Posterior Lateral Plate Mesoderm Research Articles

Hypoxia-sonic hedgehog axis as a driver of primitive hematopoiesis development and evolution in cavefish

The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and exhibit a subset of phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish.

Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.

The Cdx transcription factors and retinoic acid play parallel roles in antero-posterior position of the pectoral fin field during gastrulation

The molecular regulators that determine the precise position of the vertebrate limb along the anterio-posterior axis have not been identified. One model suggests that a combination of hox genes in the lateral plate mesoderm (LPM) promotes formation of the limb field, however redundancy among duplicated paralogs has made this model difficult to confirm. In this study, we identify an optimal window during mid-gastrulation stages when transient mis-regulation of retinoic acid signaling or the caudal related transcription factor, Cdx4, both known regulators of hox genes, can alter the position of the pectoral fin field. We show that increased levels of either RA or Cdx4 during mid-gastrulation are sufficient to rostrally shift the position of the pectoral fin field at the expense of surrounding gene expression in the anterior lateral plate mesoderm (aLPM). Alternatively, embryos deficient for both Cdx4 and Cdx1a (Cdx-deficient) form pectoral fins that are shifted towards the posterior and reveal an additional effect on size of the pectoral fin buds. Prior to formation of the pectoral fin buds, the fin field in Cdx-deficient embryos is visibly expanded into the posterior LPM (pLPM) region at the expense of surrounding gene expression. The effects on gene expression immediately post-gastrulation and during somitogenesis support a model where RA and Cdx4 act in parallel to regulate the position of the pectoral fin. Our transient method is a potentially useful model for studying the mechanisms of limb positioning along the AP axis.

Dual role of Jam3b in early hematopoietic and vascular development.

In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.

Localization of β-Catenin and Islet in the Pelvic Fin Field in Zebrafish.

In zebrafish, pelvic fin buds appear at 3 weeks post fertilization (wpf) during the larval to juvenile transition (metamorphosis), but their fate is already determined during embryogenesis. Thus, presumptive pelvic fin cells appear to memorize their positional information for three weeks, but no factors expressed in the pelvic fin field from the embryonic to the metamorphic stages have been identified. In mice, Islet1 is proposed to promote nuclear accumulation of β-catenin in the hindlimb field, which leads to the initiation of hindlimb bud outgrowth through activation of the Wnt/βcatenin pathway. Here, we examined the distribution of β-catenin and islet proteins in the pelvic fin field of zebrafish from the embryonic to the metamorphic stages. We found that transcripts of islet2a, but not islet1, are detected in the posterior lateral plate mesoderm, including the presumptive pelvic fin field, at the embryonic stage as well as in the pelvic fin bud at the metamorphic stage. Immunolocalization revealed that β-catenin and islet proteins, which are synthesized during the embryonic stage, remain in the cytoplasm of the presumptive pelvic fin cells during the larval stage, and are then translocated into the nuclei of the pelvic fin bud at the metamorphic stage. We propose that cytoplasmic localization of these proteins in the presumptive pelvic fin cells that remained during the larval stage may underlie the mechanism by which pelvic fin cells memorize their positional information from the embryonic stage to the metamorphic stage.

Rap1b Promotes Notch-Signal-Mediated Hematopoietic Stem Cell Development by Enhancing Integrin-Mediated Cell Adhesion

Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) within the ventral portion of the dorsal aorta during vertebrate development. In zebrafish, Notch signaling induces HE specification from posterior lateral plate mesoderm (PLPM) cells as they migrate over the ventral surface of the somite. During migration, PLPM cells make close contact with Notch-ligand-expressing somitic cells to acquire HE identity. Herein, we show in zebrafish that the small GTPase Rap1b regulates HSC development by potentiating Notch-mediated HE specification. PLPM cells migrate toward the midline alongthe somite boundary where fibronectin accumulates. Rap1b stimulates integrin β1 to enhance PLPM cell adhesion to fibronectin localized at thesomite boundary. Rap1b-induced integrin-β1-mediated adhesion to fibronectin leads to the spreading of PLPM cells to facilitate their physical contact with the Notch-ligand-expressing somitic cells, thereby promoting Notch-mediated HE specification. Thus, we have revealed an unexpected role of Rap1-induced integrin-mediated cell adhesion in HSC development.

