RESEARCH QUESTIONS
Background: Since it drives the formation of anterior sensory organs in several metazoan embryos, specification of the anterior neuroectoderm (ANE) is a defining feature of the animal body plan; however, it is still unclear how this territory is created or evolved. Although there are significant morphological differences among deuterostome anterior sensory structures, recent comparative gene expression and functional studies show that the initial gene regulatory networks (GRNs) underlying the specification and patterning of ANE territories are remarkably similar among deuterostomes, including vertebrates. These findings strongly suggest that the early ANE territory is both ancient and homologous. In addition, studies in the sea urchin and several vertebrate embryos, including human embryonic stem cells, indicate that an unknown, broadly active early regulatory network(s) is used to activate their respective ANE GRNs. Yet, very little is known about the components of these essential ANE activation networks in any species. Our aim is to identify the early regulatory factors necessary to activate the ANE GRN in the sea urchin embryo.
Approaches: We are using differential ATAC-seq, cis-regulatory reporter analysis, qPCR, whole mount in situ hybridization and functional perturbations to test the role of putative early broad activators of the ANE GRN in sea urchin embryos.
1. What is the early, broadly expressed regulatory mechanism that activates the anterior neuroectoderm gene regulatory network?
Q1 Figure. Conservation of the ANE GRN and evidence for an early, broadly expressed regulatory network necessary for ANE specification. (A) Shared expression of orthologs necessary for ANE specification in sea urchin and zebrafish embryos. The upper-level GRN factor Six3 activates a progression of gene expression. Boxes represent groups of genes expressed around the same relative time after Six3 in both species during ANE positioning. (B) Blocking Wnt/β-catenin signaling leads to ectopic expression of the ANE gene foxq2 in posterior blastomeres and expression of ANE markers throughout the embryo. (a, b) Control embryos showing expression of foxq2 at the 32- and late -blastula stages. (d, e) Expression of foxq2 is seen in posterior blastomeres when Wnt/β-catenin signaling is blocked by Axin overexpression. (c, f) Expression of the ANE differentiation marker serotonin in control and Wnt/β-catenin (-) embryos.
Background: During the initial stages of anterior-posterior patterning in many deuterostome embryos the ANE GRN is initially broadly expressed in the ectoderm, and then a posterior-to-anterior mechanism initiated by Wnt/β-catenin restricts ANE GRN activity to a territory around the anterior pole where it drives development of anterior sensory structures in many species, including vertebrates. As a result of this Wnt/β-catenin dependent process three early domains are established along the AP axis: endomesoderm around the posterior end; an equatorial epidermal ectoderm band and the ANE domain around the anterior pole. Until recently, the Wnt/β-catenin pathway was the only Wnt signaling pathway shown to be necessary for ANE restriction in any deuterostome embryo; however, our recent work has provided evidence that the sea urchin embryo uses an interconnected network of Wnt signaling that includes the Wnt/β-catenin pathway and the “non-canonical”, or “alternative”, Wnt/JNK and Wnt/PKC pathways. This is one of the few examples of a Wnt network involving three different Wnt pathways working on the same developmental process. Our goal is to use a systems biology approach to elucidate how this Wnt signaling network is integrated at the extracellular, intracellular, and transcriptional level in order to pattern the anterior-posterior axis.
Approaches: To identify the signal transduction components and transcriptional targets involved in the AP Wnt signaling network, we have carried out RNA-seq analysis of Wnt/β-catenin, Wnt/JNK and Wnt/PKC knockdown embryos at multiple stages of development. We validate putative components with qPCR, whole mount in situ hybridization, and/or antibody staining. Then we perform functional manipulations to build the regulatory connections in the network.
2. How do the Wnt/β-catenin, Wnt/JNK and Fzl1/2/7-PKC signaling pathways interact at the extracellular, intracellular and transcriptional levels to pattern the early anterior-posterior axis?
