Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

PTBP1 mediates Sertoli cell actin cytoskeleton organization by regulating alternative splicing of actin regulators.

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics is controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms.

Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Solubility phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains largely unclear. In this work, we discover the dynamic solubility phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere, and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus identifies important remodeling mechanisms that act on RNAs to control vertebrate OET.

Single-Cell RNA Sequencing (scRNA-seq) Identifies L1CAM as a Key Mediator between Epithelial Tuft Cell and Innate Lymphoid Cell in the Colon of Hnrnp I Knockout Mice.

(1) Background: Knockout (KO) of heterogeneous nuclear ribonucleoprotein I (Hnrnp I) in mouse intestinal epithelial cells (IECs) induced a severe inflammatory response in the colon, followed by hyperproliferation. This study aimed to investigate the epithelial lineage dynamics and cell-cell communications that underlie inflammation and colitis. (2) Methods: Single cells were isolated from the colons of wildtype (WT) and KO mice and used in scRNA-seq. Whole colons were collected for immunofluorescence staining and cytokine assays. (3) Results: from scRNA-seq, the number of DCLK1 + colonic tuft cells was significantly higher in the Hnrnp I KO mice compared to the WT mice. This was confirmed by immunofluorescent staining of DCLK1. The DCLK1 + colonic tuft cells in KO mice developed unique communications with lymphocytes via interactions between surface L1 cell adhesion molecule (L1CAM) and integrins. In the KO mice colons, a significantly elevated level of inflammatory cytokines IL4, IL6, and IL13 were observed, which marks type-2 immune responses directed by group 2 innate lymphoid cells (ILC2s). (4) Conclusions: This study demonstrates one critical cellular function of colonic tuft cells, which facilitates type-2 immune responses by communicating with ILC2s via the L1CAM-integrins interaction. This communication promotes pro-inflammatory signaling pathways in ILC2, leading to the increased secretion of inflammatory cytokines.

Infrared spectroscopic laser scanning confocal microscopy for whole-slide chemical imaging.

Chemical imaging, especially mid-infrared spectroscopic microscopy, enables label-free biomedical analyses while achieving expansive molecular sensitivity. However, its slow speed and poor image quality impede widespread adoption. We present a microscope that provides high-throughput recording, low noise, and high spatial resolution where the bottom-up design of its optical train facilitates dual-axis galvo laser scanning of a diffraction-limited focal point over large areas using custom, compound, infinity-corrected refractive objectives. We demonstrate whole-slide, speckle-free imaging in ~3 min per discrete wavelength at 10× magnification (2 µm/pixel) and high-resolution capability with its 20× counterpart (1 µm/pixel), both offering spatial quality at theoretical limits while maintaining high signal-to-noise ratios (>100:1). The data quality enables applications of modern machine learning and capabilities not previously feasible - 3D reconstructions using serial sections, comprehensive assessments of whole model organisms, and histological assessments of disease in time comparable to clinical workflows. Distinct from conventional approaches that focus on morphological investigations or immunostaining techniques, this development makes label-free imaging of minimally processed tissue practical.

Increasing dietary nutrient levels modulates colon immune adaptation and alleviates inflammation in the epithelial heterogeneous nuclear ribonucleoprotein I (Hnrnp I) knockout mice.

Heterogeneous nuclear ribonucleoprotein I (HNRNP I) is an RNA-binding protein essential for neonatal immune adaptation by downregulating interleukin-1 receptor-associated kinase (IRAK1) in toll-like receptor (TLR)-mediated NF-κB signaling pathways. TLR-mediated NF-κB is associated with chronic inflammation, including the development of inflammatory bowel diseases. Meanwhile, dietary protein intake is one of the major concerns for individuals with inflammatory bowel diseases. The present study aims to investigate the effects of a protein-enriched diet on intestinal inflammation and immune responses in a mouse model with aberrant NF-κB signaling in the colon. A transgenic mouse model with intestinal-epithelial-cell (IEC) specific Hnrnp I knocked out was used to investigate the effects of protein intake on the immune system in the colon. A control diet (CON) and a nutrient-dense modified diet (MOD) were fed to both the wild-type (WT) and the knockout (KO) male mice for 14 weeks. Inflammatory markers and colonic immune responses were examined, with gene expression and protein expression levels analyzed. IEC-specific Hnrnp I knocked out mice had significantly increased expression of the active NF-κB subunit, P65, in their colons. There was a concomitant induction of mRNA expression of Il1ß, Il6, Cxcl1, and Ccl2. The number of CD4+ T cells in the distal colon was also increased in the KO mice. The results confirmed that KO mice had proinflammatory responses with aberrant NF-κB signaling in the colon. Importantly, increased nutrient density in their diets attenuated colon inflammation by decreasing the expression of proinflammatory cytokines, reducing P65 translocation, downregulating IRAK1, and limiting the number of CD4+ T cells recruited in Hnrnp I KO mice colon. In summary, this study found that a diet with increased nutrient density relieved the inflammation induced by knockout of Hnrnp I, attributable partially to the reduced expression of inflammatory and immune-modulating cytokines in the mouse distal colon.

Phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains an open question. In this work, we discover the dynamic phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus uncovers novel remodeling mechanisms that act on RNAs to regulate vertebrate OET.

PTBP1 controls intestinal epithelial regeneration through post-transcriptional regulation of gene expression.

The intestinal epithelial regeneration is driven by intestinal stem cells under homeostatic conditions. Differentiated intestinal epithelial cells, such as Paneth cells, are capable of acquiring multipotency and contributing to regeneration upon the loss of intestinal stem cells. Paneth cells also support intestinal stem cell survival and regeneration. We report here that depletion of an RNA-binding protein named polypyrimidine tract binding protein 1 (PTBP1) in mouse intestinal epithelial cells causes intestinal stem cell death and epithelial regeneration failure. Mechanistically, we show that PTBP1 inhibits neuronal-like splicing programs in intestinal crypt cells, which is critical for maintaining intestinal stem cell stemness. This function is achieved at least in part through promoting the non-productive splicing of its paralog PTBP2. Moreover, PTBP1 inhibits the expression of an AKT inhibitor PHLDA3 in Paneth cells and permits AKT activation, which presumably maintains Paneth cell plasticity and function in supporting intestinal stem cell niche. We show that PTBP1 directly binds to a CU-rich region in the 3' UTR of Phlda3, which we demonstrate to be critical for downregulating the mRNA and protein levels of Phlda3. Our results thus reveal the multifaceted in vivo regulation of intestinal epithelial regeneration by PTBP1 at the post-transcriptional level.

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

Subacute exposure to di-isononyl phthalate alters the morphology, endocrine function, and immune system in the colon of adult female mice.

Di-isononyl phthalate (DiNP), a common plasticizer used in polyvinyl chloride products, exhibits endocrine-disrupting capabilities. It is also toxic to the brain, reproductive system, liver, and kidney. However, little is known about how DiNP impacts the gastrointestinal tract (GIT). It is crucial to understand how DiNP exposure affects the GIT because humans are primarily exposed to DiNP through the GIT. Thus, this study tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. To test this hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 to 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. DiNP exposure significantly increased histological damage in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels, increased Tnf expression, decreased a cell cycle regulator (Ccnb1), and increased apoptotic factors (Aifm1 and Bcl2l10) in the colon compared to control. Colon-extracted lipids revealed that DiNP exposure significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in adult female mice.

The Impact of Environmental Chemicals on the Gut Microbiome.

Since the surge of microbiome research in the last decade, many studies have provided insight into the causes and consequences of changes in the gut microbiota. Among the multiple factors involved in regulating the microbiome, exogenous factors such as diet and environmental chemicals have been shown to alter the gut microbiome significantly. Although diet substantially contributes to changes in the gut microbiome, environmental chemicals are major contaminants in our food and are often overlooked. Herein, we summarize the current knowledge on major classes of environmental chemicals (bisphenols, phthalates, persistent organic pollutants, heavy metals, and pesticides) and their impact on the gut microbiome, which includes alterations in microbial composition, gene expression, function, and health effects in the host. We then discuss health-related implications of gut microbial changes, which include changes in metabolism, immunity, and neurological function.

Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development.

In most species, early germline development occurs in the absence of transcription with germline determinants subject to complex translational and post-translational regulations. Here, we report for the first time that early germline development is influenced by dynamic regulation of the proteasome system, previously thought to be ubiquitously expressed and to serve 'housekeeping' roles in controlling protein homeostasis. We show that proteasomes are present in a gradient with the highest levels in the animal hemisphere and extending into the vegetal hemisphere of Xenopus oocytes. This distribution changes dramatically during the oocyte-to-embryo transition, with proteasomes becoming enriched in and restricted to the animal hemisphere and therefore separated from vegetally localized germline determinants. We identify Dead-end1 (Dnd1), a master regulator of vertebrate germline development, as a novel substrate of the ubiquitin-independent proteasomes. In the oocyte, ubiquitin-independent proteasomal degradation acts together with translational repression to prevent premature accumulation of Dnd1 protein. In the embryo, artificially increasing ubiquitin-independent proteasomal degradation in the vegetal pole interferes with germline development. Our work thus reveals novel inhibitory functions and spatial regulation of the ubiquitin-independent proteasome during vertebrate germline development.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

hnRNP I regulates neonatal immune adaptation and prevents colitis and colorectal cancer.

