The limb dorsoventral axis: Lmx1b's role in development, pathology, evolution, and regeneration.

The limb anatomy displays well-defined dorsal and ventral compartments, housing extensor, and flexor muscles, which play a crucial role in facilitating limb locomotion and manipulation. Despite its importance, the study of limb dorsoventral patterning has been relatively neglected compared to the other two axes leaving many crucial questions about the genes and developmental processes implicated unanswered. This review offers a thorough overview of the current understanding of limb dorsoventral patterning, synthesizing classical literature with recent research. It covers the specification of dorsal fate in the limb mesoderm and its subsequent translation into dorsal morphologies-a process directed by the transcription factor Lmx1b. We also discuss the potential role of dorsoventral patterning in the evolution of paired appendages and delve into the involvement of LMX1B in Nail-Patella syndrome, discussing the molecular and genetic aspects underlying this condition. Finally, the potential role of dorsoventral polarity in digit tip regeneration, a prominent instance of multi-tissue regeneration in mammals is also considered. We anticipate that this review will renew interest in a process that is critical to limb function and evolutionary adaptations but has nonetheless been overlooked.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP-associated variants in embryonic zebrafish.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins. RESULTS: Here, we performed in vivo functional characterization of human ACVR1R206H and orthologous zebrafish Acvr1lR203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1R206H and zebrafish Acvr1lR203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1R206H signaling in early embryonic zebrafish. CONCLUSIONS: Together, these data provide new insight into ACVR1R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Rnf220 is Implicated in the Dorsoventral Patterning of the Hindbrain Neural Tube in Mice.

Rnf220 is reported to regulate the patterning of the ventral spinal neural tube in mice. The brainstem has divergent connections with peripheral and central targets and contains unique internal neuronal groups, but the role of Rnf220 in the early development of the hindbrain has not been explored. In this study, Nestin-Cre-mediated conditional knockout (Rnf220 Nestin CKO) mice were used to examine if Rnf220 is involved in the early morphogenesis of the hindbrain. Rnf220 showed restricted expression in the ventral half of ventricular zone (VZ) of the hindbrain at embryonic day (E) 10.5, and as development progressed, Rnf220-expressing cells were also present in the mantle zone outside the VZ at E12.5. In Rnf220 Nestin CKO embryos, alterations of progenitor domains in the ventral VZ were observed at E10.5. There were significant reductions of the p1 and p2 domains shown by expression of Dbx1, Olig2, and Nkx6.1, accompanied by a ventral expansion of the Dbx1+ p0 domain and a dorsal expansion of the Nkx2.2+ p3 domain. Different from the case in the spinal cord, the Olig2+ pMN (progenitors of somatic motor neuron) domain shifted and expanded dorsally. Notably, the total range of the ventral VZ and the extent of the dorsal tube were unchanged. In addition, the post-mitotic cells derived from their corresponding progenitor domain, including oligodendrocyte precursor cells (OPCs) and serotonergic neurons (5-HTNs), were also changed in the same trend as the progenitor domains do in the CKO embryos at E12.5. In summary, our data suggest similar functions of Rnf220 in the hindbrain dorsoventral (DV) patterning as in the spinal cord with different effects on the pMN domain. Our work also reveals novel roles of Rnf220 in the development of 5-HTNs and OPCs.

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning.

Dorsoventral (DV) patterning is a key landmark of embryonic development that is primarily regulated by bone morphogenetic protein (BMP) signaling. Disruption of DV patterning can result in downstream effects on cell specification and organogenesis. Zebrafish embryos have been extensively used to understand signaling pathways that regulate DV patterning because zebrafish embryos develop ex utero and, in contrast to mammalian embryos, which develop in utero, can be observed in real time using brightfield and fluorescence microscopy. Embryos with disrupted DV patterning are either dorsalized or ventralized, with lack of development of head or trunk/tail structures, respectively. Although these phenotypes are typically accompanied by effects on BMP signaling, exceptions exist where some drugs or environmental chemicals can disrupt DV patterning in the absence of effects on BMP signaling. Therefore, assessments of DV patterning should be accompanied by BMP signaling-specific readouts to confirm the role of BMP disruption. Here, we describe an exposure paradigm and steps for phenotyping zebrafish embryos for two types of DV defects, dorsalization and ventralization, with a range of severities. In addition, we describe a strategy for whole-mount immunohistochemistry of zebrafish embryos with an antibody specific for phospho-SMAD 1/5/9 (pSMAD 1/5/9), as disruption in pSMAD 1/5/9 localization is indicative of an effect on BMP signaling. Taken together, these protocols describe an initial strategy for evaluating DV patterning defects under various experimental conditions and confirming BMP-mediated DV patterning disruptions, which can be followed by additional studies that aim to uncover mechanisms leading to these adverse phenotypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping for dorsalization and ventralization Basic Protocol 2: Whole-mount immunohistochemistry with antibody to phospho-SMAD 1/5/9.