3078 - Jam3b Regulates Hemato-Vascular Development Through the Repression of the Erk Signaling Pathway

Hematopoietic and endothelial cells develop in close proximity to each other within the mesoderm. It has been proposed that these two cell types share a common precursor, termed the hemangioblast. The zebrafish cloche mutant, in which both hematopoietic and endothelial development are defective, has been identified as a unique model for the study of hemangioblast development. Recently, it was determined that the cloche gene encodes a PAS-domain-containing bHLH transcription factor, Npas4l, and plays a central role in the regulation of hemangioblast specification. Here, we show in zebrafish that Junctional adhesion molecule 3b (Jam3b) plays dual roles in hemato-vascular development during early embryogenesis: (1) promotes hemato-vascular differentiation via the up-regulation of npas4l, and (2) confers the hematopoietic identity in endothelial precursors through the regulation of Leucine rich-repeat containing 15 (Lrrc15). Mutation of jam3b (jam3bsa37) led to the down-regulation of npas4l in the posterior lateral plate mesoderm (PLPM) and the cloche-like phenotype, a defect of both hematopoietic and endothelial development. We found that jam3bsa37 embryos remained the high level of Erk activation in the PLPM. Inhibition of Erk signaling by chemical treatment in jam3bsa37 embryos rescued the expression of npas4l in the PLPM and led to the recovery of the vascular formation, but not hematopoietic stem cell (HSC) formation. Transcriptome analysis and loss-of-function studies revealed that a membrane protein gene, lrrc15, is essential for HSC specification and is largely reduced in endothelial precursors in jam3bsa37 embryos. Enforced expression of lrrc15 in combination with Erk inhibition in jam3bsa37 embryos rescued HSCs in the dorsal aorta. Collectively, our data reveal a novel molecular mechanism for HSC specification, in which Jam3b mediates the repression of the Erk signaling pathway to promote npas4l expression followed by the up-regulation of lrrc15 in endothelial precursors.

Wnt signaling positively regulates endothelial cell fate specification in the Fli1a-positive progenitor population via Lef1

During vertebrate embryogenesis, vascular endothelial cells (ECs) and primitive erythrocytes become specified within close proximity in the posterior lateral plate mesoderm (LPM) from a common progenitor. However, the signaling cascades regulating the specification into either lineage remain largely elusive. Here, we analyze the contribution of β-catenin dependent Wnt signaling to EC and erythrocyte specification during zebrafish embryogenesis.We generated novel β-catenin dependent Wnt signaling reporters which, by using destabilized fluorophores (Venus-Pest, dGFP), specifically allow us to detect Wnt signaling responses in narrow time windows as well as in spatially restricted domains, defined by Cre recombinase expression (Tg(axin2BAC:Venus-Pest)mu288; Tg(14TCF:loxP-STOP-loxP-dGFP)mu202). We therefore can detect β-catenin dependent Wnt signaling activity in a subset of the Fli1a-positive progenitor population. Additionally, we show that mesodermal Wnt3a-mediated signaling via the transcription factor Lef1 positively regulates EC specification (defined by kdrl expression) at the expense of primitive erythrocyte specification (defined by gata1 expression) in zebrafish embryos.Using mesoderm derived from human embryonic stem cells, we identified the same principle of Wnt signaling dependent EC specification in conjunction with auto-upregulation of LEF1.Our data indicate a novel role of β-catenin dependent Wnt signaling in regulating EC specification during vasculogenesis.

Developmental Mechanism of Limb Field Specification along the Anterior-Posterior Axis during Vertebrate Evolution.

In gnathostomes, limb buds arise from the lateral plate mesoderm at discrete positions along the body axis. Specification of these limb-forming fields can be subdivided into several steps. The lateral plate mesoderm is regionalized into the anterior lateral plate mesoderm (ALPM; cardiac mesoderm) and the posterior lateral plate mesoderm (PLPM). Subsequently, Hox genes appear in a nested fashion in the PLPM and provide positional information along the body axis. The lateral plate mesoderm then splits into the somatic and splanchnic layers. In the somatic layer of the PLPM, the expression of limb initiation genes appears in the limb-forming region, leading to limb bud initiation. Furthermore, past and current work in limbless amphioxus and lampreys suggests that evolutionary changes in developmental programs occurred during the acquisition of paired fins during vertebrate evolution. This review presents these recent advances and discusses the mechanisms of limb field specification during development and evolution, with a focus on the role of Hox genes in this process.

