Activation of the Notch signaling pathway segregates the non-skeletogenic mesoderm (NSM) from the endomesoderm during sea urchin embryo development. Subsequently, Notch signaling helps specify the four subpopulations of NSM, and influences endoderm specification. To gain further insight into how the Notch signaling pathway is regulated during these cell specification events, we identified a sea urchin homologue of Numb (LvNumb). Previous work in other model systems showed that Numb functions as a Notch signaling pathway antagonist, possibly by mediating the endocytosis of other key Notch interacting proteins. In this study, we show that the vegetal endomesoderm expresses lvnumb during the blastula and gastrula stages, and that the protein is localized to the presumptive NSM. Injections of lvnumb mRNA and antisense morpholinos demonstrate that LvNumb is necessary for the specification of mesodermal cell types, including pigment cells, blastocoelar cells and muscle cells. Functional analysis of the N-terminal PTB domain and the C-terminal PRR domain of LvNumb shows that the PTB domain, but not the PRR domain, is sufficient to recapitulate the demonstrable function of full-length LvNumb. Experiments show that LvNumb requires an active Notch signal to function during NSM specification and that LvNumb functions in the cells responding to Delta and not in the cells presenting the Delta ligand. Furthermore, injection of mRNA encoding the intracellular domain of Notch rescues the LvNumb morpholino phenotype, suggesting that the constitutive intracellular Notch signal overcomes, or bypasses, the absence of Numb during NSM specification.
Numb; Notch; Delta; Sea urchin; Endomesoderm specification
The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations.
Ena/VASP; Filopodia; Branching; Guidance; Axon; Retina; Xenopus
Fibroblast growth factor (FGF) signalling has been implicated during several phases of early embryogenesis, including the patterning of the embryonic axes, the induction and/or maintenance of several cell lineages and the coordination of morphogenetic movements. Here, we summarise our current understanding of the regulation and roles of FGF signalling during early vertebrate development.
Fibroblast growth factor; Embryogenesis; Mesoderm; Morphogenesis; Patterning; Stem cells
Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.
Sphingosine 1-phosphate; Retinal ganglion cell; Axon pathfinding; Growth cone; Collapse; Guidance cue
Macrophages have a critical function in the recognition and engulfment of dead cells. In some settings, macrophages also actively signal programmed cell death. Here we show that during developmentally scheduled vascular regression, resident macrophages are an obligatory participant in a signaling switch that favors death over survival. This switch occurs when the signaling ligand angiopoietin 2 has the dual effect of suppressing survival signaling in vascular endothelial cells (VECs) and stimulating Wnt ligand production by macrophages. In response to the Wnt ligand, VECs enter the cell cycle and in the absence of survival signals, die from G1 phase of the cell cycle. We propose that this mechanism represents an adaptation to ensure that the macrophage and its disposal capability are on hand when cell death occurs.
Macrophage; Angiopoietin; Wnt; Programmed cell death; Vascular regression; Cell cycle
During testis development, fetal Leydig cells increase their population from a pool of progenitor cells rather than from proliferation of a differentiated cell population. However, the mechanism that regulates Leydig stem cell self-renewal and differentiation is unknown. Here, we show that blocking Notch signaling, by inhibiting γ-secretase activity or deleting the downstream target gene Hairy/Enhancer-of-split 1, results in an increase in Leydig cells in the testis. By contrast, constitutively active Notch signaling in gonadal somatic progenitor cells causes a dramatic Leydig cell loss, associated with an increase in undifferentiated mesenchymal cells. These results indicate that active Notch signaling restricts fetal Leydig cell differentiation by promoting a progenitor cell fate. Germ cell loss and abnormal testis cord formation were observed in both gain- and loss-of-function gonads, suggesting that regulation of the Leydig/interstitial cell population is important for male germ cell survival and testis cord formation.
Notch; Stem cells; Leydig cell; Germ cell; Testis cord; Hes1; Mouse
The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.
