Automatic segmentation of 15 critical anatomical labels and measurements of cardiac axis and cardiothoracic ratio in fetal four chambers using nnU-NetV2.

BACKGROUND: Accurate segmentation of critical anatomical structures in fetal four-chamber view images is essential for the early detection of congenital heart defects. Current prenatal screening methods rely on manual measurements, which are time-consuming and prone to inter-observer variability. This study develops an AI-based model using the state-of-the-art nnU-NetV2 architecture for automatic segmentation and measurement of key anatomical structures in fetal four-chamber view images. METHODS: A dataset, consisting of 1,083 high-quality fetal four-chamber view images, was annotated with 15 critical anatomical labels and divided into training/validation (867 images) and test (216 images) sets. An AI-based model using the nnU-NetV2 architecture was trained on the annotated images and evaluated using the mean Dice coefficient (mDice) and mean intersection over union (mIoU) metrics. The model's performance in automatically computing the cardiac axis (CAx) and cardiothoracic ratio (CTR) was compared with measurements from sonographers with varying levels of experience. RESULTS: The AI-based model achieved a mDice coefficient of 87.11% and an mIoU of 77.68% for the segmentation of critical anatomical structures. The model's automated CAx and CTR measurements showed strong agreement with those of experienced sonographers, with respective intraclass correlation coefficients (ICCs) of 0.83 and 0.81. Bland-Altman analysis further confirmed the high agreement between the model and experienced sonographers. CONCLUSION: We developed an AI-based model using the nnU-NetV2 architecture for accurate segmentation and automated measurement of critical anatomical structures in fetal four-chamber view images. Our model demonstrated high segmentation accuracy and strong agreement with experienced sonographers in computing clinically relevant parameters. This approach has the potential to improve the efficiency and reliability of prenatal cardiac screening, ultimately contributing to the early detection of congenital heart defects.

Multiple trait comparison and global intestine transcriptional provide new insights into bases of heterosis in hybrid tilapia (Oreochromis niloticus × Oreochromis aureus).

Heterosis has been utilized in aquaculture for many years, yet its molecular basis remains elusive. Therefore, a comprehensive analysis of heterosis was conducted by comparing growth, digestion and biochemistry indices, as well as the intestinal gene expression profiles of Nile tilapia, blue tilapia and their hybrids. The results revealed that hybrid tilapia demonstrated an enhanced growth traits and elevated digestive enzyme activity compared to Nile and blue tilapia. Additionally, the hybrid tilapia displayed superior antioxidants and non-specific immune levels, with increased levels of catalase (CAT), alkaline phosphatase (AKP), acid phosphatase (ACP), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (TAOC), lysozyme, and immunoglobulin M (IgM) relative to Nile and blue tilapia. Moreover, 3392, 2470 and 1261 differentially expressed genes (DEGs) were identified in the intestinal tissues when comparing Nile tilapia to blue tilapia, hybrid tilapia to blue tilapia, and hybrid tilapia to Nile tilapia. Upon classifying the differentially expressed genes (DEGs), non-additively expressed DEGs accounted for 68.1 % of the total DEGs, with dominant and over-dominant expressed DEGs comprising 63.7 % and 4.4 % in the intestines, respectively. These non-additively expressed DEGs were primarily associated with metabolic, digestive, growth, and developmental pathways. This enrichment enhances our comprehension of the molecular underpinnings of growth heterosis in aquatic species.

Non-Additive and Asymmetric Allelic Expression of p38 mapk in Hybrid Tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

Hybridization is a widely used breeding technique in fish species that enhances desirable traits in cultured species through heterosis. However, the mechanism by which hybrids alter gene expression to form heterosis remains unclear. In this study, a group of hybrid tilapia was used to elucidate heterosis through interspecies crossing. Specifically, p38 was analyzed to describe the regulation of gene expression variation in hybrid tilapia. Transcripts from the Nile tilapia allele were found to be significantly higher than those from the blue tilapia allele in hybrid individuals, indicating that the expression of p38 was dominated by Nile tilapia sub-genomic alleles. The study also found a compensatory interaction of cis- and trans-acting elements of the Nile tilapia and blue tilapia sub-genomes, inducing a non-additive expression of p38 in hybrids. Eight specific SNPs were identified in the p38 promoter regions of Nile tilapia and blue tilapia, and were found to be promoter differences leading to differences in gene expression efficiencies between parental alleles using a dual-luciferase reporter system. This study provides insights into the non-additive expression patterns of key functional genes in fish hybrids related to growth and immunity response.

Single nucleotide polymorphisms in the MRFs gene family associated with growth in Nile tilapia.

BACKGROUND: Muscle occupies most of the fish body, promoting the proliferation of fish muscle fibers can facilitate rapid growth and increase the body weight of fish. Some studiesSeveral previous suggest that Myogenic regulatory factors (MRFs) play an important role in the growth of fish. OBJECTIVE: To investigate the association between the polymorphism of MRFs gene family and growth traits in Nile tilapia (Oreochromis niloticus), get more molecular markers for growth. METHODS: Amplified the Nile tilapia MRFs family gene, including Myogenic determination 1 (Myod1), Myogenic determination 2 (Myod2), Myogenin (Myog), Myogenic factor 5 (Myf5), and Myogenic factor 6 (Myf6), single nucleotide polymorphism (SNP) were screened by Sanger sequencing. RESULTS: A total of 16 SNP loci were screened, including six for Myf5, six for Myf6, one for Myog, one for Myod1 and two for Myod2. The growth traits were analyzed in relation to these 16 SNP loci, and the results indicated significant associations between all 16 SNP loci and the growth traits (P < 0.05). The linkage disequilibrium analysis revealed that D1 and D2 diplotypes of Myf5 gene, E1, E2, E3 and E4 of Myf6 gene, and F1 diplotype of Myod2 gene were significantly associated with superior growth traits. CONCLUSION: There were 6, 6, 1, 1 and 2 growth-related molecular markers in Myf5, Myf6, Myog, Myod1 and Myod2 genes, respectively, which could be applied to the breeding of Nile tilapia.