The Spliceosomal Component Sf3b1 Is Essential for Hematopoietic Differentiation

Myelodysplastic syndrome (MDS) is a disorder arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis. A multitude of recurrent somatic mutations in spliceosomal components were recently identified in MDS that likely contribute to the pathogenesis of the disease. The lack of in vivo models to study cell-type specific effects of spliceosomal mutations limits our understanding of why such mutations lead to hematopoietic abnormalities. Using a zebrafish with a loss-of-function mutation in sf3b1 (sf3b1hi3394), an essential member of the spliceosome, we demonstrate hematopoietic cell differentiation and hematopoietic stem and progenitor cell (HSPC) specification are processes sensitive to spliceosomal malfunction. Primitive erythropoiesis initiates normally in sf3b1 mutants as evidenced by expression of scl in the posterior lateral plate mesoderm at 14 hours post fertilization (hpf) as well as gata1 and beta-globin at 24 hpf. Flow cytometry quantification of gata1:gfp positive erythrocytes showed sf3b1 mutants have 25% more cells at 24 hpf, but greater than 3-fold fewer cells at 36 and 48 hpf, time points when wild type erythroblasts are expanding and differentiating. At 48 hpf, we also observed decreased levels of o-dianisidine positive erythrocytes, low numbers of morphologically mature erythroblasts, and higher levels of immature erythroblasts in sf3b1 mutants. Similarly, we observed normal initiation of primitive myelopoiesis marked by stem cell leukemia (scl) expression in the anterior lateral plate mesoderm at 14 hpf, but diminished expression of more differentiated markers, l-plastin and myeloperoxidase at 24 and 28 hpf in sf3b1 mutants. Quantification of lysozyme C:dsred positivemyeloid cells using flow cytometry also showed 24-fold fewer mature myeloid cells in sf3b1 mutants at 36 hpf. Our data on primitive erythropoiesis and myelopoiesis indicate sf3b1 is required for hematopoietic cell differentiation. Additionally, sf3b1 mutants have diminished expression of the definitive HSPC marker runx1 within the aorta at 28 hpf. In contrast, we observed normal expression of the pan-endothelial marker kinase insert domain receptor-like (kdrl) and aorta-specific markers notch1b and notch3 at 24 hpf. Flow cytometry quantification of kdrl:gfp endothelial cells at 24 hpf shows no difference in the frequency of endothelial cells in sf3b1 mutants. Moreover, we observed fewer cmyb:gfp; kdrl:dsred double positive HSPCs along the dorsal aorta. The data suggest that decreased HSPC formation in sf3b1 mutants is due to a failure in hemogenic induction. From these studies, we show sf3b1 is required at specific stages of hematopoietic cell development. These results provide novel insight into the role of splicing in blood cell development, and can afford a deeper understanding of the mechanism of splicing regulation on the origins of MDS. DisclosuresNo relevant conflicts of interest to declare.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Distinct regulation of the anterior and posterior myeloperoxidase expression by Etv2 and Gata1 during primitive Granulopoiesis in zebrafish

Neutrophilic granulocytes are the most abundant type of myeloid cells and form an essential part of the innate immune system. In vertebrates the first neutrophils are thought to originate during primitive hematopoiesis, which precedes hematopoietic stem cell formation. In zebrafish embryos, it has been suggested that primitive neutrophils may originate in two distinct sites, the anterior (ALPM) and posterior lateral plate mesoderm (PLPM). An ETS-family transcription factor Etsrp/Etv2/ER71 has been implicated in vasculogenesis and hematopoiesis in multiple vertebrates. However, its role during neutrophil development is not well understood. Here we demonstrate using zebrafish embryos that Etv2 has a specific cell-autonomous function during primitive neutropoiesis in the anterior lateral plate mesoderm (ALPM) but has little effect on erythropoiesis or the posterior lateral plate mesoderm (PLPM) expression of neutrophil marker myeloperoxidase mpo/mpx. Our results argue that ALPM-derived neutrophils originate from etv2-expressing cells which downregulate etv2 during neutropoiesis. We further show that Scl functions downstream of Etv2 in anterior neutropoiesis. Additionally, we demonstrate that mpx expression within the PLPM overlaps with gata1 expression, potentially marking the cells with a dual myelo-erythroid potential. Intriguingly, initiation of mpx expression in the PLPM is dependent on gata1 but not etv2 function. Our results demonstrate that mpx expression is controlled differently in the ALPM and PLPM regions and describe novel roles for etv2 and gata1 during primitive neutropoiesis.

Protein tyrosine phosphatase PTPN9 regulates erythroid cell development through STAT3 dephosphorylation in zebrafish.

