Benjamin L. Martin

Regulation of Canonical Wnt Signaling by Brachyury Is Essential for Posterior Mesoderm Formation

The T box transcription factor Brachyury is essential for the formation of the posterior body in all vertebrates, although its critical transcriptional targets have remained elusive. Loss-of-function studies of mouse Brachyury and the zebrafish Brachyury ortholog Ntl indicated that Brachyury plays a more significant role in higher vertebrates than lower vertebrates. We have identified a second zebrafish Brachyury ortholog (Bra), and show that a combined loss of Ntl and Bra recapitulates the mouse phenotype, demonstrating an ancient role for Brachyury in patterning all but the most anterior somites. Using cell transplantation, we show that the only essential role for Brachyury during somite formation is non-cell autonomous, and demonstrate that Ntl and Bra are required for and can induce expression of the canonical Wnts wnt8 and wnt3a. We propose that a positive autoregulatory loop between Ntl/Bra and canonical Wnt signaling maintains the mesodermal progenitors to facilitate posterior somite development in chordates.

Recurrent de novo mutations implicate novel genes underlying simplex autism risk

Autism spectrum disorder (ASD) has a strong but complex genetic component. Here we report on the resequencing of 64 candidate neurodevelopmental disorder risk genes in 5,979 individuals: 3,486 probands and 2,493 unaffected siblings. We find a strong burden of de novo point mutations for these genes and specifically implicate nine genes. These include CHD2 and SYNGAP1, genes previously reported in related disorders, and novel genes TRIP12 and PAX5. We also show that mutation carriers generally have lower IQs and enrichment for seizures. These data begin to distinguish genetically distinct subtypes of autism important for etiological classification and future therapeutics.

Wnt Signaling and the Evolution of Embryonic Posterior Development

During vertebrate embryogenesis, most of the mesodermal tissue posterior to the head forms from a progenitor population that continuously adds blocks of muscles (the somites) from the back end of the embryo. Recent work in less commonly studied arthropods--the flour beetle Tribolium and the common house spider--provides evidence suggesting that this posterior growth process might be evolutionarily conserved, with canonical Wnt signaling playing a key role in vertebrates and invertebrates. We discuss these findings as well as other evidence that suggests that the genetic network controlling posterior growth was already present in the last common ancestor of the Bilateria. We also highlight other interesting commonalities as well as differences between posterior growth in vertebrates and invertebrates, suggest future areas of research, and hypothesize that posterior growth may facilitate evolution of animal body plans.

Canonical Wnt Signaling Dynamically Controls Multiple Stem Cell Fate Decisions during Vertebrate Body Formation

The vertebrate body forms in an anterior-to-posterior progression, driven by a population of undifferentiated cells at the posterior-most end of the embryo. Recent studies have demonstrated that these undifferentiated cells are multipotent stem cells, suggesting that local signaling factors specify cell fate. However, the mechanism of cell fate specification during this process is unknown. Using a combination of single cell transplantation and newly developed cell-autonomous inducible Wnt inhibitor and activator transgenic zebrafish lines, we show that canonical Wnt signaling is continuously necessary and sufficient to specify mesoderm from a bipotential neural/mesodermal precursor. Surprisingly, we also find that Wnt signaling functions subsequently within the mesoderm to specify somites instead of posterior vascular endothelium. Our results demonstrate that dynamic local Wnt signaling cues specify germ layer contribution and mesodermal tissue type specification of multipotent stem cells throughout the formation of the early vertebrate embryonic body.

Brachyury establishes the embryonic mesodermal progenitor niche

Formation of the early vertebrate embryo depends on a Brachyury/Wnt autoregulatory loop within the posterior mesodermal progenitors. We show that exogenous retinoic acid (RA), which dramatically truncates the embryo, represses expression of the zebrafish brachyury ortholog no tail (ntl), causing a failure to sustain the loop. We found that Ntl functions normally to protect the autoregulatory loop from endogenous RA by directly activating cyp26a1 expression. Thus, the embryonic mesodermal progenitors uniquely establish their own niche--with Brachyury being essential for creating a domain of high Wnt and low RA signaling--rather than having a niche created by separate support cells.

