Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes, adopting a shape named 'scutoid' that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here, we use live imaging of the sea star embryo coupled with deep learning-based segmentation to dissect the relative contributions of cell density, tissue compaction and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing immediately after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Localization and origins of juvenile skeletogenic cells in the sea urchin Lytechinuspictus.

The development of the sea urchin larval body plan is well understood from extensive studies of embryonic patterning. However, fewer studies have investigated the late larval stages during which the unique pentaradial adult body plan develops. Previous work on late larval development highlights major tissue changes leading up to metamorphosis, but the location of specific cell types during juvenile development is less understood. Here, we improve on technical limitations by applying highly sensitive hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) to the fast-developing and transparent sea urchin Lytechinus pictus, with a focus on skeletogenic cells. First, we show that HCR-FISH can be used in L. pictus to precisely localize skeletogenic cells in the rudiment. In doing so, we provide a detailed staging scheme for the appearance of skeletogenic cells around the rudiment prior to and during biomineralization and show that many skeletogenic cells unassociated with larval rods localize outside of the rudiment prior to localizing inside. Second, we show that downstream biomineralization genes have similar expression patterns during larval and juvenile skeletogenesis, suggesting some conservation of skeletogenic mechanisms during development between stages. Third, we find co-expression of blastocoelar and skeletogenic cell markers around juvenile skeleton located outside of the rudiment, which is consistent with data showing that cells from the non-skeletogenic mesoderm embryonic lineage contribute to the juvenile skeletogenic cell lineage. This work sets the foundation for subsequent studies of other cell types in the late larva of L. pictus to better understand juvenile body plan development, patterning, and evolution.

Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus.

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Treatment methods for cervical intraepithelial neoplasia in England: A cost-effectiveness analysis.

OBJECTIVE: To compare the cost-effectiveness of different treatments for cervical intraepithelial neoplasia (CIN). DESIGN: A cost-effectiveness analysis based on data available in the literature and expert opinion. SETTING: England. POPULATION: Women treated for CIN. METHODS: We developed a decision-analytic model to simulate the clinical course of 1000 women who received local treatment for CIN and were followed up for 10 years after treatment. In the model we considered surgical complications as well as oncological and reproductive outcomes over the 10-year period. The costs calculated were those incurred by the National Health Service (NHS) of England. MAIN OUTCOME MEASURES: Cost per one CIN2+ recurrence averted (oncological outcome); cost per one preterm birth averted (reproductive outcome); overall cost per one adverse oncological or reproductive outcome averted. RESULTS: For young women of reproductive age, large loop excision of the transformation zone (LLETZ) was the most cost-effective treatment overall at all willingness-to-pay thresholds. For postmenopausal women, LLETZ remained the most cost-effective treatment up to a threshold of £31,500, but laser conisation became the most cost-effective treatment above that threshold. CONCLUSIONS: LLETZ is the most cost-effective treatment for both younger and older women. However, for older women, more radical excision with laser conisation could also be considered if the NHS is willing to spend more than £31,500 to avert one CIN2+ recurrence.

The intelligent knife (iKnife) and its intraoperative diagnostic advantage for the treatment of cervical disease.

Clearance of surgical margins in cervical cancer prevents the need for adjuvant chemoradiation and allows fertility preservation. In this study, we determined the capacity of the rapid evaporative ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), to discriminate between healthy, preinvasive, and invasive cervical tissue. Cervical tissue samples were collected from women with healthy, human papilloma virus (HPV) ± cervical intraepithelial neoplasia (CIN), or cervical cancer. A handheld diathermy device generated surgical aerosol, which was transferred into a mass spectrometer for subsequent chemical analysis. Combination of principal component and linear discriminant analysis and least absolute shrinkage and selection operator was employed to study the spectral differences between groups. Significance of discriminatory m/z features was tested using univariate statistics and tandem MS performed to elucidate the structure of the significant peaks allowing separation of the two classes. We analyzed 87 samples (normal = 16, HPV ± CIN = 50, cancer = 21 patients). The iKnife discriminated with 100% accuracy normal (100%) vs. HPV ± CIN (100%) vs. cancer (100%) when compared to histology as the gold standard. When comparing normal vs. cancer samples, the accuracy was 100% with a sensitivity of 100% (95% CI 83.9 to 100) and specificity 100% (79.4 to 100). Univariate analysis revealed significant MS peaks in the cancer-to-normal separation belonging to various classes of complex lipids. The iKnife discriminates healthy from premalignant and invasive cervical lesions with high accuracy and can improve oncological outcomes and fertility preservation of women treated surgically for cervical cancer. Larger in vivo research cohorts are required to validate these findings.