Protein tyrosine phosphatases (PTPs) are involved in hematopoiesis, but the function of many PTPs is not well characterized in vivo. Here, we have identified Ptpn9a, an ortholog of human PTPN9, as a crucial regulator of erythroid cell development in zebrafish embryos. ptpn9a, but not ptpn9b, was expressed in the posterior lateral plate mesoderm and intermediate cell mass - two primitive hematopoietic sites during zebrafish embryogenesis. Morpholino-mediated knockdown of ptpn9a caused erythrocytes to be depleted by inhibiting erythroid cell maturation without affecting erythroid proliferation and apoptosis. Consistently, both dominant-negative PTPN9 (with mutation C515S) and siRNA against PTPN9 inhibited erythroid differentiation in human K562 cells. Mechanistically, depletion of ptpn9 in zebrafish embryos in vivo or in K562 cells in vitro increased phosphorylated STAT3, and the hyper-phosphorylated STAT3 entrapped and prevented the transcription factors GATA1 and ZBP-89 (also known as ZNF148) from regulating erythroid gene expression. These findings imply that PTPN9 plays an important role in erythropoiesis by disrupting an inhibitory complex of phosphorylated STAT3, GATA1 and ZBP-89, providing new cellular and molecular insights into the role of ptpn9a in developmental hematopoiesis.

Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis, Angiogenesis and Hematopoiesis

Mice lacking the 4th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.

Zic3 is required in the extra-cardiac perinodal region of the lateral plate mesoderm for left–right patterning and heart development

Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of Zic3 demonstrates an early role for Zic3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in Zic3(null) mice and the relatively broad expression pattern of Zic3 suggest its important roles in multiple developmental processes. Here, we report that Zic3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in Zic3 deficiency occur not because Zic3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for Zic3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of Zic3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of Zic3 with signaling pathways involved in the early establishment of laterality.

Caudal genes in blood development and leukemia

Members of the caudal gene family (in mice and humans: Cdx1, Cdx2, and Cdx4) have been studied during early development as regulators of axial elongation and anteroposterior patterning. In the adult, Cdx1 and Cdx2, but not Cdx4, have been intensively explored for their function in intestinal tissue homeostasis and the pathogenesis of gastrointestinal cancers. Involvement in embryonic hematopoiesis was first demonstrated in zebrafish, where cdx genes render posterior lateral plate mesoderm competent to respond to genes specifying hematopoietic fate, and compound mutations in cdx genes thus result in a bloodless phenotype. Parallel studies performed in zebrafish embryos and murine embryonic stem cells (ESCs) delineate conserved pathways between fish and mammals, corroborating a BMP/Wnt-Cdx-Hox axis during blood development that can be employed to augment derivation of blood progenitors from pluripotent stem cells in vitro. The molecular regulation of Cdx genes appears complex, as more recent data suggest involvement of non-Hox-related mechanisms and the existence of auto- and cross-regulatory loops governed by morphogens. Here, we will review the role of Cdx genes during hematopoietic development by comparing effects in zebrafish and mice and discuss their participation in malignant blood diseases.

β-Catenin 1 and β-catenin 2 play similar and distinct roles in left-right asymmetric development of zebrafish embryos

β-Catenin-mediated canonical Wnt signaling has been found to be required for left-right (LR) asymmetric development. However, the implication of endogenous β-catenin in LR development has not been demonstrated by loss-of-function studies. In zebrafish embryos, two β-catenin genes, β-catenin 1 (ctnnb1) and β-catenin 2 (ctnnb2) are maternally expressed and their zygotic expression occurs in almost all types of tissues, including Kupffer's vesicle (KV), an essential organ that initiates LR development in teleost fish. We demonstrate here that morpholino-mediated knockdown of ctnnb1, ctnnb2, or both, in the whole embryo or specifically in dorsal forerunner cells (DFCs) interrupts normal asymmetry of the heart, liver and pancreas. Global knockdown of ctnnb2 destroys the midline physical and molecular barrier, while global knockdown of ctnnb1 impairs the formation of the midline molecular barrier. Depletion of either gene or both in DFCs/KV leads to poor KV cell proliferation, abnormal cilia formation and disordered KV fluid flow with downregulation of ntl and tbx16 expression. ctnnb1 and ctnnb2 in DFCs/KV differentially regulate the expression of charon, a Nodal antagonist, and spaw, a key Nodal gene for laterality development in zebrafish. Loss of ctnnb1 in DFCs/KV inhibits the expression of charon around KV and of spaw in the posterior lateral plate mesoderm, while ctnnb2 knockdown results in loss of spaw expression in the anterior lateral plate mesoderm with little alteration of charon expression. Taken together, our findings suggest that ctnnb1 and ctnnb2 regulate multiple processes of laterality development in zebrafish embryos through similar and distinct mechanisms.