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk.

Graded levels of Pax2a and Pax8 regulate cell differentiation during sensory placode formation

Pax gene haploinsufficiency causes a variety of congenital defects. Renal-coloboma syndrome, resulting from mutations in Pax2, is characterized by kidney hypoplasia, optic nerve malformation, and hearing loss. Although this underscores the importance of Pax gene dosage in normal development, how differential levels of these transcriptional regulators affect cell differentiation and tissue morphogenesis is still poorly understood. We show that differential levels of zebrafish Pax2a and Pax8 modulate commitment and behavior in cells that eventually contribute to the otic vesicle and epibranchial placodes. Initially, a subset of epibranchial placode precursors lie lateral to otic precursors within a single Pax2a/8-positive domain; these cells subsequently move to segregate into distinct placodes. Using lineage-tracing and ablation analyses, we show that cells in the Pax2a/8+ domain become biased towards certain fates at the beginning of somitogenesis. Experiments involving either Pax2a overexpression or partial, combinatorial Pax2a and Pax8 loss of function reveal that high levels of Pax favor otic differentiation whereas low levels increase cell numbers in epibranchial ganglia. In addition, the Fgf and Wnt signaling pathways control Pax2a expression: Fgf is necessary to induce Pax2a, whereas Wnt instructs the high levels of Pax2a that favor otic differentiation. Our studies reveal the importance of Pax levels during sensory placode formation and provide a mechanism by which these levels are controlled.

A novel role for lbx1 in Xenopus hypaxial myogenesis.

We have examined lbx1 expression in early X. laevis tadpoles. In contrast to amniotes, lbx1 is expressed in all of the myoblasts that contribute to the body wall musculature, as well as in a group of cells that migrate into the head. Despite this different expression, the function of lbx1 appears to be conserved. Morpholino (MO) knockdown of lbx1 causes a specific reduction of body wall muscles and hypoglossal muscles originating from the somites. Although myoblast migratory defects are observed in antisense MO injected tadpoles targeting lbx1, this results at least in part from a lack of myoblast proliferation in the hypaxial muscle domain. Conversely, overexpression of lbx1 mRNA results in enlarged somites, an increase in cell proliferation, but a lack of differentiated muscle. The control of cell proliferation is linked to a strong downregulation of myoD expression in gain-of-function experiments. Co-injection of myoD mRNA with lbx1 mRNA eliminates the overproliferation phenotype observed when lbx1 is injected alone. The results indicate that a primary function of lbx1 in hypaxial muscle development is to repress myoD, allowing myoblasts to proliferate before the eventual onset of terminal differentiation.

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy.

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos.

Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail

The process of gastrulation is highly conserved across vertebrates on both the genetic and morphological levels, despite great variety in embryonic shape and speed of development. This mechanism spatially separates the germ layers and establishes the organizational foundation for future development. Mesodermal identity is specified in a superficial layer of cells, the epiblast, where cells maintain an epithelioid morphology. These cells involute to join the deeper hypoblast layer where they adopt a migratory, mesenchymal morphology. Expression of a cascade of related transcription factors orchestrates the parallel genetic transition from primitive to mature mesoderm. Although the early and late stages of this process are increasingly well understood, the transition between them has remained largely mysterious. We present here the first high resolution in vivo observations of the blebby transitional morphology of involuting mesodermal cells in a vertebrate embryo. We further demonstrate that the zebrafish spadetail mutation creates a reversible block in the maturation program, stalling cells in the transition state. This mutation creates an ideal system for dissecting the specific properties of cells undergoing the morphological transition of maturing mesoderm, as we demonstrate with a direct measurement of cell-cell adhesion.