Lineage tracing shows that cell size asymmetries predict the dorsoventral axis in the sea star embryo.

BACKGROUND: Cell size asymmetries are often linked to cell fate decisions, due to cell volumes and cell fate determinants being unequally partitioned during asymmetric cell divisions. A clear example is found in the sea urchin embryo, where a characteristic and obvious unequal 4th cleavage generates micromeres, which are necessary for mesendoderm cell fate specification. Unlike sea urchin development, sea star development is generally thought to have only equal cleavage. However, subtle cell size asymmetries can be observed in sea star embryos; whether those cell size asymmetries are consistently produced during sea star development and if they are involved in cell fate decisions remains unknown. RESULTS: Using confocal live imaging of early embryos we quantified cell size asymmetries in 16-cell stage embryos of two sea star species, Patiria miniata and Patiriella regularis. Using photoconversion to perform lineage tracing, we find that the position of the smallest cells of P. miniata embryos is biased toward anterior ventral tissues. However, both blastomere dissociation and mechanical removal of one small cell do not prevent dorsoventral (DV) axis formation, suggesting that embryos compensate for the loss of those cells and that asymmetrical partitioning of maternal determinants is not strictly necessary for DV patterning. Finally, we show that manipulating cell size to introduce artificial cell size asymmetries is not sufficient to direct the positioning of the future DV axis in P. miniata embryos. CONCLUSIONS: Our results show that although cell size asymmetries are consistently produced during sea star early cleavage and are predictive of the DV axis, they are not necessary to instruct DV axis formation.

Comparative effectiveness and risk of preterm birth of local treatments for cervical intraepithelial neoplasia and stage IA1 cervical cancer: a systematic review and network meta-analysis.

