In the nematode Caenorhabditis elegans, sperm entry into the oocyte triggers the completion of meiosis and the establishment of the embryonic anteroposterior (AP) axis. How the early embryo makes the transition from a meiotic to a mitotic zygote and coordinates cell cycle changes with axis formation remains unclear. We have discovered roles for the C. elegans puromycin-sensitive aminopeptidase PAM-1 in both cell cycle progression and AP axis formation, further implicating proteolytic regulation in these processes. pam-1 mutant embryos exhibit a delay in exit from meiosis: thus, this peptidase is required for progression to mitotic interphase. In addition, the centrosomes associated with the sperm pronucleus fail to closely associate with the posterior cortex in pam-1 mutants, and the AP axis is not specified. The meiotic exit and polarity defects are separable, as inactivation of the B-type cyclin CYB-3 in pam-1 mutants rescues the meiotic exit delay but not the polarity defects. Thus PAM-1 may regulate CYB-3 during meiotic exit but presumably targets other protein(s) to regulate polarity. We also show that the pam-1 gene is expressed both maternally and paternally, providing additional evidence that sperm-donated gene products have important roles during early embryogenesis in C. elegans. The degradation of proteins through ubiquitin-mediated proteolysis has been previously shown to regulate the cell cycle and AP axis formation in the C. elegans zygote. Our analysis of PAM-1 requirements shows that a puromycin-sensitive aminopeptidase is also required for proteolytic regulation of the oocyte to embryo transition.

Programmed cell death, or apoptosis, plays an important role during normal development, and is disrupted in a range of disease states. Although the key molecular events that occur during apoptosis are well characterized, less is known about the regulatory inputs that influence whether a cell will live or die. Work in mouse and human cells has shown that Pax transcription factors can influence cell death and promote cell survival, but the mechanism for their activity is not clear. Here, we show that two Pax2/5/8-related genes (egl-38 and pax-2) influence both somatic and germline cell death in C. elegans. Using genetic and molecular experiments, we show that the Pax proteins act as transcriptional regulators of ced-9, the C. elegans bcl-2 gene. These results identify a mechanism for Pax2/5/8-mediated regulation of cell death, and underscore the importance of transcriptional regulation of core apoptotic pathway genes in influencing cell survival.

The mouse Kcnq1 imprinted domain is located on distal chromosome 7 and contains several imprinted genes that are paternally repressed. Repression of these genes is regulated by a non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. Maternal repression of Kcnq1ot1 is controlled by DNA methylation originating in the oocyte. Some genes in the region are imprinted only in the placenta, whereas others are imprinted in both extra-embryonic and embryonic lineages. Here, we show that Kcnq1ot1 is paternally expressed in preimplantation embryos from the two-cell stage, and that ubiquitously imprinted genes proximal to Kcnq1ot1 are already repressed in blastocysts, ES cells and TS cells. Repressive histone marks such as H3K27me3 are present on the paternal allele of these genes in both ES and TS cells. Placentally imprinted genes that are distal to Kcnq1ot1, by contrast, are not imprinted in blastocysts, ES or TS cells. In these genes, paternal silencing and differential histone marks arise during differentiation of the trophoblast lineage between E4.5 and E7.5. Our findings show that the dynamics during preimplantation development of gene inactivation and acquisition of repressive histone marks in ubiquitously imprinted genes of the Kcnq1 domain are very similar to those of imprinted X inactivation. By contrast, genes that are only imprinted in the placenta, while regulated by the same non-coding RNA transcript Kcnq1ot1, undergo epigenetic inactivation during differentiation of the trophoblast lineage. Our findings establish a model for how epigenetic gene silencing by non-coding RNA may depend on distance from the non-coding RNA and on lineage and differentiation specific factors.

Myocardin is a transcriptional co-activator of serum response factor (Srf), which is a key regulator of the expression of smooth and cardiac muscle genes. Consistent with its role in regulating cardiovascular development, myocardin is the earliest known marker specific to both the cardiac and smooth muscle lineages during embryogenesis. To understand how the expression of this early transcriptional regulator is initiated and maintained, we scanned 90 kb of genomic DNA encompassing the myocardin gene for cis-regulatory elements capable of directing myocardin transcription in cardiac and smooth muscle lineages in vivo. Here, we describe an enhancer that controls cardiovascular expression of the mouse myocardin gene during mouse embryogenesis and adulthood. Activity of this enhancer in the heart and vascular system requires the combined actions of the Mef2 and Foxo transcription factors. In addition, the Tead transcription factor is required specifically for enhancer activation in neural-crest-derived smooth muscle cells and dorsal aorta. Notably, myocardin also regulates its own enhancer, but in contrast to the majority of myocardin target genes, which are dependent on Srf, myocardin acts through Mef2 to control its enhancer. These findings reveal an Srf-independent mechanism for smooth and cardiac muscle-restricted transcription and provide insight into the regulatory mechanisms responsible for establishing the smooth and cardiac muscle phenotypes during development.

Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components.

In flowering plants, diploid sporophytic tissues in ovules and anthers support meiosis and subsequent haploid gametophyte development. These analogous reproductive functions suggest that common mechanisms may regulate ovule and anther development. Two Arabidopsis Auxin Response Factors, ARF6 and ARF8, regulate gynoecium and stamen development in immature flowers. Wild-type pollen grew poorly in arf6 arf8 gynoecia, correlating with ARF6 and ARF8 expression in style and transmitting tract. ARF6 and ARF8 transcripts are cleavage targets of the microRNA miR167, and overexpressing miR167 mimicked arf6 arf8 phenotypes. Mutations in the miR167 target sites of ARF6 or ARF8 caused ectopic expression of these genes in domains of both ovules and anthers where miR167 was normally present. As a result, ovule integuments had arrested growth, and anthers grew abnormally and failed to release pollen. Thus, miR167 is essential for correct patterning of gene expression, and for fertility of both ovules and anthers. The essential patterning function of miR167 contrasts with cases from animals in which miRNAs reinforce or maintain transcriptionally established gene expression patterns.

The transcription factor COUP-TFI (NR2F1), an orphan member of the nuclear receptor superfamily, is an important regulator of neurogenesis, cellular differentiation and cell migration. In the forebrain, COUP-TFI controls the connectivity between thalamus and cortex and neuronal tangential migration in the basal telencephalon. Here, we show that COUP-TFI is required for proper axonal growth and guidance of all major forebrain commissures. Fibres of the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants. Moreover, hippocampal neurons lacking COUP-TFI have a defect in neurite outgrowth and show an abnormal axonal morphology. To search for downstream effectors, we used microarray analysis and showed that, in the absence of COUP-TFI, expression of various cytoskeleton molecules involved in neuronal morphogenesis is affected. Diminished protein levels of the microtubule-associated protein MAP1B and increased levels of the GTP-binding protein RND2 were confirmed in the developing cortex in vivo and in primary hippocampal neurons in vitro. Therefore, based on morphological studies, gene expression profiling and primary cultured neurons, the present data uncover a previously unappreciated intrinsic role for COUP-TFI in axonal growth in vivo and supply one of the premises for COUP-TFI coordination of neuronal morphogenesis in the developing forebrain.

Chromatin assembly factor CAF-1 facilitates the formation of nucleosomes on newly replicated DNA in vitro. However, the role of CAF-1 in development is poorly understood because mutants are not available in most multicellular model organisms. Biochemical evidence suggests that FASCIATA1, FASCIATA2 and MSI1 form CAF-1 in Arabidopsis thaliana. Because fasciata mutants are viable, CAF-1 is not essential for cell division in plants. Arabidopsis CAF-1 mutants have defects in shoot apical meristems; in addition, CAF-1 is required to establish seedling architecture, leaf size and trichome differentiation. CAF-1 is needed to restrict branching of trichomes on rosette leaves. Increased trichome branching in CAF-1 mutants is not strictly correlated with increased nuclear DNA content. In addition, fas2 glabra3 double mutants show an additive genetic interaction, demonstrating that CAF-1 acts genetically parallel to the GLABRA3-containing, endoreduplication-coupled trichome branching pathway. However, CAF-1 is often needed to restrict endoreduplication, because seedlings of most CAF-1 mutants have increased ploidy. Notably, in the Landsberg erecta background, loss of CAF-1 does not affect ploidy, demonstrating that loss of CAF-1 can be compensated in some Arabidopsis accessions. These results reveal that the functions of FAS1, FAS2 and MSI1 are not restricted to meristems, but are also needed to control genome replication at multiple steps of development.

Cilia are highly conserved organelles that have diverse motility and sensory functions. Recent discoveries have revealed that cilia also have crucial roles in cell signaling pathways and in maintaining cellular homeostasis. As such, defects in cilia formation or function have profound effects on the development of body pattern and the physiology of multiple organ systems. By categorizing syndromes that are due to cilia dysfunction in humans and from studies in vertebrate model organisms, molecular pathways that intersect with cilia formation and function have come to light. Here, we summarize an emerging view that in order to understand some complex developmental pathways and disease etiologies, one must consider the molecular functions performed by cilia.

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal 'trigger', the signaling molecule Hedgehog, is controlled. Here, we show that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field.

