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Purpose: Metastatic progression and treatment resistance of breast cancer has been associated with epithelial-mesenchymal transition (EMT), including downregulation of E-cadherin (CDH1) gene expression, which can be initiated by inflammatory signaling such as by COX-2 (PTGS2). However, E-cadherin expression is maintained in many advanced breast cancers, including inflammatory BC (IBC), where it plays an essential role in forming tumor cell emboli within the cancer parenchyma and dermal lymph vasculature and which predict poor outcomes. Thus, the study of IBC offers an opportunity to understand the mechanisms of IBC and other aggressive BCs that lead to E-cadherin-associated cluster-based metastasis, which has recently received heightened recognition. Here, we have investigated the mechanisms that sustain E-cadherin expression in metastatic breast cancer to identify new targeted treatment options. Study Design and Methods: In vitro emboli formation assays and gene/protein expression studies with IBC (SUM149, IBC-3) and non-IBC (SUM159, MCF-7) cell lines along with gene silencing, overexpression, and pharmacological interventions. Analysis of experimental (SUM149) and spontaneous (orthotopic patient-derived xenograft, PDX) lung metastases and of circulating tumor cells (CTCs) in xenograft models treated with celecoxib (Cxb) and/or paclitaxel by imaging cytometry, immunohistochemistry, and/or Western analysis. Results: By analyzing the transcription factor C/EBPδ (CEBPD) and cells derived from inflammatory breast cancer (IBC), an aggressive breast cancer subtype that often presents with E-cadherin-dependent tumor cell emboli, we discovered that COX-2, unexpectedly, sustained E-cadherin protein expression without changing its mRNA levels. Using an in vitro tumor cell emboli culture paradigm (3D), we found that COX-2 or its metabolite PGE2 increased AKT activity and the inhibitory phosphorylation on GSK3β that prevents degradation of p120 catenin (CTNND1), a stabilizer of E-cadherin adhesion complexes. Conversely, the COX-2 inhibitor celecoxib downregulated E-cadherin specifically at the protein level and caused cell death in 3D. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcomes and, along with inhibitory GSK3β phosphorylation, in patient-derived xenografts (PDX) of metastatic triple-negative breast cancers (TNBC). Celecoxib alone decreased E-cadherin protein expression within xenograft primary tumors, reduced circulating tumor cells (CTCs) and clusters, and sensitized lung metastases to paclitaxel treatment. Conclusions: Our study uncovered a novel function of COX-2/PGE2 in promoting E-cadherin protein expression and cell-cell adhesions that are relevant for tumor cell cluster-based metastasis. Indeed, COX-2 inhibition reduced CTC clusters in a xenograft model, and sentized established metastases to chemotherapy. These results suggest that patients with COX-2+/E-cadherin+ metastastic BC, including IBC, may specifically benefit from targeting the PGE2 pathway in cobniation therapy approaches. Citation Format: Kuppusamy Balamurugan, Saadiya Sehareen, Savitri Krishnamurthy, Shikha Sharan, Wei Tang, Naoto Ueno, Stefan Ambs, Dipak Poria, Esta Sterneck. Promotion of E-cadherin-mediated tumor cell adhesion by COX-2/GSK3β signaling is a targetable mechanism of metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-03.