Heart development; Conduction system; Purkinje fiber; Mouse; Optical mapping
Sox2 is expressed in developing foregut endoderm, with highest levels in the future esophagus and anterior stomach. By contrast, Nkx2.1 (Titf1) is expressed ventrally, in the future trachea. In humans, heterozygosity for SOX2 is associated with anopthalmiaesophageal-genital syndrome (OMIM 600992), a condition including esophageal atresia (EA) and tracheoesophageal fistula (TEF), in which the trachea and esophagus fail to separate. Mouse embryos heterozygous for the null allele, Sox2EGFP, appear normal. However, further reductions in Sox2, using Sox2LP and Sox2COND hypomorphic alleles, result in multiple abnormalities. Approximately 60% of Sox2EGFP/COND embryos have EA with distal TEF in which Sox2 is undetectable by immunohistochemistry or western blot. The mutant esophagus morphologically resembles the trachea, with ectopic expression of Nkx2.1, a columnar, ciliated epithelium, and very few p63+ basal cells. By contrast, the abnormal foregut of Nkx2.1-null embryos expresses elevated Sox2 and p63, suggesting reciprocal regulation of Sox2 and Nkx2.1 during early dorsal/ventral foregut patterning. Organ culture experiments further suggest that FGF signaling from the ventral mesenchyme regulates Sox2 expression in the endoderm. In the 40% Sox2EGFP/COND embryos in which Sox2 levels are ~18% of wild type there is no TEF. However, the esophagus is still abnormal, with luminal mucus-producing cells, fewer p63+ cells, and ectopic expression of genes normally expressed in glandular stomach and intestine. In all hypomorphic embryos the forestomach has an abnormal phenotype, with reduced keratinization, ectopic mucus cells and columnar epithelium. These findings suggest that Sox2 plays a second role in establishing the boundary between the keratinized, squamous esophagus/forestomach and glandular hindstomach.
Sox2; Nkx2.1; p63; Mouse embryo; Mutant; Foregut development; Tracheoesophageal fistula; Metaplasia
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.
Notch; Hairy-related transcription factor; HRT; HES; Hey; Gridlock; Atrioventricular canal; T-box; Tbx
Previous work has detailed the histological and biochemical changes associated with mammary development and remodeling. We have now made use of gene expression profiling, and in particular of the previously described signatures of cell signaling pathway activation, to explore the events associated with mammary gland development. We find that there is elevated E2F-specific pathway activity prior to lactation and relatively low levels of other important signaling pathways, such as RAS, MYC and SRC. Upon lactation and continuing into the involution phase, these patterns reverse with a dramatic increase in RAS, SRC and MYC pathway activity and a decline in E2F activity. At the end of involution, these patterns return to that of the adult non-lactating mammary gland. The importance of the changes in E2F pathway activity, particularly during the proliferative phase of mammary development, was confirmed through the analysis of mice deficient for various E2F proteins. Taken together, these results reveal a complex pattern of pathway activity in relation to the various phases of mammary gland development.
E2F transcription factor; Mammary gland; Microarray; Mouse
Skeletal syndromes are among the most common birth defects. Vertebrate skeletogenesis involves two major cell types: cartilage-forming chondrocytes and bone-forming osteoblasts. In vitro, both are under the control of retinoic acid (RA), but its exact in vivo effects remained elusive. Here, based on the positional cloning of the dolphin mutation, we have studied the role of the RA-oxidizing enzyme Cyp26b1 during cartilage and bone development in zebrafish. cyp26b1 is expressed in condensing chondrocytes as well as in osteoblasts and their precursors. cyp26b1 mutants and RA-treated wild-type fish display a reduction in midline cartilage and the hyperossification of facial and axial bones, leading to fusions of vertebral primordia, a defect not previously described in the context of RA signaling. Fusions of cervical vertebrae were also obtained by treating mouse fetuses with the specific Cyp26 inhibitor R115866. Together with data on the expression of osteoblast markers, our results indicate that temporal and spatial restriction of RA signaling by Cyp26 enzymes is required to attenuate osteoblast maturation and/or activity in vivo. cyp26b1 mutants may serve as a model to study the etiology of human vertebral disorders such as Klippel-Feil anomaly.
Cyp26b1; Retinoic acid; Bmp2; Cartilage; Bone; Chondrocyte; Osteoblast; Osteopontin; Osterix; Craniofacial development; Vertebra; Zebrafish
This essay is about the 1975 JEEM paper by Françoise Dieterlen-Lièvre (Dieterlen-Lièvre, 1975) and the studies that followed it, which indicated that the adult hematopoietic system in the avian embryo originates, not from the blood islands of the extraembryonic yolk sac as was then believed, but from the body of the embryo itself. Dieterlen-Lièvre’s 1975 paper created a paradigm shift in hematopoietic research, and provided a new and lasting focus on hematopoietic activity within the embryo body.
