In the field: an interview with Roger Hanlon

Roger Hanlon is a Senior Scientist at the Marine Biological Laboratory, USA, where he investigates body patterning and colour change in cephalopods. After his undergraduate degree in 1969 at Florida State University, USA, he joined the US Army and travelled before completing his MSc (1975) and PhD (1978) at the University of Miami Rosenstiel School of Marine and Atmospheric Sciences, USA. After a NATO Postdoctoral Fellowship at the University of Cambridge, UK, in 1981, Hanlon joined the Marine Biomedical Institute, University of Texas Medical Branch, where he became a full Professor, before joining the Marine Biological Laboratory in 1995. Hanlon talks about the seminal experience in his early 20s that inspired his career and the methods and equipment he uses to study cephalopod camouflage and communication across the globe.

Quantifying the Speed of Chromatophore Activity at the Single-Organ Level in Response to a Visual Startle Stimulus in Living, Intact Squid

The speed of adaptive body patterning in coleoid cephalopods is unmatched in the natural world. While the literature frequently reports their remarkable ability to change coloration significantly faster than other species, there is limited research on the temporal dynamics of rapid chromatophore coordination underlying body patterning in living, intact animals. In this exploratory pilot study, we aimed to measure chromatophore activity in response to a light flash stimulus in seven squid, Doryteuthis pealeii. We video-recorded the head/arms, mantle, and fin when squid were presented with a light flash startle stimulus. Individual chromatophores were detected and tracked over time using image analysis. We assessed baseline and response chromatophore surface area parameters before and after flash stimulation, respectively. Using change-point analysis, we identified 4,065 chromatophores from 185 trials with significant surface area changes elicited by the flash stimulus. We defined the temporal dynamics of chromatophore activity to flash stimulation as the latency, duration, and magnitude of surface area changes (expansion or retraction) following the flash presentation. Post stimulation, the response’s mean latency was at 50 ms (± 16.67 ms), for expansion and retraction, across all body regions. The response duration ranged from 217 ms (fin, retraction) to 384 ms (heads/arms, expansion). While chromatophore expansions had a mean surface area increase of 155.06%, the retractions only caused a mean reduction of 40.46%. Collectively, the methods and results described contribute to our understanding of how cephalopods can employ thousands of chromatophore organs in milliseconds to achieve rapid, dynamic body patterning.

DLX Genes: Roles in Development and Cancer

Homeobox genes control body patterning and cell-fate decisions during development. The homeobox genes consist of many families, only some of which have been investigated regarding a possible role in tumorigenesis. Dysregulation of HOX family genes have been widely implicated in cancer etiology. DLX homeobox genes, which belong to the NK-like family, exert dual roles in development and cancer. The DLX genes are the key transcription factors involved in regulating the development of craniofacial structures in vertebrates. The three DLX bigenes have overlapping expression in the branchial arches. Disruption of DLX function has destructive consequences in organogenesis and is associated with certain congenital disorders in humans. The role of DLX genes in oncogenesis is only beginning to emerge. DLX2 diminishes cellular senescence by regulating p53 function, whereas DLX4 has been associated with metastasis in breast cancer. In human ovarian cancer cells, DLX5 is essential for regulating AKT signaling, thereby promoting cell proliferation and survival. We previously implicated Dlx5 as an oncogene in murine T-cell lymphoma driven by a constitutively active form of Akt2. In this mouse model, overexpression of Dlx5 was caused by a chromosomal rearrangement that juxtaposed the Tcr-beta promoter region near the Dlx5 locus. Moreover, transgenic mice overexpressing Dlx5, specifically in immature T-cells, develop spontaneous thymic lymphomas. Oncogenesis in this mouse model involves binding of Dlx5 to the Notch1 and Notch3 gene loci to activate their transcription. Dlx5 also cooperates with Akt signaling to accelerate lymphomagenesis by activating Wnt signaling. We also discuss the fact that human DLX5 is aberrantly expressed in several human malignancies.

Comparison of the Zebrafish Embryo Toxicity Assay and the General and Behavioral Embryo Toxicity Assay as New Approach Methods for Chemical Screening

The movement away from mammalian testing of potential toxicants and new chemical entities has primarily led to cell line testing and protein-based assays. However, these assays may not yet be sufficient to properly characterize the toxic potential of a chemical. The zebrafish embryo model is widely recognized as a potential new approach method for chemical testing that may provide a bridge between cell and protein-based assays and mammalian testing. The Zebrafish Embryo Toxicity (ZET) model is increasingly recognized as a valuable toxicity testing platform. The ZET assay focuses on the early stages of embryo development and is considered a more humane model compared to adult zebrafish testing. A complementary model has been developed that exposes larvae to toxicants at a later time point during development where body patterning has already been established. Here we compare the toxicity profiles of 20 compounds for this General and Behavioral Toxicity (GBT) assay to the ZET assay. The results show partially overlapping toxicity profiles along with unique information provided by each assay. It appears from this work that these two assays applied together can strengthen the use of zebrafish embryos/larvae as standard toxicity testing models.