Background: Novel mechanism-oriented targeted therapies are needed for aggressive breast cancers such as triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). We previously showed the oncogenic receptor tyrosine kinase AXL crucially contributes to the aggressiveness of TNBC and TN-IBC, which suggests that AXL is a potential therapeutic target. However, AXL-targeted therapy is insufficient to control the progress of aggressive breast cancer in preclinical models. Therefore, to further enhance the efficacy of AXL-targeted therapy in aggressive breast cancer, we conducted a synthetic lethality screen of pooled kinome siRNA and identified TANK-binding kinase 1 (TBK1) as a potential target. TBK1 is a necessary serine/threonine protein kinase that regulates innate and adaptive immune responses, T cell migration, and anti-tumor immune responses. In the present work, we validated that AXL and TBK1 inhibition have a synergistic anti-tumor effect by modulating the tumor microenvironment (TME). Methods: To determine the synergistic effect of AXL and TBK1 inhibition on cell proliferation and colony formation in vitro, we first used siRNA-mediated knockdown (KD) or CRISPR/Cas9 knockout (KO) to independently silence AXL and TBK1 expression. We also used pharmacological inhibitors to inactivate AXL and TBK1 signaling and conducted similar studies. To determine the effect of targeting TBK1 and AXL pathways in vivo, we injected control or TBK1 KO murine 4T1.2 TNBC cells into BALB/c mice and assessed the efficacy of the AXL inhibitor TP-0903 in reducing the tumor growth. We used multicolor flow cytometry and multiplexed immunostaining to examine the population of TME components in control and TBK1 KO 4T1.2 tumors treated with vehicle or TP-0903. We also used an in vitro migration assay to test the effect of targeting AXL and TBK1 on the recruitment of CD8+ T cells. We performed a mouse chemokine array to identify intratumoral chemokines involved in regulating infiltrating immune cells and used RT-PCR to validate these findings. Results: Compared with AXL or TBK1 suppression alone, the genomic or pharmacological suppression of both AXL and TBK1 resulted in a significantly greater reduction of TNBC and TN-IBC cell growth and colony-forming ability (P < 0.01). In our syngeneic 4T1.2 TNBC mouse tumor model, TP-0903 was more effective at controlling tumor growth from TBK1-KO cells than from control cells (P < 0.0005). TBK1-KO 4T1.2 tumors treated with TP-0903 had a significantly higher population of functional cytotoxic T cells (GranzymeB+CD3+CD8+) than control KO tumors treated with vehicle or TP-0903. Furthermore, our migration assay showed that TBK1-KO/AXL-KD SUM149 cell-conditioned media attracted more CD8+ T cells than control cell-conditioned media, suggesting the inhibition of AXL/TBK1 signaling enhances the recruitment of CD8+ T cells through a paracrine effect. Our chemokine array revealed increased CXCL16 levels in TP-0903-treated TBK1-KO tumors. Conclusion: Targeting TBK1 could enhance the sensitivity of aggressive breast cancer cells to AXL-targeted therapy in vitro and in vivo. Our preliminary TME data suggest that combined AXL and TBK1 inhibition enhances the anti-tumor immune environment by upregulating secretion of CXCL16 by tumor cells, resulting in the increased recruitment of cytotoxic CD8+ T cells. In future studies, we will further elucidate the role that CXCL16 upregulation plays in this novel synergy. Significance of this findings: This study will enable us to develop hypothesis-driven novel combination therapies for patients with aggressive breast cancer that can be rapidly tested as a clinical trial, leading to an improvement of treatment and patients’ outcome. Citation Format: Lan Phi, Takashi Semba, Jason Foulks, Steven Warner, David Bearss, Savitri Krishnamurthy, James Long, James Reuben, Debu Tripathy, Naoto Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-10.

Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.

Summary: The physiological importance of biomolecular condensates is widely recognized, but how it is controlled in time and space during development is largely unknown. Here, we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass because they receive weaker tensile forces. Furthermore, analyses using MDCK cells demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to the establishment of robust cell-cell adhesion during early development.

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.