BACKGROUND: The trade-off between comparative effectiveness and reproductive morbidity of different treatment methods for cervical intraepithelial neoplasia (CIN) remains unclear. We aimed to determine the risks of treatment failure and preterm birth associated with various treatment techniques. METHODS: In this systematic review and network meta-analysis, we searched MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials database for randomised and non-randomised studies reporting on oncological or reproductive outcomes after CIN treatments from database inception until March 9, 2022, without language restrictions. We included studies of women with CIN, glandular intraepithelial neoplasia, or stage IA1 cervical cancer treated with excision (cold knife conisation [CKC], laser conisation, and large loop excision of the transformation zone [LLETZ]) or ablation (radical diathermy, laser ablation, cold coagulation, and cryotherapy). We excluded women treated with hysterectomy. The primary outcomes were any treatment failure (defined as any abnormal histology or cytology) and preterm birth (<37 weeks of gestation). The network for preterm birth also included women with untreated CIN (untreated colposcopy group). The main reference group was LLETZ for treatment failure and the untreated colposcopy group for preterm birth. For randomised controlled trials, we extracted group-level summary data, and for observational studies, we extracted relative treatment effect estimates adjusted for potential confounders, when available, and we did random-effects network meta-analyses to obtain odds ratios (ORs) with 95% CIs. We assessed within-study and across-study risk of bias using Cochrane tools. This systematic review is registered with PROSPERO, CRD42018115495 and CRD42018115508. FINDINGS: 7880 potential citations were identified for the outcome of treatment failure and 4107 for the outcome of preterm birth. After screening and removal of duplicates, the network for treatment failure included 19 240 participants across 71 studies (25 randomised) and the network for preterm birth included 68 817 participants across 29 studies (two randomised). Compared with LLETZ, risk of treatment failure was reduced for other excisional methods (laser conisation: OR 0·59 [95% CI 0·44-0·79] and CKC: 0·63 [0·50-0·81]) and increased for laser ablation (1·69 [1·27-2·24]) and cryotherapy (1·84 [1·33-2·56]). No differences were found for the comparison of cold coagulation versus LLETZ (1·09 [0·68-1·74]) but direct data were based on two small studies only. Compared with the untreated colposcopy group, risk of preterm birth was increased for all excisional techniques (CKC: 2·27 [1·70-3·02]; laser conisation: 1·77 [1·29-2·43]; and LLETZ: 1·37 [1·16-1·62]), whereas no differences were found for ablative methods (laser ablation: 1·05 [0·78-1·41]; cryotherapy: 1·01 [0·35-2·92]; and cold coagulation: 0·67 [0·02-29·15]). The evidence was based mostly on observational studies with their inherent risks of bias, and the credibility of many comparisons was low. INTERPRETATION: More radical excisional techniques reduce the risk of treatment failure but increase the risk of subsequent preterm birth. Although there is uncertainty, ablative treatments probably do not increase risk of preterm birth, but are associated with higher failure rates than excisional techniques. Although we found LLETZ to have balanced effectiveness and reproductive morbidity, treatment choice should rely on a woman's age, size and location of lesion, and future family planning. FUNDING: National Institute for Health and Care Research: Research for Patient Benefit.

Movement and storage of nematocysts across development in the nudibranch Berghia stephanieae (Valdés, 2005).

BACKGROUND: Intracellular sequestration requires specialized cellular and molecular mechanisms allowing a predator to retain and use specific organelles that once belonged to its prey. Little is known about how common cellular mechanisms, like phagocytosis, can be modified to selectively internalize and store foreign structures. One form of defensive sequestration involves animals that sequester stinging organelles (nematocysts) from their cnidarian prey. While it has been hypothesized that nematocysts are identified by specialized phagocytic cells for internalization and storage, little is known about the cellular and developmental mechanisms of this process in any metazoan lineage. This knowledge gap is mainly due to a lack of genetically tractable model systems among predators and their cnidarian prey. RESULTS: Here, we introduce the nudibranch Berghia stephanieae as a model system to investigate the cell, developmental, and physiological features of nematocyst sequestration selectivity. We first show that B. stephanieae, which feeds on Exaiptasia diaphana, selectively sequesters nematocysts over other E. diaphana tissues found in their digestive gland. Using confocal microscopy, we document that nematocyst sequestration begins shortly after feeding and prior to the formation of the appendages (cerata) where the organ responsible for sequestration (the cnidosac) resides in adults. This finding is inconsistent with previous studies that place the formation of the cnidosac after cerata emerge. Our results also show, via live imaging assays, that both nematocysts and dinoflagellates can enter the nascent cnidosac structure. This result indicates that selectivity for nematocysts occurs inside the cnidosac in B. stephanieae, likely in the cnidophage cells themselves. CONCLUSIONS: Our work highlights the utility of B. stephanieae for future research, because: (1) this species can be cultured in the laboratory, which provides access to all developmental stages, and (2) the transparency of early juveniles makes imaging techniques (and therefore cell and molecular assays) feasible. Our results pave the way for future studies using live imaging and targeted gene editing to identify the molecular mechanisms involved in nematocyst sequestration. Further studies of nematocyst sequestration in B. stephanieae will also allow us to investigate how common cellular mechanisms like phagocytosis can be modified to selectively internalize and store foreign structures.