Alternative splicing (AS) is the primary mechanism by which a limited number of protein coding genes can generate the proteome diversity. We have investigated the role of an alternative splicing factor (ASF), Sfrs1, an arginine/serine (SR) rich-protein family member, during retinal development. Here we report that loss of Sfrs1 function during embryonic retinal development had a profound effect such that it led to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early and mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. In contrast, Sfrs1 was not required for the survival of the neurons generated later, including later born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated AS for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated. In all, this is the first description addressing the function of an ASF in vertebrate retinal development.
CudA, a nuclear protein required for Dictyostelium prespore-specific gene expression, binds in vivo to the promoter of the cotC prespore gene. A 14 nucleotide region of the cotC promoter binds CudA in vitro and ECudA, an Entamoeba CudA homologue, also binds to this site. The CudA and ECudA DNA-binding sites contain a dyad and, consistent with a symmetrical binding site, CudA forms a homodimer in the yeast two-hybrid system. Mutation of CudA binding sites within the cotC promoter reduces expression from cotC in prespore cells. The CudA and ECudA proteins share a 120 amino acid core of homology, and clustered point mutations introduced into two highly conserved motifs within the ECudA core region decrease its specific DNA binding in vitro. This region, the presumptive DNA-binding domain, is similar in sequence to domains in two Arabidopsis proteins and one Oryza protein. Significantly, these are the only proteins in the two plant species that contain an SH2 domain. Such a structure, with a DNA-binding domain located upstream of an SH2 domain, suggests that the plant proteins are orthologous to metazoan STATs. Consistent with this notion, the DNA sequence of the CudA half site, GAA, is identical to metazoan STAT half sites, although the relative positions of the two halves of the dyad are reversed. These results define a hitherto unrecognised class of transcription factors and suggest a model for the evolution of STATs and their DNA-binding sites.
Dictyostelium; CudA; Amoeboza; Plant STATs; SH2 domains
The first axis to be specified during vertebrate development is that between the site where gastrulation will begin and the opposite pole of the embryo (dorso-ventral axis in amphibians and fish, anterior-posterior in amniotes). This relies on Nodal activity, but different vertebrates differ in how this activity is positioned. In chick, the earliest known asymmetry is posterior expression of the TGFβ-related factor Vg1, close to the future Nodal expression domain. Here we show that the transcription factor GATA2 is expressed anteriorly before this stage. GATA2 influences the site of primitive streak formation and its role is independent from, and upstream of, Vg1 and Wnt. However while Vg1 is required for streak formation, GATA2 does not act as an absolute anterior specifier, but provides an anterior bias. These findings point to previously unsuspected global determinants of polarity of the early amniote embryo.
embryonic polarity; GATA factors; chick embryo; gastrulation; primitive streak formation; Nodal; Vg1; GDF-1; embryonic regulation; regeneration
Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active KrasG12D. The KrasG12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature KrasG12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.
Ovary; Ovulation; Granulosa cell; Kras (K-ras); Signal transduction; MKP3 (DUSP6)
The signalling molecule DIF-1 is required for normal cell fate choice and patterning in Dictyostelium. To understand how these developmental processes are regulated will require knowledge of how cells receive and respond to the DIF-1 signal. Previously, we have described a bZIP transcription factor, DimA, which is required for cells to respond to DIF-1. However, it was unknown whether DimA activity is required to activate the DIF response pathway in certain cells or is a component of the response pathway itself. In this study, we describe the identification of a DimA-related bZIP transcription factor, DimB. Rapid changes in the subcellular localisation of both DimA and DimB in response to DIF-1 suggest that they are directly downstream of the DIF-1 signal. Genetic and biochemical interactions between DimA and DimB provides evidence that their ability to regulate diverse targets in response to DIF-1 is partly due to their ability to form homo- and heterodimeric complexes. DimA and DimB are therefore direct regulators of cellular responses to DIF-1.