The leukodystrophies cause severe neurodevelopmental defects from birth and follow an incurable and progressive course that often leads to premature death. It has recently been reported that abnormalities in aminoacyl t-RNA synthetase (ARS) genes are linked to various unique leukodystrophies and leukoencephalopathies. Aminoacyl t-RNA synthetase proteins are fundamentally known as the first enzymes of translation, catalysing the conjugation of amino acids to cognate tRNAs for protein synthesis. It is known that certain aminoacyl t-RNA synthetase have multiple non-canonical roles in both transcription and translation, and their disruption results in varied and complicated phenotypes. We clinically and genetically studied seven patients (six male and one female; aged 2 to 12 years) from five unrelated families who all showed the same phenotypes of severe developmental delay or arrest (7/7), hypotonia (6/7), deafness (7/7) and inability to speak (6/7). The subjects further developed intractable epilepsy (7/7) and nystagmus (6/6) with increasing age. They demonstrated characteristic laboratory data, including increased lactate and/or pyruvate levels (7/7), and imaging findings (7/7), including calcification and abnormal signals in the white matter and pathological involvement (2/2) of the corticospinal tracts. Through whole-exome sequencing, we discovered genetic abnormalities in lysyl-tRNA synthetase (KARS). All patients harboured the variant [c.1786C>T, p.Leu596Phe] KARS isoform 1 ([c.1702C>T, p.Leu568Phe] of KARS isoform 2) either in the homozygous state or compound heterozygous state with the following KARS variants, [c.879+1G>A; c.1786C>T, p.Glu252_Glu293del; p.Leu596Phe] ([c.795+1G>A; c.1702C>T, p.Glu224_Glu255del; p.Leu568Phe]) and [c.650G>A; c.1786C>T, p.Gly217Asp; p.Leu596Phe] ([c.566G>A; c.1702C>T, p.Gly189Asp; p.Leu568Phe]). Moreover, similarly disrupted lysyl-tRNA synthetase (LysRS) proteins showed reduced enzymatic activities and abnormal CNSs in Xenopus embryos. Additionally, LysRS acts as a non-canonical inducer of the immune response and has transcriptional activity. We speculated that the complex functions of the abnormal LysRS proteins led to the severe phenotypes in our patients. These KARS pathological variants are novel, including the variant [c.1786C>T; p.Leu596Phe] (c.1702C>T; p.Leu568Phe) shared by all patients in the homozygous or compound-heterozygous state. This common position may play an important role in the development of severe progressive leukodystrophy. Further research is warranted to further elucidate this relationship and to investigate how specific mutated LysRS proteins function to understand the broad spectrum of KARS-related diseases.

Background Expert knowledge is often shared among multidisciplinary academic teams at tumor boards (TBs) across the country, but these conversations exist in silos and do not reach the wider oncology community. Objective Using an oncologist-only question and answer (Q&A) website, we sought to document expert insights from TBs at National Cancer Institute–designated Comprehensive Cancer Centers (NCI-CCCs) to provide educational benefits to the oncology community. Methods We designed a process with the NCI-CCCs to document and share discussions from the TBs focused on areas of practice variation on theMednet, an interactive Q&A website of over 13,000 US oncologists. The faculty translated the TB discussions into concise, non–case-based Q&As on theMednet. Answers were peer reviewed and disseminated in email newsletters to registered oncologists. Reach and engagement were measured. Following each Q&A, a survey question asked how the TB Q&As impacted the readers’ practice. Results A total of 23 breast, thoracic, gastrointestinal, and genitourinary programs from 16 NCI-CCC sites participated. Between December 2016 and July 2021, the faculty highlighted 368 questions from their TBs. Q&As were viewed 147,661 times by 7381 oncologists at 3515 institutions from all 50 states. A total of 277 (75%) Q&As were viewed every month. Of the 1063 responses to a survey question on how the Q&A affected clinicians’ practices, 646 (61%) reported that it confirmed their current practice, 163 (20%) indicated that a Q&A would change their future practice, and 214 (15%) reported learning something new. Conclusions Through an online Q&A platform, academics at the NCI-CCCs share knowledge outside the walls of academia with oncologists across the United States. Access to up-to-date expert knowledge can reassure clinicians’ practices, significantly impact patient care in community practices, and be a source of new knowledge and education.

Behavioral responses to environmental factors at the planktonic larval stage can have a crucial influence on habitat selection and therefore adult distributions in many benthic organisms. Reef-building corals show strong patterns of zonation across depth or underwater topography, with different suites of species aggregating in different light environments. One potential mechanism driving this pattern is the response of free-swimming larvae to light. However, there is little experimental support for this hypothesis; in particular, there are few direct and quantitative observations of larval behavior in response to light. Here, we analyzed the swimming behavior of larvae of the common reef coral Acropora tenuis under various light conditions. Larvae exhibited a step-down photophobic response, i.e. a marked decrease in swimming speed, in response to a rapid attenuation (step-down) of light intensity. Observations of larvae under different wavelengths indicated that only the loss of blue light (wavelengths between 400 and 500 nm) produced a significant response. Mathematical simulations of this step-down photophobic response indicate that larvae will aggregate in the lighter areas of two-dimensional large rectangular fields. These results suggest that the step-down photophobic response of coral larvae may play an important role in determining where larval settle on the reef.

SummaryAlthough the physiological importance of biomolecular condensates is widely recognized, how it is controlled in time and space during development is largely unknown. Here we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass, as they receive weaker tensile forces. Furthermore, analyses using MDCK cells have demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to establishing robust cell-cell adhesion during early development.