BMP signaling plays a role in anterior-neural/head development, but not organizer activity, in the gastropod Crepidula fornicata.

BMP signaling is involved in many aspects of metazoan development, with two of the most conserved functions being to pattern the dorsal-ventral axis and to specify neural versus epidermal fates. An active area of research within developmental biology asks how BMP signaling was modified over evolution to build disparate body plans. Animals belonging to the superclade Spiralia/Lophotrochozoa are excellent experimental subjects for studying the evolution of BMP signaling because a highly conserved, stereotyped early cleavage program precedes the emergence of distinct body plans. In this study we examine the role of BMP signaling in one representative, the slipper snail Crepidula fornicata. We find that mRNAs encoding BMP pathway components (including the BMP ligand decapentaplegic, and BMP antagonists chordin and noggin-like proteins) are not asymmetrically localized along the dorsal-ventral axis in the early embryo, as they are in other species. Furthermore, when BMP signaling is perturbed by adding ectopic recombinant BMP4 protein, or by treating embryos with the selective Activin receptor-like kinase-2 (ALK-2) inhibitor Dorsomorphin Homolog 1 (DMH1), we observe no obvious effects on dorsal-ventral patterning within the posterior (post-trochal) region of the embryo. Instead, we see effects on head development and the balance between neural and epidermal fates specifically within the anterior, pre-trochal tissue derived from the 1q1 lineage. Our experiments define a window of BMP signaling sensitivity that ends at approximately 44-48 â€‹hours post fertilization, which occurs well after organizer activity has ended and after the dorsal-ventral axis has been determined. When embryos were exposed to BMP4 protein during this window, we observed morphogenetic defects leading to the separation of the anterior, 1q lineage from the rest of the embryo. The 1q-derived organoid remained largely undifferentiated and was radialized, while the post-trochal portion of the embryo developed relatively normally and exhibited clear signs of dorsal-ventral patterning. When embryos were exposed to DMH1 during the same time interval, we observed defects in the head, including protrusion of the apical plate, enlarged cerebral ganglia and ectopic ocelli, but otherwise the larvae appeared normal. No defects in shell development were noted following DMH1 treatments. The varied roles of BMP signaling in the development of several other spiralians have recently been examined. We discuss our results in this context, and highlight the diversity of developmental mechanisms within spiral-cleaving animals.

The Impact of Focality and Centricity on Vulvar Intraepithelial Neoplasia on Disease Progression in HIV+ Patients: A 10-Year Retrospective Study.

BACKGROUND: The impact of lesion focality and centricity in relation to patient outcome and disease recurrence of vulvar intraepithelial neoplasia (VIN) is an understudied area of research, especially in immunocompromised women. The prevalence and incidence of VIN have increased steadily since the 1980s because of the co-existence of human papillomavirus (HPV) and human immunodeficiency virus (HIV). In this study, we retrospectively examined the records of VIN patients to determine the effect of lesion focality and centricity with respect to the interval to disease recurrence. MATERIALS AND METHODS: All women diagnosed with VIN and managed between January 2002 and December 2011 were included (n = 90) and followed up until December 2017. Symptoms at the time of presentation, including HIV positivity (n = 75), were collated, including the influences of multifocality and multicentricity on time to disease recurrence. RESULTS: Multicentricity caused a more rapid recurrence of disease than unicentricity (p = 0.006), whereas multifocality increased the risk of recurrence more than unifocality (p < 0.0001). Viral load in the HIV+ patients was not associated with time to disease recurrence, but the reduced number of CD4+ lymphocytes present in HIV+ patients was. Treatment modalities had no effect on disease recurrence. CONCLUSION: Both focality and centricity have effects on interval to recurrence and final patient outcome, with multifocal disease having a poorer prognosis. Centricity and focality should be recorded at the time of diagnosis and act as a warning for disease recurrence. HIV+ VIN patients with multifocal disease and/or known immunosuppression (low CD4+ lymphocyte counts) should be regarded as "high-risk" patients and treated accordingly.

