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Results for “Developmental Biology”

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bioRxiv2018

tbx5 and tbx15 mediate zebrafish posterior lateral line migration and development via a negative feedback loop

The zebrafish posterior lateral line (pLL) is a mechanosensory organ enabling detection of movement in the aqueous environment. Evidence suggests that two T-box transcription factors, tbx5 and tbx15, participate in pLL migration and development. Lateral line migration and deposition defects are observed following tbx5 or tbx15 morpholino knockdown. Additional studies demonstrate that the tbx5 phenotype is partially rescued when tbx15 is also knocked down. pLL defects similar to those of tbx5 are noted following knockdown of camKII-{beta}2, a known downstream target of Tbx5, suggesting a potential mechanistic pathway. Ectopic expression of human CamKII-{partial}C partially rescues the tbx5 phenotype but exacerbates the tbx15 phenotype. These results, combined with in situ hybridization and qRT-PCR profiling, indicate a negative feedback loop controlling multiple primordium patterning markers, including cxcr4b, cxcr7b, fgf10a and notch3, which ultimately affects pLL neuromast migration and deposition.

developmental biology↗

bioRxiv2018

Anti-tumor effects of antimicrobial peptides, targets of the innate immune system, against hematopoietic tumors in Drosophila mxc mutants

The innate immune response is the first line of defense against microbial infections. In Drosophila, three immune pathways induce the synthesis of antimicrobial peptides (AMPs) in the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells. However, little is known about their anti-tumor effects. Drosophila mxcmbn1 mutants exhibit malignant hyperplasia in a larval hematopoietic organ called the lymph gland (LG). Here, using RNA-Seq analysis, we found that many immunoresponsive genes, including AMP genes, were up-regulated in the mutants. Down-regulation of these pathways by either a Toll or an imd mutation enhanced the tumor phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress progression of hematopoietic tumors by inducing AMP gene expression. Overexpression of any one of these five AMPs resulted in enhanced apoptosis in the mutant LGs, while no apoptosis signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating hemocyte-like cells, which were associated with LG regions showing reduced cell-to-cell adhesion in the mutants; another AMP, diptericin, was directly localized on the tumors without intermediating hemocytes. These results lead us to conclude that the AMPs have a specific cytotoxic effect that enhance apoptosis exclusively in the tumor cells.\n\nSummary statementAntimicrobial peptides can be associated with tumor cells generated in a hematopoietic tissue in Drosophila mxc mutants and have an anti-tumor effect in suppressing their growth.

developmental biology↗

bioRxiv2018

Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning

The generation of patterns during development is generally viewed as a direct process. In the mouse jaw, however, the sequential patterning of molars initiates with abortive tooth signaling centers called MS and R2, thought to be vestiges of the lost rodent premolars. Moreover, the mature signaling center of the first molar (M1) is formed from the fusion of two signaling centers (R2 and early M1). Here, we report that Edar expression reveals the hidden dynamics of signalling centers patterning. First, Edar expression evidenced a hidden two-step patterning process that we modelled with a single activator-inhibitor pair: the epithelium is initially broadly activated, then activation becomes restricted in space to give rise to the signalling centers. Second, Edar expression unveils successive phases of pattern making and pattern erasing events, a phenomenon that we called a developmental palimpsest. MS is erased by a broad activation for the benefit of R2, which itself is erased before it recovers when the first molar signaling center forms. In the lower but not the upper jaw, the two neighboring signaling centers then fuse into a single elongated center. Our model recapitulated the erasure of the R2 signaling center by the wave of activation that precedes the formation of M1 signaling center, and predicted the surprising rescue of R2 in the context of an Edar mutant with reduced activation. It suggested that R2 was not intrinsically defective, but actively outcompeted by M1 formation. We confirmed this by cultivating R2 separately from the posterior tissue and showing it could then generate a tooth. Finally, by introducing chemotaxis as a secondary process of tooth germ maturation, we recapitulated the fusion of R2 and M1 in the lower jaw only, and the loss of fusion when Edar function is impaired in organ cultures. In conclusion, we have uncovered a highly indirect and dynamic nature of pattern formation in the molar field that could nevertheless be simulated with simple mathematical models. Our study argues for viewing embryonic patterns as dynamical objects rather than as fixed endpoints, where dynamics is essential to the outcome of the patterning process.