Dictyostelium; DIF-1; bZIP; DimB
The molecular basis of vertebrate germ layer formation has been the focus of intense scrutiny for decades, and the inductive interactions underlying this process are well-defined; only recently, however, have studies demonstrated that the regulated inhibition of ectopic germ layer formation is also critical for patterning the early vertebrate embryo. We report here the characterization of Xema (Xenopus Ectodermally-expressed Mesendoderm Antagonist), a novel member of the Foxi-subclass of winged-helix transcription factors that is involved in the suppression of ectopic germ layer formation in the frog, Xenopus laevis. Xema transcripts are restricted to the animal pole ectoderm during early Xenopus development. Ectopic expression of Xema RNA inhibits mesoderm induction, both by growth factors and in the marginal zone, in vivo. Conversely, introduction of antisense morpholino oligos directed against the Xema transcript stimulates the expression of a broad range of mesodermal and endodermal marker genes in the animal pole. Our studies demonstrate that Xema is both necessary and sufficient for the inhibition of ectopic mesendoderm in the cells of the presumptive ectoderm, and support a model in which Fox proteins function in part to restrict inappropriate germ layer development throughout the vertebrate embryo.
We use non-invasive muscle imaging to study onset of motor activity and emergence of coordinated movement in Drosophila embryos. Earliest movements are myogenic and neurally controlled muscle contractions first appear with the onset of bursting activity 17 hours after egg laying. Initial episodes of activity are poorly organised and coordinated crawling sequences only begin to appear after a further hour of bursting. Thus network performance improves during this first period of activity. The embryo continues to exhibit bursts of crawling like sequences until shortly before hatching, while other reflexes also mature. Bursting does not begin as a reflex response to sensory input but appears to reflect the onset of spontaneous activity in the motor network. It does not require GABA-ergic transmission, and using a light activated channel to excite the network we demonstrate activity dependent depression that may cause burst termination.
Drosophila; embryo; movement; muscle; coordination; development
Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels due to cell cycle-dependent proteolysis.
Here, we show that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes together with their cell cycle spatiotemporal dynamics strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after Mid Blastula Transition. However functional knockdown of geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos.
Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation.
Cdt1:Geminin; DNA replication; Xenopus embryos
The number of muscle fibers in the vocal organ of the adult male African clawed frog, Xenopus laevis, exceeds that of adult females. This sex difference is the result of rapid fiber addition in males between the end of metamorphosis, post-metamorphic stage 0 (PM0) and PM2. At PM0, male and female frogs have similar numbers of laryngeal muscle fibers. Males then add more muscle fibers than females and achieve an adult value that is 1.7 times the female number. Males castrated at PM0 have the same fiber number as females. Ovariectomy at PM0 does not alter muscle fiber addition in females. Gonadectomy at PM2 has no effect on fiber addition in either sex. Females attain masculine muscle fiber number if their ovaries are replaced with a testis at metamorphosis. Exogenous testosterone treatment at PM0 significantly increases fiber number in females but not in males. Exogenous testosterone given at PM2 has no effect on fiber number in females but decreases fiber number in males. We conclude that the testes are necessary for the marked addition of laryngeal muscle fibers seen in male X. laevis between PM0 and PM2. The masculine pattern of muscle fiber addition can be induced in females provided with a testis. Androgen secretion from the testes most probably accounts for masculinization of laryngeal muscle fiber number. After PM2, androgens are no longer necessary for muscle fiber addition and cannot increase fiber number in females.
myogenesis; androgen; sexual differentiation
In contrast to cyclin D1 nulls (cD1−/−), mice without cyclin D2 (cD2−/−) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2−/− mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2−/− mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2−/− and cD1−/− suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.
Cyclin D2 (Ccnd2); Parvalbumin interneurons; Cell cycle; Cortical excitability
Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf−/−mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.
CCN; CTGF; Chondrogenesis; Angiogenesis; Mutant
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.
Drosophila; Cell signaling; Feedback modulation; Transcriptional repression
The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. While emerging evidence shows that the 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, an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates critical aspects of FGF-dependent OFT development. Ablation of Frs2α 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, Frs2α mutants had defective endothelial-mesenchymal-transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. The results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.
receptor tyrosine kinase; cell signaling; heart development; second heart field; mouse model