The expression of C/EBP, which may govern transcription of mature hepatocyte marker genes, was suppressed in periportal hepatoblasts in mouse liver development, leading to biliary cell differentiation. This study was undertaken to analyze how inactivation of the Cebpa gene affects biliary cell differentiation and gene expression of the regulatory genes for that differentiation, including Hnf1b and Hnf6. In the knockout mouse liver at midgestation stages, pseudoglandular structures were abundantly induced in the parenchyma with elevated expression of Hnf6 and Hnf1b mRNAs. The wild-type liver parenchyma expressed mRNAs of these transcription factors at low levels, though periportal biliary progenitors had strong expression of them. These results suggest that expression of Hnf6 and Hnf1b is downstream of C/EBP action in fetal liver development, and that the suppression of C/EBP expression in periportal hepatoblasts may lead to expression of Hnf6 and Hnf1b mRNAs. Immunohistochemical studies with biliary cell markers in knockout livers demonstrated that differentiated biliary epithelial cells were confined to around the portal veins. The suppression of C/EBP expression may result in upregulation of Hnf6 and Hnf1b gene expression, but be insufficient for biliary cell differentiation. When liver fragments of Cebpa-knockout fetuses, in which hepatoblasts were contained as an endodermal component, were transplanted in the testis of Scid (Prkdc) male mice, almost all hepatoblasts gave rise to biliary epithelial cells. Wild-type hepatoblasts constructed mature hepatic tissue accompanied by biliary cell differentiation. These results also demonstrate that the suppression of C/EBP expression may stimulate biliary cell differentiation.

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination

The NK homeobox gene tinman (tin) is required for the specification of the cardiac, visceral muscle and somatic muscle progenitors in the early dorsal mesoderm of Drosophila. Like its vertebrate counterpart Nkx2.5, the expression of tin is maintained in cardiac cells during cardiac maturation and differentiation; however, owing to the complete lack of a dorsal vessel in tin mutant embryos, the function of tin in these cells has not been defined. Here we show that myocardial cells and dorsal vessels can form even though they lack Tin, and that viable adults can develop, as long as Tin is provided in the embryonic precardiac mesoderm. However, embryos in which tin expression is specifically missing from cardial cells show severe disruptions in the normal diversification of the myocardial cells, and adults exhibit severe defects in cardiac remodeling and function. Our study reveals that the normal expression and activity of Tin in four of the six bilateral cardioblasts within each hemisegment of the heart allows these cells to adopt a cell fate as 'working' myocardium, as opposed to a fate as inflow tract (ostial) cells. This function of tin involves the repression of Dorsocross (Doc) T-box genes and, hence, the restriction of Doc to the Tin-negative cells that will form ostia. We conclude that tin has a crucial role within myocardial cells that is required for the proper diversification, differentiation, and post-embryonic maturation of cardiomyocytes, and we present a pathway involving regulatory interactions among seven-up, midline, tinman and Dorsocross that establishes these developmental events upon myocardial cell specification.

Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina.

Shroom family proteins have been implicated in the control of the actin cytoskeleton, but so far only a single family member has been studied in the context of developing embryos. Here, we show that the Shroom-family protein, Shroom2 (previously known as APXL) is both necessary and sufficient to govern the localization of pigment granules at the apical surface of epithelial cells. In Xenopus embryos that lack Shroom2 function, we observed defects in pigmentation of the eye that stem from failure of melanosomes to mature and to associate with the apical cell surface. Ectopic expression of Shroom2 in naïve epithelial cells facilitates apical pigment accumulation, and this activity specifically requires the Rab27a GTPase. Most interestingly, we find that Shroom2, like Shroom3 (previously called Shroom), is sufficient to induce a dramatic apical accumulation of the microtubule-nucleating protein -tubulin at the apical surfaces of naïve epithelial cells. Together, our data identify Shroom2 as a central regulator of RPE pigmentation, and suggest that, despite their diverse biological roles, Shroom family proteins share a common activity. Finally, because the locus encoding human SHROOM2 lies within the critical region for two distinct forms of ocular albinism, it is possible that SHROOM2 mutations may be a contributing factor in these human visual system disorders.

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice we found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions.

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (TCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of TCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by TCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.

The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. Here, we examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism - rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation - controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted 'landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). We believe that this is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting.

Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, we identified piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. We further show that loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a muscle-derived attractant. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process.

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, we have established the long germ parasitic wasp Nasonia vitripennis as a model for comparative studies with Drosophila. Here we report that, in Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudal mRNA. Loss of caudal function in Nasonia results in severe segmentation defects. We show that Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies, together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila.

Tudor domains are found in many organisms and have been implicated in protein-protein interactions in which methylated protein substrates bind to these domains. Here, we present evidence for the involvement of specific Tudor domains in germline development. Drosophila Tudor, the founder of the Tudor domain family, contains 11 Tudor domains and is a component of polar granules and nuage, electron-dense organelles characteristic of the germline in many organisms, including mammals. In this study, we investigated whether the 11 Tudor domains fulfil specific functions for polar granule assembly, germ cell formation and abdomen formation. We find that even a small number of non-overlapping Tudor domains or a substantial reduction in overall Tudor protein is sufficient for abdomen development. In stark contrast, we find a requirement for specific Tudor domains in germ cell formation, Tudor localization and polar granule architecture. Combining genetic analysis with structural modeling of specific Tudor domains, we propose that these domains serve as 'docking platforms' for polar granule assembly.