BMP Signaling: Lighting up the Way for Embryonic Dorsoventral Patterning.

One of the most significant events during early embryonic development is the establishment of a basic embryonic body plan, which is defined by anteroposterior, dorsoventral (DV), and left-right axes. It is well-known that the morphogen gradient created by BMP signaling activity is crucial for DV axis patterning across a diverse set of vertebrates. The regulation of BMP signaling during DV patterning has been strongly conserved across evolution. This is a remarkable regulatory and evolutionary feat, as the BMP gradient has been maintained despite the tremendous variation in embryonic size and shape across species. Interestingly, the embryonic DV axis exhibits robust stability, even in face of variations in BMP signaling. Multiple lines of genetic, molecular, and embryological evidence have suggested that numerous BMP signaling components and their attendant regulators act in concert to shape the developing DV axis. In this review, we summarize the current knowledge of the function and regulation of BMP signaling in DV patterning. Throughout, we focus specifically on popular model animals, such as Xenopus and zebrafish, highlighting the similarities and differences of the regulatory networks between species. We also review recent advances regarding the molecular nature of DV patterning, including the initiation of the DV axis, the formation of the BMP gradient, and the regulatory molecular mechanisms behind BMP signaling during the establishment of the DV axis. Collectively, this review will help clarify our current understanding of the molecular nature of DV axis formation.

The forkhead box containing transcription factor FoxB is a potential component of dorsal-ventral body axis formation in the spider Parasteatoda tepidariorum.

In the spider, determination of the dorsal-ventral body (DV) axis depends on the interplay of the dorsal morphogen encoding gene decapentaplegic (Dpp) and its antagonist, short gastrulation (sog), a gene that is involved in the correct establishment of ventral tissues. Recent work demonstrated that the forkhead domain encoding gene FoxB is involved in dorsal-ventral axis formation in spider limbs. Here, Dpp likely acts as a dorsal morphogen, and FoxB is likely in control of ventral tissues as RNAi-mediated knockdown of FoxB causes dorsalization of the limbs. In this study, we present phenotypes of FoxB knockdown that demonstrate a function in the establishment of the DV body axis. Knockdown of FoxB function leads to embryos with partially duplicated median germ bands (Duplicitas media) that are possibly the result of ectopic activation of Dpp signalling. Another class of phenotypes is characterized by unnaturally slim (dorsal-ventrally compressed) germ bands in which ventral tissue is either not formed, or is specified incorrectly, likely a result of Dpp over-activity. These results suggest that FoxB functions as an antagonist of Dpp signalling during body axis patterning, similarly as it is the case in limb development. FoxB thus represents a general player in the establishment of dorsal-ventral structures during spider ontogeny.

A single cell transcriptome atlas of the developing zebrafish hindbrain.

Segmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.

Ciglitazone-a human PPARγ agonist-disrupts dorsoventral patterning in zebrafish.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that regulates lipid/glucose homeostasis and adipocyte differentiation. While the role of PPARγ in adipogenesis and diabetes has been extensively studied, little is known about PPARγ function during early embryonic development. Within zebrafish, maternally-loaded pparγ transcripts are present within the first 6 h post-fertilization (hpf), and de novo transcription of zygotic pparγ commences at ~48 hpf. Since maternal pparγ transcripts are elevated during a critical window of cell fate specification, the objective of this study was to test the hypothesis that PPARγ regulates gastrulation and dorsoventral patterning during zebrafish embryogenesis. To accomplish this objective, we relied on (1) ciglitazone as a potent PPARγ agonist and (2) a splice-blocking, pparγ-specific morpholino to knockdown pparγ. We found that initiation of ciglitazone-a potent human PPARγ agonist-exposure by 4 hpf resulted in concentration-dependent effects on dorsoventral patterning in the absence of epiboly defects during gastrulation, leading to ventralized embryos by 24 hpf. Interestingly, ciglitazone-induced ventralization was reversed by co-exposure with dorsomorphin, a bone morphogenetic protein signaling inhibitor that induces strong dorsalization within zebrafish embryos. Moreover, mRNA-sequencing revealed that lipid- and cholesterol-related processes were affected by exposure to ciglitazone. However, pparγ knockdown did not block ciglitazone-induced ventralization, suggesting that PPARγ is not required for dorsoventral patterning nor involved in ciglitazone-induced toxicity within zebrafish embryos. Our findings point to a novel, PPARγ-independent mechanism of action and phenotype following ciglitazone exposure during early embryonic development.

Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes.

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medialhigh/laterallow expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.

BMP controls dorsoventral and neural patterning in indirect-developing hemichordates providing insight into a possible origin of chordates.

A defining feature of chordates is the unique presence of a dorsal hollow neural tube that forms by internalization of the ectodermal neural plate specified via inhibition of BMP signaling during gastrulation. While BMP controls dorsoventral (DV) patterning across diverse bilaterians, the BMP-active side is ventral in chordates and dorsal in many other bilaterians. How this phylum-specific DV inversion occurs and whether it is coupled to the emergence of the dorsal neural plate are unknown. Here we explore these questions by investigating an indirect-developing enteropneust from the hemichordate phylum, which together with echinoderms form a sister group of the chordates. We found that in the hemichordate larva, BMP signaling is required for DV patterning and is sufficient to repress neurogenesis. We also found that transient overactivation of BMP signaling during gastrulation concomitantly blocked mouth formation and centralized the nervous system to the ventral ectoderm in both hemichordate and sea urchin larvae. Moreover, this mouthless, neurogenic ventral ectoderm displayed a medial-to-lateral organization similar to that of the chordate neural plate. Thus, indirect-developing deuterostomes use BMP signaling in DV and neural patterning, and an elevated BMP level during gastrulation drives pronounced morphological changes reminiscent of a DV inversion. These findings provide a mechanistic basis to support the hypothesis that an inverse chordate body plan emerged from an indirect-developing ancestor by tinkering with BMP signaling.

Horizontal Boundary Cells, a Special Group of Somitic Cells, Play Crucial Roles in the Formation of Dorsoventral Compartments in Teleost Somite.

Establishment of robust gene expression boundary is crucial for creating elaborate morphology during development. However, mechanisms underlying boundary formation have been extensively studied only in a few model systems. We examined the establishment of zic1/zic4-expression boundary demarcating dorsoventral boundary of the entire trunk of medaka fish (Oryzias latipes) and identified a subgroup of dermomyotomal cells called horizontal boundary cells (HBCs) as crucial players for the boundary formation. Embryological and genetic analyses demonstrated that HBCs play crucial roles in the two major events of the process, i.e., refinement and maintenance. In the refinement, HBCs could serve as a chemical barrier against Wnts from the neural tube by expressing Hhip. At later stages, HBCs participate in the maintenance of the boundary by differentiating into the horizontal myoseptum physically inhibiting cell mixing across the boundary. These findings reveal the mechanisms underlying the dorsoventral boundary in the teleost trunk by specialized boundary cells.

Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain.

BACKGROUND: Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear. METHODS: We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation. RESULTS: We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1. CONCLUSIONS: We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain - suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically needed to promote p1 fates.

Ankyrin domain encoding genes from an ancient horizontal transfer are functionally integrated into Nasonia developmental gene regulatory networks.

BACKGROUND: How regulatory networks incorporate additional components and how novel genes are functionally integrated into well-established developmental processes are two important and intertwined questions whose answers have major implications for understanding the evolution of development. We recently discovered a set of lineage-restricted genes with strong and specific expression patterns along the dorsal-ventral (DV) axis of the embryo of the wasp Nasonia that may serve as a powerful system for addressing these questions. We sought to both understand the evolutionary history of these genes and to determine their functions in the Nasonia DV patterning system. RESULTS: We have found that the novel DV genes are part of a large family of rapidly duplicating and diverging ankyrin domain-encoding genes that originated most likely by horizontal transfer from a prokaryote in a common ancestor of the wasp superfamily Chalcidoidea. We tested the function of those ankyrin-encoding genes expressed along the DV axis and found that they participate in early embryonic DV patterning. We also developed a new wasp model system (Melittobia) and found that some functional integration of ankyrin genes have been preserved for over 90 million years. CONCLUSIONS: Our results indicate that regulatory networks can incorporate novel genes that then become necessary for stable and repeatable outputs. Even a modest role in developmental networks may be enough to allow novel or duplicate genes to be maintained in the genome and become fully integrated network components.

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors.

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Zic Genes in Teleosts: Their Roles in Dorsoventral Patterning in the Somite.

The medaka contains seven zic genes, two of which, zic1 and zic4, have been studied extensively. The analyses are mainly based on the double anal fin (Da) mutant, which was isolated from the wild. Da is an enhancer mutant of zic1/zic4, and the expression of zic1/zic4 is specifically lost in the dorsal half of the somites, which leads to a mirror-image duplication of the ventral half across the lateral midline from larva to adult. The studies of medaka Da give us important insights into the function of zic1/zic4 in mesodermal tissues and also the mechanism of dorsoventral patterning in the vertebrate trunk region occurring during late development, which is a long-standing mystery in developmental biology. In this chapter, we introduce genomic organization of medaka zic genes and discuss their function, mainly focusing on zic1 and zic4 in dorsoventral patterning of the trunk region and possible connections to human congenital disorders.