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca2+ signals play an essential role in the active cell migration during gastrulation.

The stress distribution inside a Xenopus laevis tailbud embryo was estimated to examine the cause of the straightening and elongation. The embryos were cut in the middle, yielding a cross-section perpendicular to the body axis. The section was not flat, owing to the residual stress relief. The stress needed to restore the flatness corresponded to the stress inside the embryo and was calculated using the surface topography and Young's-moduli in the section. We found the areas of the notochord (Nc), neural tube (NT), and abdominal tissue (AT) bulged in the cross-section, which revealed that compressive forces acted in these tissues. The moduli of the Nc, NT, and AT were in the order of several thousand, hundred, and tens of pascals, respectively. In the Nc, the compressive force was largest and increased with the development, suggesting Nc playing a central role in the elongation. The bending moment generated by the AT was 10 times higher than that by the Nc in the early stages of the tailbud formation, and the two were similar in the latter stages, suggesting that the compressive force in the AT was the major cause of the straightening during the early stage. The straightening and elongation could be orchestrated by changes in the compressive forces acting on the Nc, NT, and AT over time. For the sake of simplicity, we calculated the compressive force only and neglected the tensile force. Thus, it should be noted that the amount of the compressive force was somewhat overestimated.

Cell proliferation is strictly regulated by the dosage balance among cell-cycle regulators such as CDK/cyclin complexes and CDK-Inhibitors. Even in the allotetraploid genome of Xenopus laevis, the dosage balance must be maintained for animals to stay alive, and the duplicated homeologous genes seem to have gradually changed, through evolution, resulting in the best genes for them to thrive. In the Xenopus laevis genome, while homeologous gene pairs of CDKs are fundamentally maintained and a few cyclin genes are amplified, homeologous gene pairs of the important CDK-Inhibitors, CDKn1c and CDKn2a, are deleted from chromosomes L and S. Although losses of CDKn1c and CDKn2a can lead to diseases in humans, their loss in X. laevis does not affect the animals' health. Also, another gene coding CDKn1b is lost besides CDKn1c and CDKn2a in the genome of Xenopus tropicalis. These findings suggest a high resistance of Xenopus to diseases. We also found that CDKn2c.S expression is higher than that of CDKn2c.L, and a conserved noncoding sequence (CNS) of CDKn2c genomic loci on X. laevis chromosome S and X. tropicalis has an enhancement activity in regulating the different expression. These findings together indicate a surprising fragility of CDK inhibitor gene loci in the Xenopus genome in spite of their importance, and may suggest that factors other than CDK-inhibitors decelerate cell-cycling in Xenopus.

Stable endosymbiotic relationships between cnidarian animals and dinoflagellate algae are vital for sustaining coral reef ecosystems. Recent studies have shown that elevated seawater temperatures can cause the collapse of their endosymbiosis, known as ‘bleaching’, and result in mass mortality. However, the molecular interplay between temperature responses and symbiotic states still remains unclear. To identify candidate genes relevant to the symbiotic stability, we performed transcriptomic analyses under multiple conditions using the symbiotic and apo-symbiotic (symbiont free) Exaiptasia diaphana, an emerging model sea anemone. Gene expression patterns showed that large parts of differentially expressed genes in response to heat stress were specific to the symbiotic state, suggesting that the host sea anemone could react to environmental changes in a symbiotic state-dependent manner. Comparative analysis of expression profiles under multiple conditions highlighted candidate genes potentially important in the symbiotic state transition under heat-induced bleaching. Many of these genes were functionally associated with carbohydrate and protein metabolisms in lysosomes. Symbiont algal genes differentially expressed in hospite encode proteins related to heat shock response, calcium signaling, organellar protein transport, and sugar metabolism. Our data suggest that heat stress alters gene expression in both the hosts and symbionts. In particular, heat stress may affect the lysosome-mediated degradation and transportation of substrates such as carbohydrates through the symbiosome (phagosome-derived organelle harboring symbiont) membrane, which potentially might attenuate the stability of symbiosis and lead to bleaching-associated symbiotic state transition.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