Evi-1 Regulates Myelopoiesis and Hematopoietic Stem Cell Development in Zebrafish and Human Pluripotent Stem Cells

Abstract 1281The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Over the last years, Evi-1 evolved as one of the most potent oncogenes associated with human and murine myeloid leukemia. More recent studies in knockout mice suggest also involvement of Evi-1 in the regulation of developmental hematopoiesis, but the role of Evi-1 in this context is poorly understood. Here, we analyzed zebrafish embryo and human pluripotent stem cells (PSC) to understand how Evi-1 modulates early hematopoietic development.We examined the hematopoietic development in zebrafish embryo by in situ hybridization (ISH) for hematopoietic markers. The zebrafish homologue evi-1 was shown to be expressed in co-localization with scl in the posterior blood islands, indicating a role during early blood development. We also performed loss-of-function studies were by injecting morpholino oligonucleotides (MO) in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. Inhibition of evi-1 was confirmed in MO-injected versus control embryos. N=100 zebrafish embryos were analyzed per experiment in each group. To control for off-target effects, two separate MO were designed and injected. MO mediated evi-1 knockdown severely reduced numbers of circulating blood cells and induced hemorrhages. ISH performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells (measured by pu.1 expression), while no changes were observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Moreover, analyses at 36 hours and 5 days post fertilization showed strong reduction of runx1+/cmyb+ cells and rag1+ lymphoid cells, indicating a role of evi-1 in developing hematopoietic stem cells (HSC).Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects hematopoietic stem cell proliferation through regulation of Gata2. To test whether Gata2 is a putative downstream regulator of Evi-1 in our system, we performed a rescue experiment and co-injected gata2 mRNA in evi-1 MO treated fish. Indeed, ectopic gata2 rescued the impaired myeloid phenotype, as shown by re-occurrence of mpo, l-plastin as well as pu.1 expressing cells.To assess whether these molecular interactions are conserved during human developmental hematopoiesis, we surveyed in vitro differentiating human pluripotent stem cells (PSC) genetically modified to suppress EVI-1. EVI-1 expression was detected during differentiation of human PSC in embryoid bodies, especially around day 9 when hematopoietic progenitors start to emerge in this system. Treatment with EVI-1 shRNA strongly reduced the generation of myeloid colonies from human PSC in vitro as well as the numbers of emerging CD34+ and CD45+ cells. Molecularly, EVI-1 suppression inhibited the expression of PU.1 and GATA2 during the course of development, while leaving SCL and GATA1 expression unaltered.Taken together, our data suggest that, in both fish and human, Evi-1 regulates embryonic myelopoiesis through interactions with Gata2 and independently of Gata1 and embryonic erythropoiesis. Moreover, Evi-1 appears crucial for HSC development. Currently ongoing experiments in our laboratory focus on the further elucidation of the molecular mechanisms underlying the Evi-1 effects during developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Two additional midline barriers function with midlinelefty1expression to maintain asymmetric Nodal signaling during left-right axis specification in zebrafish

Left-right (L/R) patterning is crucial for the proper development of all vertebrates and requires asymmetric expression of nodal in the lateral plate mesoderm (LPM). The mechanisms governing asymmetric initiation of nodal have been studied extensively, but because Nodal is a potent activator of its own transcription, it is also crucial to understand the regulation required to maintain this asymmetry once it is established. The 'midline barrier', consisting of lefty1 expression, is a conserved mechanism for restricting Nodal activity to the left. However, the anterior and posterior extremes of the LPM are competent to respond to Nodal signals yet are not adjacent to this barrier, suggesting that lefty1 is not the only mechanism preventing ectopic Nodal activation. Here, we demonstrate the existence of two additional midline barriers. The first is a 'posterior barrier' mediated by Bmp signaling that prevents nodal propagation through the posterior LPM. In contrast to previous reports, we find that Bmp represses Nodal signaling independently of lefty1 expression and through the activity of a ligand other than Bmp4. The 'anterior barrier' is mediated by lefty2 expression in the left cardiac field and prevents Nodal activation from traveling across the anterior limit of the notochord and propagating down the right LPM. Both barriers appear to be conserved across model systems and are thus likely to be present in all vertebrates.