Establishment and activity of the D quadrant organizer in the marine gastropod Crepidula fornicata.

During development in metazoan embryos, the fundamental embryonic axes are established by organizing centers that influence the fates of nearby cells. Among the spiralians, a large and diverse branch of protostome metazoans, studies have shown that an organizer sets up the dorsal-ventral axis, which arises from one of the four basic cell quadrants during development (the dorsal, D quadrant). Studies in a few species have also revealed variation in terms of how and when the D quadrant and the organizer are established. In some species the D quadrant is specified conditionally, via cell-cell interactions, while in others it is specified autonomously, via asymmetric cell divisions (such as those involving the formation of polar lobes). The third quartet macromere (3D) typically serves as the spiralian organizer; however, other cells born earlier or later in the D quadrant lineage can serve as the organizer, such as the 2d micromere in the annelid Capitella teleta or the 4d micromere in the mollusc Crepidula fornicata. Here we present work carried out in the snail C. fornicata to show that establishment of a single D quadrant appears to rely on a combination of both autonomous (via inheritance of the polar lobe) and conditional mechanisms (involving induction via the progeny of the first quartet micromeres). Through systematic ablation of cells, we show that D quadrant identity is established between 5th and 6th cleavage stages, as it is in other spiralians that use conditional specification. Subsequently, following the next cell cycle, organizer activity takes place soon after the birth of the 4d micromere. Therefore, unlike the case in other spiralians that use conditional specification, the specification of the D quadrant and the activity of the dorso-ventral organizer are temporally and spatially uncoupled. We also present data on organizer function in naturally-occurring and experimentally-induced twin embryos, which possess multiple D quadrants. We show that supernumerary D quadrants can arise in C. fornicata (either spontaneously or following polar lobe removal); when multiple D quadrants are present these do not exhibit effective organizer activity. We conclude that the polar lobe is not required for D quadrant specification, though it could play a role in effective organizer activity. We also tested whether the inheritance of the small polar lobe by the D quadrant is associated with the ability to laterally inhibit neighboring quadrants by direct contact in order to normally prevent supernumerary organizers from arising. Finally, we discuss the variation of spiralian organizers in a phylogenetic context.

Morphogenesis along the animal-vegetal axis: fates of primary quartet micromere daughters in the gastropod Crepidula fornicata.

BACKGROUND: The Spiralia are a large, morphologically diverse group of protostomes (e.g. molluscs, annelids, nemerteans) that share a homologous mode of early development called spiral cleavage. One of the most highly-conserved features of spiralian development is the contribution of the primary quartet cells, 1a-1d, to the anterior region of the embryo (including the brain, eyes, and the anterior ciliary band, called the prototroch). Yet, very few studies have analyzed the ultimate fates of primary quartet sub-lineages, or examined the morphogenetic events that take place in the anterior region of the embryo. RESULTS: This study focuses on the caenogastropod slipper snail, Crepidula fornicata, a model for molluscan developmental biology. Through direct lineage tracing of primary quartet daughter cells, and examination of these cells during gastrulation and organogenesis stages, we uncovered behaviors never described before in a spiralian. For the first time, we show that the 1a2-1d2 cells do not contribute to the prototroch (as they do in other species) and are ultimately lost before hatching. During gastrulation and anterior-posterior axial elongation stages, these cells cleavage-arrest and spread dramatically, contributing to a thin provisional epidermis on the dorsal side of the embryo. This spreading is coupled with the displacement of the animal pole, and other pretrochal cells, closer to the ventrally-positioned mouth, and the vegetal pole. CONCLUSIONS: This is the first study to document the behavior and fate of primary quartet sub-lineages among molluscs. We speculate that the function of 1a2-1d2 cells (in addition to two cells derived from 1d12, and the 2b lineage) is to serve as a provisional epithelium that allows for anterior displacement of the other progeny of the primary quartet towards the anterior-ventral side of the embryo. These data support a new and novel mechanism for axial bending, distinct from canonical models in which axial bending is suggested to be driven primarily by differential proliferation of posterior dorsal cells. These data suggest also that examining sub-lineages in other spiralians will reveal greater variation than previously assumed.