Broadcast-spawning scleractinian corals annually release their gametes with high levels of synchrony, both within and among species. However, the timing of spawning can vary inter-annually. In particular, the night of spawning relative to the full moon phase can vary considerably among years at some locations. Although multiple environmental factors can affect the night of spawning, their effects have not been quantitatively assessed at the multi-regional level. In this study, we analysed environmental factors that are potentially correlated with spawning day deviation, in relation to the full moon phase, in Acropora corals inhabiting seven reefs in Australia and Japan. We accordingly found that sea surface temperature and wind speed within one to two months prior to the full moon of the spawning month were strongly correlated with spawning day deviations. In addition, solar flux had a weak effect on the night of spawning. These findings indicate that Acropora have the capacity to adjust their development and physiology in response to environmental factors for fine-tuning the timing of synchronous spawning, thereby maximizing reproductive success and post-fertilization survival.

Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype.

Patterning of the vertebrate anterior–posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway.

Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development., Highlights • c-FLIP/CFLAR is specific to the vertebrate lineage. • A rapid evolutionary divergence of the c-FLIP subfamily members has occurred. • The pseudocatalytic triad is formed by evolutionarily conserved amino acids. • Vertebrate c-FLIP proteins work on both apoptotic inhibition and NF-κB activation. • c-FLIP proteins play a developmental role during embryogenesis of frog and fish.

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

Neural progenitor cells (NPCs), which are apicobasally elongated and densely packed in the developing brain, systematically move their nuclei/somata in a cell cycle–dependent manner, called interkinetic nuclear migration (IKNM): apical during G2 and basal during G1. Although intracellular molecular mechanisms of individual IKNM have been explored, how heterogeneous IKNMs are collectively coordinated is unknown. Our quantitative cell-biological and in silico analyses revealed that tissue elasticity mechanically assists an initial step of basalward IKNM. When the soma of an M-phase progenitor cell rounds up using actomyosin within the subapical space, a microzone within 10 μm from the surface, which is compressed and elastic because of the apical surface’s contractility, laterally pushes the densely neighboring processes of non–M-phase cells. The pressed processes then recoil centripetally and basally to propel the nuclei/somata of the progenitor’s daughter cells. Thus, indirect neighbor-assisted transfer of mechanical energy from mother to daughter helps efficient brain development., Author summary The development of large brain structures, such as the mammalian cerebral cortex, depends on the continuous and efficient production of cells by neural progenitor cells. Neural progenitor cells are elongated and span the developing brain wall. The nuclei and bodies of these cells move cyclically between the apical and basal surfaces, and they divide every time they reach the apical surface. While we understand how individual cells achieve this cycle, how the movements of several progenitor cells are coordinated with one another remains elusive. By using a combination of live imaging and mechanical experiments, coupled with mathematical simulations, we show that cell crowding at the apical surface, where progenitor cells divide, creates a subapical microzone that is compressed and elastic. We then show that when each mother cell rounds up, preparing for division, it pushes this elastic microzone laterally, thereby storing mechanical energy. After cell division, this mechanical energy is transferred to the daughter cells, propelling them along the axis of movement in the direction of the basal surface, in an energy-saving manner. Our mathematical simulations show that timely departure of newly generated daughter cells is critical for the overall tissue structure of the cerebral proliferative zone.

Summary In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/β-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1+ cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1+ cells and confers resistance to the Wnt/β-catenin signaling. Shisa6+ cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)+ cells that are found largely overlapped with Shisa6+ cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs., Graphical Abstract, Highlights • Wnt/β-catenin signaling promotes the differentiation of GFRα1+ spermatogonia • SHISA6 is a cell-autonomous Wnt inhibitor expressed in subset GFRα1+ cells • SHISA6 confers resistance to differentiation induction by Wnt/β-catenin signaling • SHISA6+ spermatogonia show stem cell-related properties, In mouse testicular seminiferous tubules, Yoshida and colleagues questioned how sperm stem cells follow different fates (to differentiate or to self-renew) in response to homogeneously distributed extracellular signals. They showed that heterogeneous expression of Shisa6, a cell-autonomous Wnt inhibitor, confers resistance to homogeneously distributed differentiation-promoting Wnt signaling. This may be generic for stem cell regulation in a facultative niche.