The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm.

The proper development of a multicellular organism requires precise spatial and temporal coordination of cell intrinsic and cell extrinsic regulatory mechanisms. Both Notch signaling and bone morphogenetic protein (BMP) signaling function to regulate the proper development of the C. elegans postembryonic mesoderm. We have identified the C. elegans FOXD transcription factor UNC-130 as a major target functioning downstream of both BMP signaling and Notch signaling to regulate dorsoventral patterning of the postembryonic mesoderm. We showed that unc-130 expression in the postembryonic M lineage is asymmetric: its absence of expression in the dorsal side of the M lineage requires the antagonism of BMP signaling by the zinc finger transcription factor SMA-9, while its expression in the ventral side of the M lineage is activated by LIN-12/Notch signaling. We further showed that the regulation of UNC-130 expression by BMP signaling and Notch signaling is specific to the M lineage, as the ventral expression of UNC-130 in the embryonically-derived bodywall muscles was not affected in either BMP pathway or Notch pathway mutants. Finally, we showed that the function of UNC-130 in the M lineage is independent of UNC-129, a gene previously shown to function downstream of and be repressed by UNC-130 for axon guidance. Our studies uncovered a new function of UNC-130/FOXD in the C. elegans postembryonic mesoderm, and identify UNC-130 as a critical factor that integrates two independent spatial cues for the proper patterning and fate specification of the C. elegans postembryonic mesoderm.

Tris(1,3-dichloro-2-propyl) phosphate disrupts dorsoventral patterning in zebrafish embryos.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in genome-wide alterations in methylation during cleavage (2 hpf) as well as epiboly delay or arrest (at higher concentrations) during late-blastula and early-gastrula (4-6 hpf). To determine whether these TDCIPP-induced effects were associated with impacts on the transcriptome, embryos were exposed to vehicle (0.1% DMSO) or 2 µM TDCIPP from 0.75 hpf to 6 hpf, and total RNA was extracted from triplicate embryo pools per treatment and hybridized onto duplicate Affymetrix Zebrafish Gene 1.0 ST Arrays per RNA sample. Based on transcriptome-wide profiling, TDCIPP resulted in a significant impact on biological processes involved in dorsoventral patterning and bone morphogenetic protein (BMP) signaling. Consistent with these responses, TDCIPP exposure also resulted in strongly dorsalized embryos by 24 hpf-a phenotype that mimicked the effects of dorsomorphin, a potent and selective BMP inhibitor. Moreover, the majority of dorsalized embryos were preceded by epiboly arrest at 6 hpf. Our microarray data also revealed that the expression of sizzled (szl)-a gene encoding a secreted Frizzled-related protein that limits BMP signaling-was significantly decreased by nearly 4-fold at 6 hpf. Therefore, we used a splice-blocking morpholino to test the hypothesis that knockdown of szl phenocopies TDCIPP-induced delays in epiboly progression. Interestingly, contrary to our hypothesis, injection of szl MOs did not affect epiboly progression but, similar to chordin (chd) morphants, resulted in mildly ventralized embryos by 24 hpf. Overall, our findings suggest that TDCIPP-induced epiboly delay may not be driven by decreased szl expression, and that TDCIPP-induced dorsalization may-similar to dorsomorphin-be due to interference with BMP signaling during early zebrafish development.

Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish.

The vegetal pole cytoplasm represents a crucial source of maternal dorsal determinants for patterning the dorsoventral axis of the early embryo. Removal of the vegetal yolk in the zebrafish fertilised egg before the completion of the first cleavage results in embryonic ventralisation, but removal of this part at the two-cell stage leads to embryonic dorsalisation. How this is achieved remains unknown. Here, we report a novel mode of maternal regulation of BMP signalling during dorsoventral patterning in zebrafish. We identify Vrtn as a novel vegetally localised maternal factor with dorsalising activity and rapid transport towards the animal pole region after fertilisation. Co-injection of vrtn mRNA with vegetal RNAs from different cleavage stages suggests the presence of putative vegetally localised Vrtn antagonists with slower animal pole transport. Thus, vegetal ablation at the two-cell stage could remove most of the Vrtn antagonists, and allows Vrtn to produce the dorsalising effect. Mechanistically, Vrtn binds a bmp2b regulatory sequence and acts as a repressor to inhibit its zygotic transcription. Analysis of maternal-zygotic vrtn mutants further shows that Vrtn is required to constrain excessive bmp2b expression in the margin. Our work unveils a novel maternal mechanism regulating zygotic BMP gradient in dorsoventral patterning.

Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus, hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development.