developmental biology↗

bioRxiv2018

fruitless functions downstream of doublesex to promote sexual dimorphism of the gonad stem cell niche

Backgrounddoublesex (dsx) and fruitless (fru) are the two downstream transcription factors that actuate Drosophila sex determination. While dsx assists fru to regulate sex-specific behavior, whether fru collaborates with dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs.\n\nResultsHere we report that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously studied regulation of sex-specific Fru expression, which is regulated by alternative splicing by Transformer (Tra), we show that male-specific expression of fru is regulated downstream of dsx, and is independent of Tra. Regulation of fru by dsx also occurs in the nervous system. fru genetically interacts with dsx to support maintenance of the hub throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, we identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the hub, indicating that fru is likely a direct target for transcriptional regulation by Dsx.\n\nConclusionsThese findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra.

developmental biology↗

bioRxiv2018

Suppressor of Fused controls perinatal expansion and quiescence of future dentate adult neural stem cells

Adult hippocampal neurogenesis requires the quiescent neural stem cell (NSC) pool to persist lifelong. The establishment and maintenance of quiescent NSC pools during development is not understood. Here we show that Suppressor of Fused (Sufu) controls establishment of the quiescent NSC pool during mouse dentate gyrus (DG) development through regulation of Sonic Hedgehog (Shh) signaling. Deletion of Sufuin NSCs at the beginning of DG development decreases Shh signaling leading to reduced proliferation of NSCs, resulting in a small quiescent NSC pool in adult mice. We found that putative adult NSCs proliferate and increase their numbers in the first postnatal week and subsequently become quiescent towards the end of the first postnatal week. In the absence of Sufu, postnatal expansion of NSCs is compromised, and NSCs prematurely become quiescent. Thus, Sufu is required for Shh signaling activity ensuring the expansion and proper transition of NSC pools to quiescence during DG development.

developmental biology↗

bioRxiv2018

An ancient role for Collier/Olf/Ebf (COE)-type transcription factors in axial motor neuron development

BackgroundMammalian motor circuits display remarkable cellular diversity with hundreds of motor neuron (MN) subtypes innervating hundreds of different muscles. Extensive research on limb muscle-innervating MNs has begun to elucidate the genetic programs that control animal locomotion. In striking contrast, the molecular mechanisms underlying the development of axial muscle-innervating MNs, which control breathing and spinal alignment, are poorly studied.\n\nMethodsOur previous studies indicated that the function of the Collier/Olf/Ebf (COE) family of transcription factors (TFs) in axial MN development may be conserved from nematodes to simple chordates. Here, we examine the expression pattern of all four mouse COE family members (mEbf1-mEbf4) in spinal MNs and employ genetic approaches in both nematodes and mice to investigate their function in axial MN development.\n\nResultsWe report that mEbf1 and mEbf2 are expressed in distinct MN clusters (termed \"columns\") that innervate different axial muscles. Mouse Ebf1 is expressed in MNs of the hypaxial motor column (HMC), which is necessary for breathing, while mEbf2 is expressed in MNs of the medial motor column (MMC) that control spinal alignment. Our characterization of Ebf2 knock-out mice revealed a requirement for Ebf2 in the differentiation of a subset of MMC MNs, indicating molecular diversity within MMC neurons. Intriguingly, transgenic expression of mEbf1 or mEbf2 can rescue axial MN differentiation and locomotory defects in nematodes (Caenorhabditis elegans) lacking unc-3, the sole C. elegans ortholog of the COE family, suggesting functional conservation among mEbf1, mEbf2 and nematode UNC-3.\n\nConclusionsThese findings support the hypothesis that the genetic programs controlling axial MN development are deeply conserved across species, and further advance our understanding of such programs by revealing an essential role for Ebf2 in mouse axial MNs. Because human mutations in COE ortholgs lead to neurodevelopmental disorders characterized by motor developmental delay, our findings may advance our understanding of these human conditions.

developmental biology↗

bioRxiv2018

AMPK regulates germline stem cell quiescence and integrity through an endogenous small RNA pathway.

C. elegans larvae can undergo a global developmental arrest following the execution of a diapause-like state called dauer in response to unfavourable growth conditions. Survival in this stage surpasses the normal lifespan of reproductive animals quite dramatically, and without any apparent negative impact on their reproductive fitness. During this period, the germ cells become quiescent and must retain their reproductive integrity so the animal can reproduce following recovery. This germline stem cell (GSC) arrest requires the activity of AMP-activated protein kinase (AMPK) and in its absence the germ line undergoes hyperplasia. We show here that AMPK mutant animals exhibit complete sterility after recovery from dauer, suggesting that germ cell integrity is compromised during this stage in the absence of AMPK. These defects correlate with altered abundance and distribution of a number of chromatin modifications that affect gene expression. These aberrant chromatin modifications, along with the supernumerary germ cell divisions and the observed post-dauer sterility, were all corrected by disabling key effectors of the small interfering RNA pathway (dcr-1 and rde-4) and the primary Argonaute protein ergo-1, suggesting that AMPK regulates the function of these small RNA pathway components, and in its absence, the pathways become abnormally active. The aberrant regulation of the small RNA pathway components releases the germ cells from quiescence to proliferative state thereby compromising germ cell integrity. Curiously, AMPK expression in either the neurons or the excretory system is sufficient to restore the GSC quiescence and the fertility in the AMPK mutant post-dauer adults, while the fertility of these animals is also partially restored by disabling the dsRNA importer SID-1. Our data suggest that AMPK regulates a small RNA pathway in the soma to establish and/or maintain GSC quiescence and integrity cell non-autonomously in response to the energy stress associated with the dauer stage. Our findings therefore provide a unique model to better understand how the soma communicates with the germ line to establish the appropriate epigenetic modifications required to adapt to acute environmental challenges.

developmental biology↗

bioRxiv2018

Early detection of impending cryostorage tank failure using a weight-based monitoring system

ObjectTo study the ability of custom-built, web-enabled scales to monitor liquid nitrogen (LN2) levels in cryostorage dewars.\n\nDesignLaboratory study\n\nSettingA large academic fertility center in New York City.\n\nInterventionsCryostorage dewars were placed on top of the custom-engineered scales with continuous real-time monitoring, and weight and temperature data were recorded in the setting of slow, medium, and fast rate-loss of LN2 designed to mimic models of tank failures.\n\nMain Outcome MeasuresWeights were continuously monitored and recorded, with a calculated alarm trigger set at 10% weight loss. Temperature within the tanks was simultaneously monitored with probes placed near the top of the tanks, with calculated alarms using a -185 {degrees}C as the threshold. For the \"slow rate-loss\" simulations, tanks were left intact and closed in usual operating conditions, and LN2 was allowed to evaporate at the normal rate. For the \"medium rate-loss\" simulation, the foam core of the tank neck was removed and the insulating vacuum was eliminated by making a 1/16 inch hole in the outer tank wall. For the \"fast rate-loss\" simulation, a 1/16\" hole was made through the outer tank wall and LN2 was released at a rate of 0.15 L/second. All simulations were performed in duplicate.\n\nResultsWith an intact and normally functioning tank, a 10% loss in LN2 occurred in 4.2-4.9 days. Warming to -185 {degrees}C occurred in 37.8 - 43.7 days, over 30 days after the weight-based alarm was triggered. Full evaporation of LN2required 36.8 days. For the medium rate-loss simulation, a 10% loss in LN2 occurred in 0.8 h. Warming to -185 {degrees}C occurred in 3.7 - 4.8 hours, approximately 3 hours after the weight-based alarm was triggered. For the fast rate-loss simulation, a 10% weight loss occurred within 15 seconds and tanks were completely depleted in under 3 minutes. Tank temperatures began to rise immediately and at a relatively constant rate of 43.9 {degrees}C/hour and 51.6 {degrees}C/hour. Temperature alarms would have sounded within 0.37 and 0.06 hours after the breech.\n\nConclusionsThis study demonstrates that a weight-based, automated alarm system can detect tank failures prior to a temperature-based alarm system, in some cases over a month in advance. In combination with existing safety mechanisms such as temperature probes, a weight-based monitoring system could serve as a redundant safety mechanism for added protection of cryopreserved reproductive tissues.

developmental biology↗

bioRxiv2018

Intraflagellar transport protein 74 is essential for mouse spermatogenesis and male fertility by regulating axonemal microtubule assembly in mice

IFT74 is a component of the core intraflagellar transport (IFT) complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in IFT74 in male germ cells were infertile associated with low sperm counts and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axoneme in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88 and IFT140 were significantly reduced in the mutant mice, with the exception of IFT20 and IFT25. The levels of ODF2 and SPAG16L proteins were also not changed. However, the processed AKAP4 protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and highlights a potential genetic factor (IFT74) that contributes to human infertility in men.

developmental biology↗

bioRxiv2018

Hereditary Pancreatitis Model by Blastocyst Complementation in Mouse

Summary StatementThe present study is the first report for reproducing human disease by utilizing blastocyst complementation method and would lead to the development of novel therapy for human disease.\n\nThe application of pluripotent stem cell is expected to contribute to the elucidation of the unknown mechanism of human diseases. However, in vitro induction of cells in several organs, such as the pancreas and liver, remains difficult; therefore, reproduction of those diseases in a model has not been feasible. To reproduce human hereditary pancreatitis (HP), which is most frequently caused by the mutations in the PRSS1 gene, we performed the blastocyst complementation (BC) method. In the BC method, mouse embryonic stem (ES) cells harboring CRISPR/CAS9-mediated mutations in the Prss1 were injected into the blastocysts of deficient Pdx1 gene mice, which is a critical transcription factor in the pancreas. The results showed that the blastocysts injected into the Prss1-mutant ES cells induced trypsin activation. This implied that the mouse phenotype mimics that of human HP and that the BC method was useful for the reproduction and study of pancreatic disorders. The present study opens the possibility of investigating uncharacterized human diseases by utilizing the BC method.

developmental biology↗

bioRxiv2018

BmCncC/keap1-pathway is involved in high-temperature induced metamorphosis regulation of silkworm, Bombyx mori

The global warming has affected the growth, development and reproduction of insects. However, the molecular mechanism of high temperature stress-mediated metamorphosis regulation of lepidopteran insect has not been elucidated. In this study, the relationship between the insect developmental process and endogenous hormone level was investigated under high temperature (36 {degrees} C) stress in Bombyx mori (B. mori). The results showed that the duration of 5th instar larvae were shortened by 28 {+/-} 2 h, and the content of 20E was up-regulated significantly after 72 h of high temperature treatment, while the transcription levels of 20E response genes E93, Br-C, USP, E75 were up-regulated 1.35, 1.25, 1.28, and 1.27-fold, respectively. The high temperature treatment promoted the phosphorylation level of Akt and the downstream BmCncC/keap1 pathway was activated, the transcription levels of 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12, 1.51, 2.17 and 1.23-fold, respectively. After treatment with double stranded RNA of BmCncC (dsBmCncC) in BmN cells, the transcription levels of cyp302a1 and cyp306a1 were significantly decreased, whereas up-regulated by 2.15 and 1.31-fold, respectively, after treatment with CncC activator Curcumin. These results suggested that BmCncC/keap1-mediated P450 genes (cyp302a1, cyp306a1) expression resulted in the changes of endogenous hormone level, which played an important role in the regulation of metamorphosis under high temperature stress. Studies provide novel clues for understanding the CncC/keap1 pathway-mediated metamorphosis regulation mechanism in insects.\n\nAuthor SummaryMammalian nuclear transcription factor Nrf2 (NF-E2-related factor 2) plays an important role in the stress response of cells. CncC is a homolog of mammalian Nrf2 in insect, regulating the genes expression of insect antioxidant enzymes and cytochrome P450 detoxification enzyme. Evidence suggests that the CncC/Keap1 pathway also plays an important role in regulating insect development. Here, we investigated the regulatory mechanism between the CncC/Keap1 pathway and metabolism of silkworm hormones in Lepidoptera. We found that high temperature induction accelerated the development of silkworm, the ecdysone content and related metabolic genes in hemolymph were significantly up-regulated, the CncC/Keap1 pathway was activated, and the expression of BmCncC was significantly increased, indicating that the Cncc/Keap1 pathway plays an important role in this process. The expression of cyp302a1 and cyp306a1 was significantly decreased by RNA interference with BmCncC, which indicated that CncC in silkworm had a regulatory relationship with downstream 20E synthetic gene. In summary, the results indicate that the CncC/Keap1 pathway plays an important role in regulating hormone metabolism in silkworm, providing a basis for further study of the relationship between CncC/Keap1 pathway and development in insects.

developmental biology↗

bioRxiv2018

Inferring novel lncRNA associated with Ventricular septal defect by DNA methylation interaction network

Ventricular septal defect (VSD) is one of the most common types of congenital heart disease. To find more and more molecular alteration is conducive to explore the mechanism and biomarker in VSD. Herein we devised a predictive strategy to uncover novel lncRNA of VSD integrating DNA methylation, gene expression and lncRNA expression of early embryo and VSD by profiles from GEO database. In totally, 175 lncRNAs, 7290 genes and 3002 DNA methylation genes were obtained by logistic regression analysis associated with embryonic development. Moreover, 7304 DMGs were significant differential methylated by Wilcoxon rank test and Students test in VSD. We constructed the lncRNA-mRNA co-expression network in embryo (LMCNe). Then, a reconstructed co-expression weighted network (RCWN) was built integrated LMCNe and the DNA methylation associated network (DMAN) based on the correlation of the DNA methylation level and protein interaction network between embryonic development and VSD. We extracted top 10 lncRNAs with higher score performing DRaWR from the weight network, which as potential VSD related lncRNAs. Six lncRNAs showed a high level of expression in the heart tissue recorded in the NONOCOND database. Furthermore, associated lncRNA genes DCAF8L1, NIT1, SH2D7 and DOCK9-AS2 in validated samples showed a prominently association with VSD. These outcomes provide a reference for lncRNA involved in VSD initialization and a new insight for studies of VSD-associated lncRNAs. Author SummaryVentricular septal defect (VSD) is one of the most common types of congenital heart disease and has a high mortality rate in infants. Many molecular markers have proved effective as biomarker in VSD like DNA methylation and lncRNA. lncRNA is a type of non-coding RNA which has important effect in regulation gene expression and disease occurrence. VSD is an embryonic stage developmental disease. Therefore we hypothesized that lncRNA which was associated with DNA methylation and mRNA in early embryonic development may also affect the occurrence of VSD. So in this work, from the perspective of embryonic development, we devised a predictive strategy to uncovering novel lncRNA of VSD. In our result, four lncRNA associated genes were found differential expressed in VSD and normal samples by qPCR validation. The identification of lncRNA associated with ventricular septal defect is beneficial to further study the mechanism of VSD from the molecular level and also provides a good molecular marker for clinical therapeutic and diagnosis. At the same time, it also provides a new insight for the researches of lncRNA associated with VSD.

developmental biology↗

bioRxiv2018

Tfap2a is a novel gatekeeper of differentiation in renal progenitors during kidney development

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular populations with discrete physiological tasks. Knowledge about the terminal differentiation programs of each nephron segment has central importance for understanding kidney disease and to advance regenerative medicine, as mammalian nephrons grown in organoid cultures from pluripotent cells fail to terminally differentiate. Here, from a novel forward genetic screen using zebrafish we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that controls the progression of nephron distal segment progenitors into the differentiated state. Overexpression of tfap2a rescued differentiation in mutants and caused ectopic expression of distal segment markers in wild-type nephrons, indicating tfap2a is sufficient to instigate the distal segment differentiation program. tfap2a/2b deficiency exacerbated distal nephron segment differentiation defects, revealing functional redundancy where tfap2a has a dominant role upstream of its family member. With further genetic studies, we assembled a blueprint of the tfap2a gene regulatory network during nephrogenesis. We demonstrate that tfap2a acts downstream of Iroquois homeobox 3b, a conserved distal lineage transcription factor. tfap2a controls a circuit consisting of irx1a, tfap2b, and genes encoding solute transporters that dictate the specialized metabolic functions of the distal nephron segments, and we show for the first time that this regulatory node is distinct from the pathway circuits controlling aspects such as apical-basal polarity and ciliogenesis during the differentiation process. Thus, our studies reveal new insights into the genetic control of differentiation, where tfap2a regulates the suite of segment transporter traits. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to make functional units that can facilitate organoid applications such as drug discovery and regenerative therapies.\n\nSummary StatementHere, we report for the first time that transcription factor AP-2 alpha (tfap2a) controls the progression from nephron progenitor into the fully differentiated state. This fundamentally deepens our knowledge about the genetic control of kidney development.

developmental biology↗

bioRxiv2018

Stem cell differentiation trajectories in Hydra resolved at single-cell resolution

The adult Hydra polyp continuously renews all of its cells using three separate stem cell populations, but the genetic pathways enabling homeostatic tissue maintenance are not well understood. We used Drop-seq to sequence transcriptomes of 24,985 single Hydra cells and identified the molecular signatures of a broad spectrum of cell states, from stem cells to terminally differentiated cells. We constructed differentiation trajectories for each cell lineage and identified the transcription factors expressed along these trajectories, thus creating a comprehensive molecular map of all developmental lineages in the adult animal. We unexpectedly found that neuron and gland cell differentiation transits through a common progenitor state, suggesting a shared evolutionary history for these secretory cell types. Finally, we have built the first gene expression map of the Hydra nervous system. By producing a comprehensive molecular description of the adult Hydra polyp, we have generated a resource for addressing fundamental questions regarding the evolution of developmental processes and nervous system function.

developmental biology↗

bioRxiv2018

let-7 controls the transition to adulthood by releasing select transcriptional regulators from repression by LIN41

Development of multicellular organisms relies on faithful temporal control of cell fates. In Caenorhabditis elegans, the heterochronic pathway governs temporal patterning of somatic cells. This function may be phylogenetically conserved as several heterochronic genes have mammalian orthologues, and as the heterochronic let-7 miRNA and its regulator LIN28 appear to time the onset of puberty in mice and humans. Here, we have investigated how let-7 promotes the transition to adulthood in C. elegans. We find that let-7 controls each of three relevant processes, namely male and female sexual organ morphogenesis as well as changes in skin progenitor cell fates, through the same single target, lin-41. LIN41 in turn silences two pairs of targets post-transcriptionally, by binding and silencing their mRNAs. The EGR-type transcription factor LIN-29a and its co-factor, the NAB1/2 orthologous MAB-10, mediate control of progenitor cell fates and vulval integrity. By contrast, male tail development depends on regulation of the DM domain-containing transcription factors DMD-3 and MAB-3. Our results provide mechanistic insight into an exemplary temporal patterning pathway, demonstrate that let-7 - LIN41 function as a versatile regulatory module that can be connected to different outputs, and reveal how several levels of post-transcriptional regulation ultimately achieve effects through controlling transcriptional outputs.

developmental biology↗

bioRxiv2018

Pluripotent Stem Cell-derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins

The process of oligodendrogenesis has been relatively well delineated in the rodent brain. However, it remains unknown whether analogous developmental processes are manifested in the human brain. Here, we report oligodendrogenesis in forebrain organoids, generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) vs. dorsal forebrain organoids (DFOs). Interestingly, oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore, dorsally-derived oligodendroglial cells outcompete ventrally-derived oligodendroglia and become dominant in FFOs after long-term culture. Thus, our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally- and dorsally-derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects.

developmental biology↗

bioRxiv2018

Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

Basal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer1-4. Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury5-8. However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages9,10. These bud tip progenitor cells have also been described in the developing human lung11-13, but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures11, and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state ( hub progenitors) and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFT1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator of newly born basal cells in the lung may have implications for regenerative medicine, basal cell development in other organs, and understanding basal cell misregulation in disease.

developmental biology↗

bioRxiv2018

Genome-wide strategies reveal target genes of Npas4l associated with cardiovascular development in zebrafish

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we combined complementary genome-wide approaches including transcriptome analyses and chromatin immunoprecipitation (ChIP). The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes crucial for hematoendothelial specification such as etv2, tal1 and lmo2. We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.

developmental biology↗