Q2 Figure. A model for the Wnt signaling network governing ANE restriction in the sea urchin embryo and the evolutionary of sFRP3/4 and sFRP-1 in deuterostomes. (A) Early down regulation of the ANE GRN from posterior blastomeres. (Posterior half of the embryo) A broad, maternal regulatory mechanism is able to activate the ANE GRN throughout the embryo by the 32-cell stage, but n-catenin signaling prevents the expression of the ANE GRN in the posterior half of the 32-cell embryo through an unknown mechanism and activates wnt1 and wnt8 expression at the 32-cell stage. In addition around the 32- to 60-cell stage, sFRP-1 and Fzl1/2/7 signaling antagonize the down regulation of the ANE GRN in the anterior hemisphere. (B) Down regulation of the ANE GRN from cells in the lateral ectoderm domain and the establishment of the ANE around the anterior pole. (Anterior half of the embryo) Around the 32- to 60-cell stage (7-9 hpf), secreted Wnt1 and Wnt8 diffuse into more anterior ectodermal blastomeres (wnt8 expression is activated throughout the central ectoderm territory) and signal through the Fzl5/8 receptor, activating the JNK pathway. This Wnt/JNK pathway progressively down regulates ANE GRN expression from the central ectodermal territory during the early to late blastula stages (12 – 18 hpf). Within the same ectodermal cells sFRP-1 and Fzl1/2/7 signaling antagonize Wnt1/Wnt8-Fzl5/8-JNK mediated down regulation of the ANE GRN. Around the mid-blastula stage (16 hpf), an anterior signaling center activated by the cardinal ANE transcriptional regulator FoxQ2 secretes the Wnt modulators Dkk3 and sFRP1/5 within the regressing ANE territory. Dkk3 and low levels of sFRP1/5 are necessary to stimulate the Fzl5/8-JNK signaling during the later stages of ANE restriction. During the final phase of the process from early to late blastula stages (18 – 24 hpf), Fzl5/8 signaling activates the expression of the secreted Wnt antagonists sFRP-1 and Dkk1 in the anterior-most cells around the anterior pole. These antagonists establish a correctly sized ANE territory by preventing the down regulation of ANE factors by antagonizing Fzl5/8 signaling through a negative feedback mechanism.
Background: When we compared our findings in the sea urchin embryo with fragmentary expression and functional data currently available for vertebrates, cephalochordates, and hemichordates, we discovered that there were remarkable similarities in molecules involved in ANE specification and anterior-posterior patterning. Importantly, many of the orthologues of key Wnt signaling players mediating sea urchin ANE restriction are also essential for ANE restriction in vertebrates. Together, these studies raise the possibility that critical aspects of the multistep ANE positioning mechanism observed in sea urchin embryos are widely shared among deuterostome embryos (for review see Range, genesis, 2014). Our objective is to work in collaboration with the Lowe Lab at Stanford (website) to functionally characterize components of the early AP Wnt network identified in sea urchin embryos that share similar spatiotemporal expression patterns in indirect (Schizocardium californicum) and direct (Saccoglossus kowalevskii) developing hemichordate embryos.
Approaches: We use qPCR and whole mount in situ hybridization to determine the spatiotemporal expression of hemichordate orthologues of AP Wnt network components identified in the sea urchin. If orthologues have expression patterns consistent with a role in early AP patterning in hemichordates, then we perform molecular manipulations to determine their role in AP patterning.
3. Are the early regulatory factors necessary for activation of the ANE GRN and the components of the sea urchin anterior-posterior Wnt signaling network conserved in deuterostome embryos?
Q3 Figure. Conservation of sea urchin Wnt network orthologues in deuterostomes during ANE restriction. The diagram of each embryo is colored to indicate the general ANE regulatory network (blue) and the expression of wnt8 (green). The spatial expression patterns of ANE factor orthologs in the neuroectoderm along the AP axis are indicated to the left and to the right of each diagram. No expression data are available for the genes shaded light gray.