Early in the development of the central nervous system, progenitor cells undergo a shape change, called apical constriction, that triggers the neural plate to form a tubular structure. How apical constriction in the neural plate is controlled and how it contributes to tissue morphogenesis are not fully understood. In this study, we show that intracellular calcium ions (Ca2+) are required for Xenopus neural tube formation and that there are two types of Ca2+-concentration changes, a single-cell and a multicellular wave-like fluctuation, in the developing neural plate. Quantitative imaging analyses revealed that transient increases in Ca2+ concentration induced cortical F-actin remodeling, apical constriction and accelerations of the closing movement of the neural plate. We also show that extracellular ATP and N-cadherin (cdh2) participate in the Ca2+-induced apical constriction. Furthermore, our mathematical model suggests that the effect of Ca2+ fluctuations on tissue morphogenesis is independent of fluctuation frequency and that fluctuations affecting individual cells are more efficient than those at the multicellular level. We propose that distinct Ca2+ signaling patterns differentially modulate apical constriction for efficient epithelial folding and that this mechanism has a broad range of physiological outcomes.

In the last several decades, developmental biology has clarified the molecular mechanisms of embryogenesis and organogenesis. In particular, it has demonstrated that the “tool-kit genes” essential for regulating developmental processes are not only highly conserved among species, but are also used as systems at various times and places in an organism to control distinct developmental events. Therefore, mutations in many of these tool-kit genes may cause congenital diseases involving morphological abnormalities. This link between genes and abnormal morphological phenotypes underscores the importance of understanding how cells behave and contribute to morphogenesis as a result of gene function. Recent improvements in live imaging and in quantitative analyses of cellular dynamics will advance our understanding of the cellular pathogenesis of congenital diseases associated with aberrant morphologies. In these studies, it is critical to select an appropriate model organism for the particular phenomenon of interest.

Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no approved targeted therapies specifically for IBC have been approved. The epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC, with a reported pathological complete response rate of 42%. The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Therefore, delineating the cross-talk between EGFR-targeted therapy and TME components could lead to more efficient combination regimens and novel clinical trial designs for IBC. Because an immunocompetent IBC animal model is not available for our study, we established an IBC SUM149 immunocompetent mouse model (SUM149-hu-NSG-SGM3) for such studies. Here we report the effects of panitumumab (PmAb), a humanized monoclonal anti-EGFR antibody, on tumor growth and on the TME in the SUM149-hu-NSG-SMG3 mouse model. Methods: SUM149 cells were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with human hematopoietic stem cells. SUM149 tumor growth in mice treated with either isotype IgG2 or PmAb was measured. We used flow cytometry to measure the percentages of human immune cells in the peripheral blood and tumor tissues from mice treated with IgG2 and PmAb. The effects of PmAb treatment on the expression and distribution of TME components in tumor tissues was examined by multiplex immunohistochemical staining. Cytokine expression in tumor tissues from IgG2- and PmAb-treated mice was measured by cytokine antibody array, and the changes in cytokines expression were validated with RT-PCR. Results: PmAb treatment, compared with IgG2 treatment, significantly reduced SUM149 tumor growth. Flow cytometry analysis showed infiltration of lymphocytes in SUM149 tumors with an observed increase in the level of CD8+ T cells and a reduced level of Tregs in both peripheral blood and tumor tissues from PmAb-treated mice compared with IgG2-treated mice. Cytokine array and RT-PCR showed that PmAb treatment increased expression of cytokines that function as a chemoattractant for T cells, natural killer cells, and monocytes, including CXCL10, CCL5, CCL4, and CXCL9. In contrast, PmAb treatment reduced expression of cytokines involved in antitumor immune suppression, including osteoprotegerin (OPG), CCL2, CXCL5, and CCL20. Conclusion: PmAb treatment reduced IBC tumor growth by increasing cytotoxic T cells and reducing immunosuppressive Treg cells. Our data also suggest that the combination of PmAb with an immune checkpoint inhibitor may potentiate the efficacy of anti-EGFR therapy in IBC. A study of the therapeutic efficacy of the PmAb and anti-PD-L1 combination in the SUM149-hu-NSG-SMG3 mouse model is in progress. Citation Format: Xiaoping Wang, Shan Shao, Takashi Semba, Troy Pearson, James M. Re, Debu Tripathy, Naoto Ueno. Humanized anti-EGFR antibody panitumumab inhibits tumor growth of inflammatory breast cancer by inducing antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4492.