The intestinal epithelium plays a critical role in host-microbe homeostasis by sensing gut microbes and subsequently initiating proper immune responses. During the neonatal stage, the intestinal epithelium is under immune repression, allowing the transition for newborns from a relatively sterile intra-uterine environment to one that is rich in foreign antigens. The mechanism underlying such immune repression remains largely unclear, but involves downregulation of IRAK1 (interleukin-1 receptor-associated kinase), an essential component of toll-like receptor-mediated NF-κB signaling. We report here that heterogeneous nuclear ribonucleoprotein I (hnRNPI), an RNA binding protein, is essential for regulating neonatal immune adaptation. We generated a mouse model in which hnRNPI is ablated specifically in the intestinal epithelial cells, and characterized intestinal defects in the knockout mice. We found that loss of hnRNPI function in mouse intestinal epithelial cells results in early onset of spontaneous colitis followed by development of invasive colorectal cancer. Strikingly, the epithelium-specific hnRNPI knockout neonates contain aberrantly high IRAK1 protein levels in the colons and fail to develop immune tolerance to environmental microbes. Our results demonstrate that hnRNPI plays a critical role in establishing neonatal immune adaptation and preventing colitis and colorectal cancer.

Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

17ß-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17ß-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17ß-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17ß-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17ß-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17ß-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17ß-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17ß-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-ß had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17ß-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17ß-estradiol content, or steroidogenic capacity in these lymphoid organs.

Reversible optogenetic control of kinase activity during differentiation and embryonic development.

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

Members of the Rusc protein family interact with Sufu and inhibit vertebrate Hedgehog signaling.

Hedgehog (Hh) signaling is fundamentally important for development and adult tissue homeostasis. It is well established that in vertebrates Sufu directly binds and inhibits Gli proteins, the downstream mediators of Hh signaling. However, it is unclear how the inhibitory function of Sufu towards Gli is regulated. Here we report that the Rusc family of proteins, the biological functions of which are poorly understood, form a heterotrimeric complex with Sufu and Gli. Upon Hh signaling, Rusc is displaced from this complex, followed by dissociation of Gli from Sufu. In mammalian fibroblast cells, knockdown of Rusc2 potentiates Hh signaling by accelerating signaling-induced dissociation of the Sufu-Gli protein complexes. In Xenopus embryos, knockdown of Rusc1 or overexpression of a dominant-negative Rusc enhances Hh signaling during eye development, leading to severe eye defects. Our study thus uncovers a novel regulatory mechanism controlling the response of cells to Hh signaling in vertebrates.

Regulation of nuclear-cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt.

Recent genome-wide association studies reveal that the FAM13A gene is associated with human lung function and a variety of lung diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and pulmonary fibrosis. The biological functions of Fam13a, however, have not been studied. In an effort to identify novel substrates of B56-containing PP2As, we found that B56-containing PP2As and Akt act antagonistically to control reversible phosphorylation of Fam13a on Ser-322. We show that Ser-322 phosphorylation acts as a molecular switch to control the subcellular distribution of Fam13a. Fam13a shuttles between the nucleus and cytoplasm. When Ser-322 is phosphorylated by Akt, the binding between Fam13a and 14-3-3 is enhanced, leading to cytoplasmic sequestration of Fam13a. B56-containing PP2As dephosphorylate phospho-Ser-322 and promote nuclear localization of Fam13a. We generated Fam13a-knockout mice. Fam13a-mutant mice are viable and healthy, indicating that Fam13a is dispensable for embryonic development and physiological functions in adult animals. Intriguingly, Fam13a has the ability to activate the Wnt pathway. Although Wnt signaling remains largely normal in Fam13a-knockout lungs, depletion of Fam13a in human lung cancer cells causes an obvious reduction in Wnt signaling activity. Our work provides important clues to elucidating the mechanism by which Fam13a may contribute to human lung diseases.

Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification.

Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3'-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification.