We employ non-radioactive in situ hybridization techniques, which combine good tissue morphology preservation with high sensitivity of transcript detection, to map gene expression in the regenerating digestive tube of the sea cucumber Holothuria glaberrima. We investigated localization of transcripts of Wnt9, TCTP, Bmp1/Tll, the genes that have been previously known to be implicated in embryogenesis and cancer. The choice was determined by our long-term goal of trying to understand how the developmental regulatory pathways known to be involved in tumor development can be activated in post-traumatic regeneration without leading to malignant growth. The gene expression data combined with the available morphological information highlight the gut mesothelium (the outer layer of the digestive tube) as a highly dynamic tissue, whose cells undergo remarkable changes in their phenotype and gene expression in response to injury. This reversible transition of the gut mesothelium from a complex specialized tissue to a simple epithelium composed of rapidly proliferating multipotent cells seems to depend on the expression of genes from multiple developmental/cancer-related pathways.
The specification of temporal identity within single progenitor lineages is essential to generate functional neuronal diversity in Drosophila and mammals. In Drosophila, four transcription factors are sequentially expressed in neural progenitors (neuroblasts) and each regulates the temporal identity of the progeny produced during its expression window. The first temporal identity is established by the Ikaros-family zinc finger transcription factor Hunchback (Hb). Hb is detected in young (newly-formed) neuroblasts for about an hour and is maintained in the early-born neurons produced during this interval. Hb is necessary and sufficient to specify early-born neuronal or glial identity in multiple neuroblast lineages. The timing of hb expression in neuroblasts is regulated at the transcriptional level. Here we identify the cis-regulatory elements that confer proper hb expression in “young” neuroblasts and early-born neurons. We show that the neuroblast element contains clusters of predicted binding sites for the Seven-up transcription factor, which is known to limit hb neuroblast expression. We identify highly conserved sequences in the neuronal element that are good candidates for maintaining Hb transcription in neurons. Our results provide the necessary foundation for identifying trans-acting factors that establish the Hb early temporal expression domain.
Tbx2 and Tbx3 are closely related members of the T-box family of transcription factors that are important regulators during normal development as well as major contributors to human developmental syndromes when mutated. Although there is evidence for the involvement of Tbx2 and Tbx3 in pancreatic cancer, so far there are no reports characterizing the normal expression pattern of these genes in the pancreas. In this study, we examined spatial and temporal expression of Tbx2 and Tbx3 in mouse pancreas during development and in the adult using in situ hybridization and immunohistochemistry. Our results show that Tbx2 and Tbx3 are both expressed in the pancreatic mesenchyme throughout development beginning at embryonic day (E) 9.5. In addition, Tbx2 is expressed in pancreatic vasculature during development and in epithelial-derived endocrine and ductal cells during late fetal stages, postnatal development and in adult pancreas. In contrast, Tbx3 is expressed in exocrine tissue in the postnatal and adult pancreas. Further our results demonstrate that Tbx2 and Tbx3 are expressed in tumor-derived endocrine and exocrine cell lines, respectively. These dynamic changes in the expression pattern of these transcription factors lay the foundation for investigation of potential roles in pancreas development.
Tbx2; Tbx3; T-box; Pancreas; Islets
Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. In this paper, we demonstrate that sim is expressed in 3 clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in 3 clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which we term PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim+ neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5’-flanking, intronic, and 3’-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim+ brain cells, including all sim+ neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim+ brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim+ brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim+ brain neurons. Sequence analysis of existing sim mutant alleles identified 3 likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate Sim that have an early role in neuronal migration and a later role in axonogenesis.
Autoregulation; Axon guidance; Brain; Drosophila; Enhancer; Optic lobes; single-minded
The zebrafish is an ideal model for elucidating the cellular and molecular mechanisms that underlie development of the peripheral nervous system. A transgenic line that selectively labels all the sensory circuits would be a valuable tool for such investigations. In this study, we describe such a line: the enhancer trap zebrafish line Tg(SKIV2L2:gfp)j1775 which expresses green fluorescent protein (gfp) in the peripheral sensory ganglia. We show that this transgene marks all peripheral ganglia and sensory nerves, beginning at the time when the neurons are first extending their processes, but does not label the efferent nerves. The trapped reporter is inserted just upstream of a previously poorly described gene: lhfpl4 on LG6. The expression pattern of this gene by in situ hybridization reveals a different, but overlapping, pattern of expression compared to that of the transgene. This pattern also does not mimic that of the gene (skiv2l2), which provided the promoter element in the construct. These findings indicate that reporter expression is not dictated by an endogenous enhancer element, but instead arises through an unknown mechanism. Regardless, this reporter line should prove to be a valuable tool in the investigation of peripheral nervous system formation in the zebrafish.
Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.
Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are functionally important during vertebrate development. We have generated a zebrafish genetrap line that produces fluorescently tagged Crmp1 protein, which can be dynamically tracked in living fish at subcellular resolution. The results show that Crmp1 is expressed in numerous sites in the developing nervous system. Early expression is apparent in the forebrain, epiphysis, optic tectum and the developing spinal cord. In the larval brain, Crmp1 is expressed in several distinct brain regions, such as the telencephalon, habenula and cerebellum. In addition, it is expressed in the spinal cord in a manner that persists in the larva. The results suggest that this Crmp1 protein trap line offers a powerful tool to track selected neuronal populations at high resolution.
Crmp1; zebrafish; nervous system; development
Neuronal migration and growth cone motility are essential aspects of the development and maturation of the nervous system. These cellular events result from dynamic changes in the organization and function of the cytoskeleton, in part due to the activity of cytoskeletal motor proteins such as myosins. Although specific myosins such as Myo2 (conventional or muscle myosin), Myo1, and Myo5 have been well characterized for roles in cell motility, the roles of the majority of unconventional (other than Myo2) myosins in cell motility events have not been investigated. To address this issue, we have undertaken an analysis of unconventional myosins in zebrafish, a premier model for studying cellular and growth cone motility in the vertebrate nervous system. We describe the characterization and expression patterns of several members of the unconventional myosin gene family. Based on available genomic sequence data, we identified 18 unconventional myosin- and 4 Myo2-related genes in the zebrafish genome in addition to previously characterized myosin (-1, -2, -3, -5, -6, -7) genes. Phylogenetic analyses indicate that these genes can be grouped into existing classifications for unconventional myosins from mouse and man. In situ hybridization analyses using EST probes for 18 of the 22 identified genes indicate that 11/18 genes are expressed in a restricted fashion in the zebrafish embryo. Specific myosins are expressed in particular neuronal or neuroepithelial cell types in the developing zebrafish nervous system, spanning the periods of neuronal differentiation and migration, and of growth cone guidance and motility.
cytoskeleton; unconventional myosin; neuronal migration; axon guidance; growth cone motility; zebrafish; commissure; spinal cord; motor neuron; neural crest; somite; ear; eye; morphogenesis; in situ hybridization; phylogenetic tree; hindbrain; forebrain; midbrain; cranial muscles
The Lim-kinase (LIMK) proteins are important for the regulation of the actin cytoskeleton, in particular the control of actin nucleation and depolymerisation via regulation of cofilin, and hence may control a large number of processes during development, including cell tensegrity, migration, cell cycling, and axon guidance. LIMK1/LIMK2 knockouts disrupt spinal cord morphogenesis and synapse formation but other tissues and developmental processes that require LIMK are yet to be fully determined. To identify tissues and cell-types that may require LIMK, we characterised the pattern of LIMK1 protein during mouse embryogenesis. We showed that LIMK1 displays an expression pattern that is temporally dynamic and tissue-specific. In several tissues LIMK1 is detected in cell-types that also express Wilms’ tumour protein 1 and that undergo transitions between epithelial and mesenchymal states, including the pleura, epicardium, kidney nephrons, and gonads. LIMK1 was also found in a subset of cells in the dorsal retina, and in mesenchymal cells surrounding the peripheral nerves. This detailed study of the spatial and temporal expression of LIMK1 shows that LIMK1 expression is more dynamic than previously reported, in particular at sites of tissue–tissue interactions guiding multiple developmental processes.