Deployment of regulatory genes during gastrulation and germ layer specification in a model spiralian mollusc Crepidula.

BACKGROUND: During gastrulation, endoderm and mesoderm are specified from a bipotential precursor (endomesoderm) that is argued to be homologous across bilaterians. Spiralians also generate mesoderm from ectodermal precursors (ectomesoderm), which arises near the blastopore. While a conserved gene regulatory network controls specification of endomesoderm in deuterostomes and ecdysozoans, little is known about genes controlling specification or behavior of either source of spiralian mesoderm or the digestive tract. RESULTS: Using the mollusc Crepidula, we examined conserved regulatory factors and compared their expression to fate maps to score expression in the germ layers, blastopore lip, and digestive tract. Many genes were expressed in both ecto- and endomesoderm, but only five were expressed in ectomesoderm exclusively. The latter may contribute to epithelial-to-mesenchymal transition seen in ectomesoderm. CONCLUSIONS: We present the first comparison of genes expressed during spiralian gastrulation in the context of high-resolution fate maps. We found variation of genes expressed in the blastopore lip, mouth, and cells that will form the anus. Shared expression of many genes in both mesodermal sources suggests that components of the conserved endomesoderm program were either co-opted for ectomesoderm formation or that ecto- and endomesoderm are derived from a common mesodermal precursor that became subdivided into distinct domains during evolution.

Delayed transition to new cell fates during cellular reprogramming.

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.

The significance and scope of evolutionary developmental biology: a vision for the 21st century.

Evolutionary developmental biology (evo-devo) has undergone dramatic transformations since its emergence as a distinct discipline. This paper aims to highlight the scope, power, and future promise of evo-devo to transform and unify diverse aspects of biology. We articulate key questions at the core of eleven biological disciplines-from Evolution, Development, Paleontology, and Neurobiology to Cellular and Molecular Biology, Quantitative Genetics, Human Diseases, Ecology, Agriculture and Science Education, and lastly, Evolutionary Developmental Biology itself-and discuss why evo-devo is uniquely situated to substantially improve our ability to find meaningful answers to these fundamental questions. We posit that the tools, concepts, and ways of thinking developed by evo-devo have profound potential to advance, integrate, and unify biological sciences as well as inform policy decisions and illuminate science education. We look to the next generation of evolutionary developmental biologists to help shape this process as we confront the scientific challenges of the 21st century.

Left-right asymmetry in the sea urchin embryo: BMP and the asymmetrical origins of the adult.

Bilateral animals, including humans and most metazoans, are not perfectly symmetrical. Some internal structures are distributed asymmetrically to the right or left side. A conserved Nodal and BMP signaling system directs molecular pathways that impart the sidedness to those asymmetric structures. In the sea urchin embryo, one such asymmetrical structure, oddly enough, is the entire adult, which grows out of left sided structures produced in the larva. In a paper just published in PLOS Biology, BMP signaling is shown to be necessary early in larval development to initiate the asymmetric specification of one of those left-sided structures, called the left coelomic pouch. This study reports that BMP signaling activates a group of transcription factors asymmetrically in the left coelomic pouch only, which launch the pathway that eventually leads to the formation of the adult that emerges from the larva at metamorphosis.

Branching out: origins of the sea urchin larval skeleton in development and evolution.

It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production.

Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

Cephalopod vision: How to build a better eye.

Cephalopods' eyes superficially resemble our own, but because of their evolutionary and developmental history, the photoreceptors face forward, with the downstream neural circuitry in the brain, not the retina. Two new papers uncover molecular and developmental mechanisms underlying cephalopod visual development.