Lysine demethylase 7a regulates murine anterior-posterior development by modulating the transcription of Hox gene cluster

Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a−/− mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a−/− embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates.

Regulatory gene function handoff allows essential gene loss in mosquitoes

Regulatory genes are often multifunctional and constrained, which results in evolutionary conservation. It is difficult to understand how a regulatory gene could be lost from one species’ genome when it is essential for viability in closely related species. The gene paired is a classic Drosophila pair-rule gene, required for formation of alternate body segments in diverse insect species. Surprisingly, paired was lost in mosquitoes without disrupting body patterning. Here, we demonstrate that a paired family member, gooseberry, has acquired paired-like expression in the malaria mosquito Anopheles stephensi. Anopheles-gooseberry CRISPR-Cas9 knock-out mutants display pair-rule phenotypes and alteration of target gene expression similar to what is seen in Drosophila and beetle paired mutants. Thus, paired was functionally replaced by the related gene, gooseberry, in mosquitoes. Our findings document a rare example of a functional replacement of an essential regulatory gene and provide a mechanistic explanation of how such loss can occur.

The Canonical Wnt Pathway as a Key Regulator in Liver Development, Differentiation and Homeostatic Renewal

The canonical Wnt (Wnt/β-catenin) signalling pathway is highly conserved and plays a critical role in regulating cellular processes both during development and in adult tissue homeostasis. The Wnt/β-catenin signalling pathway is vital for correct body patterning and is involved in fate specification of the gut tube, the primitive precursor of liver. In adults, the Wnt/β-catenin pathway is increasingly recognised as an important regulator of metabolic zonation, homeostatic renewal and regeneration in response to injury throughout the liver. Herein, we review recent developments relating to the key role of the pathway in the patterning and fate specification of the liver, in the directed differentiation of pluripotent stem cells into hepatocytes and in governing proliferation and zonation in the adult liver. We pay particular attention to recent contributions to the controversy surrounding homeostatic renewal and proliferation in response to injury. Furthermore, we discuss how crosstalk between the Wnt/β-catenin and Hedgehog (Hh) and hypoxia inducible factor (HIF) pathways works to maintain liver homeostasis. Advancing our understanding of this pathway will benefit our ability to model disease, screen drugs and generate tissue and organ replacements for regenerative medicine.

Primary Cilia and Calcium Signaling Interactions

The calcium ion (Ca2+) is a diverse secondary messenger with a near-ubiquitous role in a vast array of cellular processes. Cilia are present on nearly every cell type in either a motile or non-motile form; motile cilia generate fluid flow needed for a variety of biological processes, such as left–right body patterning during development, while non-motile cilia serve as the signaling powerhouses of the cell, with vital singling receptors localized to their ciliary membranes. Much of the research currently available on Ca2+-dependent cellular actions and primary cilia are tissue-specific processes. However, basic stimuli-sensing pathways, such as mechanosensation, chemosensation, and electrical sensation (electrosensation), are complex processes entangled in many intersecting pathways; an overview of proposed functions involving cilia and Ca2+ interplay will be briefly summarized here. Next, we will focus on summarizing the evidence for their interactions in basic cellular activities, including the cell cycle, cell polarity and migration, neuronal pattering, glucose-mediated insulin secretion, biliary regulation, and bone formation. Literature investigating the role of cilia and Ca2+-dependent processes at a single-cellular level appears to be scarce, though overlapping signaling pathways imply that cilia and Ca2+ interact with each other on this level in widespread and varied ways on a perpetual basis. Vastly different cellular functions across many different cell types depend on context-specific Ca2+ and cilia interactions to trigger the correct physiological responses, and abnormalities in these interactions, whether at the tissue or the single-cell level, can result in diseases known as ciliopathies; due to their clinical relevance, pathological alterations of cilia function and Ca2+ signaling will also be briefly touched upon throughout this review.

Pattern regulation in a regenerating jellyfish

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around ‘hubs’ which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber ‘spokes’. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/β-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.

Hedgehog signaling controls segmentation dynamics and diversity via msx1 in a spider embryo

Hedgehog (Hh) signaling plays fundamental roles in animal body patterning. Understanding its mechanistic complexity requires simple tractable systems that can be used for these studies. In the early spider embryo, Hh signaling mediates the formation of overall anterior-posterior polarity, yet it remains unclear what mechanisms link the initial Hh signaling activity with body axis segmentation, in which distinct periodic stripe-forming dynamics occur depending on the body region. We performed genome-wide searches for genes that transcriptionally respond to altered states of Hh signaling. Characterization of genes negatively regulated by Hh signaling suggested that msx1, encoding a conserved transcription factor, functions as a key segmentation gene. Knockdown of msx1 prevented all dynamic processes causing spatial repetition of stripes, including temporally repetitive oscillations and bi-splitting, and temporally nonrepetitive tri-splitting. Thus, Hh signaling controls segmentation dynamics and diversity via msx1. These genome-wide data from an invertebrate illuminate novel mechanistic features of Hh-based patterning.