Limk; Kidney; Heart; Epithelia-to-mesenchyme transition; Mesenchyme-to-epithelia transition; Eye; Testes
During embryogenesis, the rhombic lip of the fourth ventricle is the germinal origin of a diverse collection of neuronal populations that ultimately reside in the brainstem and cerebellum. Rhombic lip neurogenesis requires the bHLH transcription factor Atoh1 (Math1), and commences shortly after neural tube closure (E9.5). Within the rhombomere 1 – isthmus region, the rhombic lip first produces brainstem and deep cerebellar neurons (E9.5-E12), followed by granule cell precursors after E12. While Atoh1 function is essential for all of these populations to be specified, the downstream genetic programs that confer specific properties to early and late born Atoh1 lineages are not well characterized. We have performed a comparative microarray analysis of gene expression within early and later born cohorts of Atoh1 expressing neural precursors purified from E14.5 embryos using a transgenic labeling strategy. We identify novel transcription factors, cell surface molecules, and cell cycle regulators within each pool of Atoh1 lineages that likely contribute to their distinct developmental trajectories and cell fates. In particular, our analysis reveals new insights into the genetic programs that regulate the specification and proliferation of granule cell precursors, the putative cell of origin for the majority of medulloblastomas.
rhombic lip; Atoh1; Math1; cerebellum; neurogenesis; rhombomere 1
Dystrophin/dystrobrevin superfamily proteins play structural and signalling roles at the plasma membrane of many cell types. Defects in them or the associated multiprotein complex cause a range of neuromuscular disorders. Members of the dystrophin branch of the family form heterodimers with members of the dystrobrevin branch, mediated by their coiled-coil domains. To determine which combinations of these proteins might interact during embryonic development, we set out to characterise the gene expression pattern of dystrophin and dystrobrevin family members in zebrafish. γ-dystrobrevin (dtng), a novel dystrobrevin recently identified in fish, is the predominant form of dystrobrevin in embryonic development. Dtng and dmd (dystrophin) have similar spatial and temporal expression patterns in muscle, where transcripts are localized to the ends of differentiated fibres at the somite borders. Dtng is expressed in the notochord while dmd is expressed in the chordo-neural hinge and then in floor plate and hypochord. In addition, dtng is dynamically expressed in rhombomeres 2 and 4-6 of the hindbrain and in the ventral midbrain. α-dystrobrevin (dtna) is expressed widely in the brain with particularly strong expression in the hypothalamus and the telencephalon; drp2 is also expressed widely in the brain. Utrophin expression is found in early pronephros and lateral line development and utrophin and dystrophin are both expressed later in the gut. β-dystrobrevin (dtnb) is expressed in the pronephric duct and widely at low levels. In summary, we find clear instances of co-expression of dystrophin and dystrobrevin family members in muscle, brain and pronephric duct development and many examples of strong and specific expression of members of one family but not the other, an intriguing finding given the presumed heterodimeric state of these molecules.
muscle; zebrafish; notochord; midbrain; rhombomere; gene expression; utrophin; dystrophin; dystrobrevin; drp2; dystrotelin
The vestigial gene has been shown to control skeletal muscle formation in Drosophila and the related Vestigial-like 2 (Vgl-2) protein plays a similar role in mice. Vgl-family proteins are thought to regulate tissue-specific gene expression by binding to members of the broadly expressed Scalloped/Tef/TEAD transcription factor family. Zebrafish have at least four Vgl genes, including two Vgl-2s, and at least three TEAD genes, including two Tead3s. We describe the cloning and expression of one member from each family in the zebrafish. A novel gene, vgl-2b, with closest homology to mouse and human vgl-2, is expressed transiently in nascent notochord and in muscle fibres as they undergo terminal differentiation during somitogenesis. Muscle cells also express a TEAD-3 homologue, a possible partner of Vgl-2b, during myoblast differentiation and early fibre assembly. Tead3a is also expressed in rhombomeres, eye and epiphysis regions.
muscle; adaxial; zebrafish; vestigial-like; transcription enhancer factor; TEAD domain
► We studied the expression of Pxn, Kcna10 and Odf2 in the developing mouse inner ear. ► We covered several ages between E14.5 and P5, and also looked at adults. ► Pxn is a focal adhesion protein expressed strongly in pillar cells. ► Kcna10 is a potassium channel expressed in hair cells. ► Odf2 (Cenexin) marks dendrites extending to and contacting hair cells.
The development of the organ of Corti and the highly specialized cells required for hearing involves a multitude of genes, many of which remain unknown. Here we describe the expression pattern of three genes not previously studied in the inner ear in mice at a range of ages both embryonic and early postnatal. Kcna10, a tetrameric Shaker-like potassium channel, is expressed strongly in the hair cells themselves. Odf2, as its centriolar isoform Cenexin, marks the dendrites extending to and contacting hair cells, and Pxn, a focal adhesion scaffold protein, is most strongly expressed in pillar cells during the ages studied. The roles of these genes are yet to be elucidated, but their specific expression patterns imply potential functional significance in the inner ear.
Odf2; Cenexin; Pxn; Kcna10; Inner ear
In most animals, the Antero-Posterior (A-P) axis requires a gradient of Wnt signaling. Wnts are expressed posteriorly in many vertebrate and invertebrate embryos, forming a gradient of canonical Wnt/β-Catenin activity that is highest in the posterior and lowest in the anterior. One notable exception to this evolutionary conservation is in the Drosophila embryo, in which the A-P axis is established by early transcription factors of maternal origin. Despite this initial axial establishment, Drosophila still expresses Wingless (Wg), the main Drosophila Wnt homologue, in a strong posterior band early in embryogenesis. Since its discovery 30 years ago this posterior band of Wg has been largely ignored. In this study, we re-examined the onset of expression of the Wg posterior band in relation to the expression of Wg in other segments, and compared the timing of its expression to that of axial regulators such as gap and pair-rule genes. It was found that the posterior band of Wg is first detected in blastoderm at mid nuclear cycle 14, before the segment-polarity stripes of Wg are formed in other segments. The onset of the posterior band of Wg expression was preceded by that of the gap gene products Hunchback (hb) and Krüppel (Kr), and the pair-rule protein Even-skipped (Eve). Although the function of the posterior band of Wg was not analyzed in this study, we note that in temperature-sensitive Wg mutants, in which Wg is not properly secreted, the posterior band of Wg expression is diminished in strength, indicating a positive feedback loop required for Wg robust expression at the cellular blastoderm stage. We propose that this early posterior expression could play a role in the refinement of A-P patterning.
Wnt; antero-posterior patterning; gap genes; pair-rule genes; evo-devo
Sizn1 (Zcchc12) is a transcriptional co-activator that positively modulates BMP (Bone Morphogenic Protein) signaling through its interaction with Smad family members and CBP. We have demonstrated a role for Sizn1 in basal forebrain cholinergic neuron specific gene expression. Furthermore, mutations in SIZN1 have been associated with X-linked mental retardation. Given the defined role of SIZN1 in mental retardation, knowing its complete forebrain expression pattern is essential to further elucidating its role in cognition. To better define the dynamic expression pattern of Sizn1 during forebrain development, we investigated its expression in mouse brain development from embryonic day 8.0 (E8.0) to adult. We found that Sizn1 is primarily restricted to the ventral forebrain including the medial ganglionic eminence, the septum, amygdala, and striatum. In addition, Sizn1 expression is detected in the cortical hem and Pallial-subpallial boundary (PSB; anti-hem); both sources of Cajal-Retzius cells. Sizn1 expression in the dorsal forebrain is restricted to a subset of cells in the marginal zone that also express Reln, indicative of Cajal-Retzius cells. These data provide novel information on brain regions and cell types that express Sizn1, facilitating further investigations into the function of Sizn1 in both development and the pathogenesis of mental retardation.
LIM-Homeodomain genes encode a family of proteins defined by the cysteine-rich protein/protein interacting (Lin-11, Isl-1, and Mec-3) LIM domain and a highly conserved DNA-binding domain. Studies in several organisms have shown that these transcriptional regulators control multiple aspects of embryonic development and are responsible for the pathogenesis of several human diseases. Here we report the expression of Islet-1 (Isl-1) in the gastrointestinal epithelium in developing and adult mice. At embryonic day (E) 9.5–10.5, Isl-1 expression was first detected in the ventral gastric mesenchyme, and expression in the dorsal mesenchyme initiated a few days later. Isl-1 expression was first observed in the gastric epithelium at E13.5 and at E14.5 was restricted to the posterior half of the stomach. In the mature stomach, Isl-1 expression was detected only in subsets of enteroendocrine cells. Furthermore, Isl-1 expression in the intestinal epithelium was first detected at E15.5 and was restricted to subpopulations of enteroendocrine cells in adult mice. These expression analyses suggest that Isl-1 might have an early broad role in stomach and intestinal cells and a secondary role in terminal differentiation and/or maintenance of mature enteroendocrine subtypes in the gastrointestinal epithelium.
Islet-1; stomach; intestine; gastrointestinal tract; development; transcriptional control; endocrine cell differentiation; somatostatin; gastrin; ghrelin
Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.
stem cell; cell lineage; embryo; trophoblast
Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24 hours that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.
Clock genes; Tooth development; Bmal1; Clock; Per1; Per2; expression pattern; immunohistochemistry
Nonmuscle myosin II (myosin hereafter) has well-established roles in generating contractile force on actin filaments during morphogenetic processes in all metazoans. Myosin activation is regulated by phosphorylation of the myosin regulatory light chain (MRCL, encoded by spaghetti squash or sqh in Drosophila) first on Ser21 and subsequently on Thr20. These phosphorylation events are positively controlled by a variety of kinases including myosin light chain kinase, Rho kinase, citron kinase, and AMP kinase and are negatively regulated by myosin phosphatase. The activation of myosin is thus highly regulated and likely developmentally controlled. In order to monitor the activity of myosin during development, we have generated antibodies against the monophosphorylated (Sqh1P) and diphosphorylated (Sqh2P) forms of Sqh. We first show that the antibodies are highly specific. We then used these antibodies to monitor myosin activation in wild type Drosophila tissues. Interestingly, Sqh1P and Sqh2P show distinct patterns of expression in embryos. Sqh1P is expressed nearly ubiquitously and outlines cells consistent with a junctional localization, whereas Sqh2P is strongly expressed on the apical surfaces and in filopodia of tissues undergoing extensive cell shape change or cell movements including the invaginating fore- and hindgut, the invaginating tracheal system, the dorsal pouch and the dorsal most row of epidermal (DME) cells during dorsal closure. In imaginal discs, Sqh1P predominantly localizes in the adherens junction, whereas Sqh2P locates to the apical domain. These antibodies thus have the potential to be very useful in monitoring myosin activation for functional studies of morphogenesis in Drosophila.
Drosophila; myosin; myosin regulatory light chain; spaghetti squash; dorsal closure; morphogenesis
Dysregulation of the transcription factor CRTC1 by a t(11;19) chromosomal rearrangement mediates the formation of mucoepidermoid salivary gland carcinoma (MEC). Although the Crtc1 promoter is consistently active in fusion-positive MEC and low levels of Crtc1 transcripts have been reported in normal adult salivary glands, the distribution of CRTC1 protein in the normal salivary gland is not known. The aim of this study was to determine if CRTC1, like many known oncogenes, is expressed during early submandibular salivary gland (SMG) development and re-expressed in an experimental tumor model. Our results indicate that CRTC1 protein is expressed in SMG epithelia during early stages of morphogenesis, disappears with differentiation, and reappears in initial tumor-like pathology. This stage-dependent expression pattern suggests that CRTC1 may play a role during embryonic SMG branching morphogenesis but not for pro-acinar/acinar differentiation, supporting a precursor cell origin for MEC tumorigenesis. Moreover, the coincident expression of CRTC1 protein and cell proliferation markers in tumor-like histopathology suggests that CRTC1-mediated cell proliferation may contribute, in part, to initial tumor formation.
CREB coactivator; CRTC1; salivary glands; development; tumorigenesis; cell proliferation; mucoepidermoid carcinoma
The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2 were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.
The critical contribution of the Notch signaling pathway to vascular morphogenesis has been underscored by loss-of-function studies in mouse and zebrafish. Nonetheless, a comprehensive understanding as to how this signaling system influences the formation of blood vessels at the cellular and molecular level is far from reached. Here, we provide a detailed analysis of the distribution of active Notch1 in relation to its DSL (Delta, Serrate, Lag2) ligands, Jagged1, Delta-like1, and Delta-like4, during progressive stages of vascular morphogenesis and maturation. Important differences in the cellular distribution of Notch ligands were found. Jagged1 (Jag1) was detected in “stalk cells” of the leading vasculature and at arterial branch points, a site where Delta-like4 (Dll4) was clearly absent. Dll4 was the only ligand expressed in “tip cells” at the end of the growing vascular sprouts. It was also present in stalk cells, capillaries, arterial endothelium, and in mural cells of mature arteries in a homogenous manner. Delta-like1 (Dll1) was observed in both arteries and veins of the developing network, but was also excluded from mature arterial branch points. These findings support alternative and distinct roles for Notch ligands during the angiogenic process.
arteries; blood vessels; capillaries; delta-like1; delta-like4; endothelial; jagged1; vascular remodeling; vasculature; veins
Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler’s, and Marshall’s syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.
minor fibrillar collagens; zebrafish; development; in situ hybridization; Danio rerio
Expression of astrocyte elevated gene-1 (AEG-1) is elevated in multiple human cancers including brain tumors, neuroblastomas, melanomas, breast cancers, non-small cell lung cancers, liver cancers, prostate cancers, and esophageal cancers. This gene plays crucial roles in tumor cell growth, invasion, angiogenesis and progression to metastasis. In addition, over-expression of AEG-1 protects primary and transformed cells from apoptosis-inducing signals by activating PI3K-Akt signaling pathways. These results suggest that AEG-1 is intimately involved in tumorigenesis and may serve as a potential therapeutic target for various human cancers. However, the normal physiological functions of AEG-1 require clarification. We presently analyzed the expression pattern of AEG-1 during mouse development. AEG-1 was expressed in mid-to-hindbrain, fronto-nasal processes, limbs, and pharyngeal arches in the early developmental period from E8.5 to E9.5. In addition, at stages of E12.5-E18.5 AEG-1 was localized in the brain, and olfactory and skeletal systems suggesting a role in neurogenesis, as well as in skin, including hair follicles, and in the liver, which are organ sites in which AEG-1 has been implicated in tumor development and progression. AEG-1 co-localized with Ki-67, indicating a role in cell proliferation, as previously revealed in tumorigenesis. Taken together, these results suggest that AEG-1 may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation, and that temporal regulation of AEG-1 expression may be required during specific stages and in specific tissues during development.
AEG-1; development; mouse embryo; cell proliferation; cancer