by membrane-localized histidine-kinase receptors and are transduced through a His-Asp phosphorelay to activate a family of transcription factors in the nucleus. Here, and in the accompanying poster, we summarize the current understanding of cytokinin metabolism, transport and signaling, and discuss how this phytohormone regulates changes in gene expression to mediate its pleiotropic effects.

microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.

The Hippo pathway has emerged as a conserved signaling pathway that is essential for the proper regulation of organ growth in Drosophila and vertebrates. Although the mechanisms of signal transduction of the core kinases Hippo/Mst and Warts/Lats are relatively well understood, less is known about the upstream inputs of the pathway and about the downstream cellular and developmental outputs. Here, we review recently discovered mechanisms that contribute to the dynamic regulation of Hippo signaling during Drosophila and vertebrate development. We also discuss the expanding diversity of Hippo signaling functions during development, discoveries that shed light on a complex regulatory system and provide exciting new insights into the elusive mechanisms that regulate organ growth and regeneration.

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequently address population-level phenomena. Sonic hedgehog (SHH) signaling activity in the vertebrate forebrain and face is thought to contribute to continuous variation in the morphology of the upper jaw, but despite its potential explanatory power, this idea has never been quantitatively assessed. Here, we test this hypothesis with an experimental design that is explicitly focused on the generation and measurement of variation in multivariate shape, tissue growth, cellular behavior and gene expression. We show that the majority of upper jaw shape variation can be explained by progressive changes in the spatial organization and mitotic activity of midfacial growth zones controlled by SHH signaling. In addition, nonlinearity between our treatment doses and phenotypic outcomes suggests that threshold effects in SHH signaling may play a role in variability in midfacial malformations such as holoprosencephaly (HPE). Together, these results provide novel insight into the generation of facial morphology, and demonstrate the value of quantifying variation for our understanding of development and disease.

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor(FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2α (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2 α in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2 α mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.

The Notch (N) signaling pathway is involved in a vast number of patterning processes in all metazoans. The regulation of the core N pathway is largely understood, but little is known about fine-tuning modulatory effects. Here, we address the role of Drosophila Krüppel-family Zn-finger transcription factor roughened eye ( roe ) in the context of N signaling. We demonstrate that during eye patterning, N signaling regulates the expression of roe . In turn, Roe negatively modulates the expression of target genes of N-signaling activation. In the absence of roe function, expression of N target genes is elevated and the resulting phenotypes during patterning of the retina are similar to those of N gain-of-function scenarios. Importantly, our data show that Roe binds regulatory DNA sequences of N target genes of the E(spl)-complex both in vitro and in vivo, independently of Su(H)-DNA interaction. Thus, our data suggest that Roe acts as a transcriptional repressor in a negative-feedback loop of the N pathway.

During Drosophila oogenesis, patterning activities of the EGFR and Dpp pathways specify several subpopulations of the follicle cells that give rise to dorsal eggshell structures. The roof of dorsal eggshell appendages is formed by the follicle cells that express Broad (Br), a zinc-finger transcription factor regulated by both pathways. EGFR induces Br in the dorsal follicle cells. This inductive signal is overridden in the dorsal midline cells, which are exposed to high levels of EGFR activation, and in the anterior cells, by Dpp signaling. We show that the resulting changes in the Br pattern affect the expression of Dpp receptor thickveins ( tkv ), which is essential for Dpp signaling. By controlling tkv , Br controls Dpp signaling in late stages of oogenesis and, as a result, regulates its own repression in a negative-feedback loop. We synthesize these observations into a model, whereby the dynamics of Br expression are driven by the sequential action of feedforward and feedback loops. The feedforward loop controls the spatial pattern of Br expression,while the feedback loop modulates this pattern in time. This mechanism demonstrates how complex patterns of gene expression can emerge from simple inputs, through the interaction of regulatory network motifs.

The mes/metencephalic boundary (isthmus) is an organizing center for the optic tectum and cerebellum. Fgf8 is accepted as a crucial organizing signal. Previously, we reported that Fgf8b could induce cerebellum in the mesencephalon, while Fgf8a transformed the presumptive diencephalon into mesencephalon. Since lower doses of Fgf8b exerted similar effects to those of Fgf8a , the type difference could be attributed to the difference in the strength of the signal. It is of great interest to uncover mechanisms of signal transduction pathways downstream of the Fgf8 signal in tectal and cerebellar development, and in this report we have concentrated on the Ras-ERK pathway. In normal embryos,extracellular-signal-regulated kinase (ERK) is activated at the site where Fgf8 mRNA is expressed. Fgf8b activated ERK while Fgf8a or a lower dose of Fgf8b did not activate ERK in the mes/metencephalon. Disruption of the Ras-ERK signaling pathway by a dominant negative form of Ras ( Ras S17N ) changed the fate of the metencephalic alar plate from cerebellum to tectum. Ras S17N canceled the effects of Fgf8b , while co-transfection of Fgf8a and Ras S17N exerted additive effects. Disruption of Fgf8b , not Fgf8a , by siRNA resulted in posterior extension of the Otx2 expression domain. Our results indicate that the presumptive metencephalon receives a strong Fgf8 signal that activates the Ras-ERK pathway and differentiates into the cerebellum.

Signaling by the hedgehog ( hh )-class gene pathway is essential for embryogenesis in organisms ranging from Drosophila to human. We have isolated a hh homolog ( Hro-hh ) from a lophotrochozoan species, the glossiphoniid leech, Helobdella robusta ,and examined its expression by reverse transcription polymerase chain reaction(RT-PCR) and whole-mount in situ hybridization. The peak of Hro-hh expression occurs during organogenesis (stages 10-11). No patterned expression was detected within the segmented portion of the germinal plate during the early stages of segmentation. In stage 10-11 embryos, Hro-hh is expressed in body wall, foregut, anterior and posterior midgut, reproductive organs and in a subset of ganglionic neurons. Evidence that Hro-hh regulates gut formation was obtained using the steroidal alkaloid cyclopamine,which specifically blocks HH signaling. Cyclopamine induced malformation of both foregut and anterior midgut in Helobdella embryos, and no morphologically recognizable gonads were seen. In contrast, no gross abnormalities were observed in the posterior midgut. Segmental ectoderm developed normally, as did body wall musculature and some other mesodermal derivatives, but the mesenchymal cells that normally come to fill most of the coelomic cavities failed to develop. Taken with data from Drosophila and vertebrates, our data suggest that the role of hh -class genes in gut formation and/or neural differentiation is ancestral to the bilaterians,whereas their role in segmentation evolved secondarily within the Ecdysozoa.

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, we have isolated and characterized the pax2.1 -promoter using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5′ region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1 -expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, which do not normally express endogenous pax2.1 , demonstrates the importance of negative regulation of pax2.1 . Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires eng2 and eng3 as known targets for Pax2.1 regulation. We suggest that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b , both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.

The bipotential Ganglion Mother Cells, or GMCs, in the Drosophila CNS asymmetrically divide to generate two distinct post-mitotic neurons. Here,we show that the midline repellent Slit (Sli), via its receptor Roundabout(Robo), promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes asymmetric division by down regulating two POU proteins, Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric localization of Inscuteable, leading to the asymmetric division of GMC-1. Consistent with this, over-expression of these POU genes in a late GMC-1 causes mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s. Similarly, increasing the dosage of the two POU genes in sli mutant background enhances the penetrance of the RP2 lineage defects whereas reducing the dosage of the two genes reduces the penetrance of the phenotype. These results tie a cell-non-autonomous signaling pathway to the asymmetric division of precursor cells during neurogenesis.