The -tubulin ring complex (TuRC) forms an essential template for microtubule nucleation in animal cells. The molecular composition of the TuRC has been described; however, the functions of the subunits proposed to form the cap structure remain to be characterized in vivo. In Drosophila, the core components of the TuRC are essential for mitosis, whereas the cap component Grip75 is not required for viability but functions in bicoid RNA localization during oogenesis. The other cap components have not been analyzed in vivo. We report the functional characterization of the cap components Grip128 and Grip75. Animals with mutations in Dgrip128 or Dgrip75 are viable, but both males and females are sterile. Both proteins are required for the formation of distinct sets of microtubules, which facilitate bicoid RNA localization during oogenesis, the formation of the central microtubule aster connecting the meiosis II spindles in oocytes and cytokinesis in male meiosis. Grip75 and Grip128 anchor the axoneme at the nucleus during sperm elongation. We propose that Grip75 and Grip128 are required to tether microtubules at specific microtubule-organizing centers, instead of being required for general microtubule nucleation. The TuRC cap structure may be essential only for non-centrosome-based microtubule functions.

Meiosis is a specialized cell cycle limited to the gametes in Metazoa. In Drosophila, oocyte determination and meiosis control are interdependent processes, and BicD appears to play a key role in both. However, the exact mechanism of how BicD-dependent polarized transport could influence meiosis and vice versa remains an open question. In this article, we report that the cell cycle regulatory kinase Polo binds to BicD protein during oogenesis. Polo is expressed in all cells during cyst formation before specifically localizing to the oocyte. This is the earliest known example of asymmetric localization of a cell-cycle regulator in this process. This localization is dependent on BicD and the Dynein complex. Loss- and gain-of-function experiments showed that Polo has two independent functions. On the one hand, it acts as a trigger for meiosis. On the other hand, it is independently required, in a cell-autonomous manner, for the activation of BicD-dependent transport. Moreover, we show that Polo overexpression can rescue a hypomorphic mutation of BicD by restoring its localization and its function, suggesting that the requirement for Polo in polarized transport acts through regulation of BicD. Taken together, our data indicate the existence of a positive feedback loop between BicD and Polo, and we propose that this loop represents a functional link between oocyte specification and the control of meiosis.

Leaf development in higher plants requires the specification of leaf initials at the flanks of a pluripotent structure termed the shoot apical meristem. In Arabidopsis, this process is facilitated by negative interactions between class I KNOTTED1-like homeobox (KNOX) and ASYMMETRIC LEAVES1 (AS1) transcription factors, such that KNOX proteins are confined to the meristem and AS1 to leaf initials. Sites of leaf inception are also defined by local accumulation of the hormone auxin; however, it is unknown how auxin and AS1 activities are integrated to control leaf development. Here, we show that auxin and AS1 pathways converge to repress expression of the KNOX gene BREVIPEDICELLUS (BP) and thus promote leaf fate. We also demonstrate that regulated auxin gradients control leaf shape in a KNOX-independent fashion and that inappropriate KNOX activity in leaves perturbs these gradients, hence altering leaf shape. We propose that regulatory interactions between auxin, AS1 and KNOX activities may both direct leaf initiation and sculpt leaf form.

Tissue-restricted transcription factors (TFs), which confer specialized cellular properties, are usually identified through sequence homology or cis-element analysis of lineage-specific genes; conventional modes of mRNA profiling often fail to report non-abundant TF transcripts. We evaluated the dynamic expression during mouse gut organogenesis of 1381 transcripts, covering nearly every known and predicted TF, and documented the expression of approximately 1000 TF genes in gastrointestinal development. Despite distinctive structures and functions, the stomach and intestine exhibit limited differences in TF genes. Among differentially expressed transcripts, a few are virtually restricted to the digestive tract, including Nr2e3, previously regarded as a photoreceptor-specific product. TFs that are enriched in digestive organs commonly serve essential tissue-specific functions, hence justifying a search for other tissue-restricted TFs. Computational data mining and experimental investigation focused interest on a novel homeobox TF, Isx, which appears selectively in gut epithelium and mirrors expression of the intestinal TF Cdx2. Isx-deficient mice carry a specific defect in intestinal gene expression: dysregulation of the high density lipoprotein (HDL) receptor and cholesterol transporter scavenger receptor class B, type I (Scarb1). Thus, integration of developmental gene expression with biological assessment, as described here for TFs, represents a powerful tool to investigate control of tissue differentiation.