Comparative transcriptomes reveal different tolerance mechanisms to Streptococcus agalactiae in hybrid tilapia, nile tilapia, and blue tilapia.

Tilapia is one of the most economically important freshwater fish farmed in China. Streptococcosis outbreaks have been extensively documented in farmed tilapia species. Hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂) exhibit greater disease resistance than Nile tilapia (O. niloticus) and blue tilapia (O. aureus). However, the molecular mechanism underlying the enhanced tolerance of hybrid tilapia is still poorly understood. In this study, comparative transcriptome analysis was performed to reveal the different tolerance mechanisms to Streptococcus agalactiae in the three tilapia lines. In total, 1982, 2355, and 2076 differentially expressed genes were identified at 48 h post-infection in hybrid tilapia, Nile tilapia, and blue tilapia, respectively. Functional enrichment analysis indicated that numerous metabolic and immune-related pathways were activated in all three tilapia lines. The differential expression of specific genes associated with phagosome, focal adhesion, cytokine-cytokine receptor interaction, and toll-like receptor signaling pathways contributed to the resistance of hybrid tilapia. Notably, immune response genes in hybrid tilapia, such as P38, TLR5, CXCR3, CXCL12, PSTPIP1, and TFR, were generally suppressed under normal conditions but selectively induced following pathogen challenge. These results expand our knowledge of the molecular mechanisms underlying S. agalactiae tolerance in hybrid tilapia and provide valuable insights for tilapia breeding programs.

Chlorogenic acid improves food allergy through the AMPK/ACC/CPT-1 pathway.

Chlorogenic acid (CGA) is a polyphenol prevalent in daily food and plants. Food allergy (FA) can lead to metabolic disorders of the immune system. The objective of this study was to investigate CGA therapeutic effect on FA and regulatory mechanism through shrimp food allergy in mice models. Here, 24 female BALB/C mice were randomly allocated into the (I) Control group, (II) Food allergy group, (III) Chlorogenic acid low (50 mg/kg), and (IV) high group (200 mg/kg). Enzyme-linked immunosorbent assay revealed that CGA decreased levels of IgE and IgG induced by food allergy significantly. Real-time PCR demonstrated that high-dose chlorogenic acid significantly reduced Acetyl-CoA carboxylase (ACC) mRNA expression and increased Carnitine palmitoyltransferase-1 (CPT-1) mRNA expression. Western blot indicated that CGA promoted a noticeable increase at the levels of AMP-activated protein kinase (AMPK) and ACC phosphorylation, resulting in a significant activation in AMPK and inhibition in ACC, and increased CPT-1 expression. Consequently, CGA improves FA by the regulation of the AMPK/ACC/CPT-1 signaling pathway in the spleen. PRACTICAL APPLICATIONS: Chlorogenic acid is a water-soluble polyphenolic substance that is widely distributed in natural plants that show a variety of pharmacological effects. At present, CGA has been developed as a weigh-reducing tonic in western countries. As one of the most widely found and most easily obtained phenolic acids from food, the diverse physiological effects of CGA (such as anti-inflammatory, antioxidant, metabolic regulation, intestinal microbial regulation, etc.) imply its potential for application in functional foods, food additives, and clinical medicine. However, the basic molecular mechanisms of its effects have not been elucidated. In this study, CGA reduced allergy in a mouse model, likely by interacting with the AMPK/ACC/CPT-1 pathway.

Comparing Transcriptomes Reveals Key Metabolic Mechanisms in Superior Growth Performance Nile Tilapia (Oreochromis niloticus).

Metabolic capacity is intrinsic to growth performance. To investigate superior growth performance in Nile tilapia, three full-sib families were bred and compared at the biochemical and transcriptome levels to determine metabolic mechanisms involved in significant growth differences between individuals under the same culture environment and feeding regime. Biochemical analysis showed that individuals in the higher growth group had significantly higher total protein, total triglyceride, total cholesterol, and high- and low-density lipoproteins, but significantly lower glucose, as compared with individuals in the lower growth group. Comparative transcriptome analysis showed 536 differentially expressed genes (DEGs) were upregulated, and 622 DEGs were downregulated. These genes were significantly enriched in three key pathways: the tricarboxylic acid cycle (TCA cycle), fatty acid biosynthesis and metabolism, and cholesterol biosynthesis and metabolism. Conjoint analysis of these key pathways and the biochemical parameters suggests that Nile tilapia with superior growth performance have higher ability to consume energy substrates (e.g., glucose), as well as higher ability to biosynthesize fatty acids and cholesterol. Additionally, the fatty acids biosynthesized by the superior growth performance individuals were less active in the catabolic pathway overall, but were more active in the anabolic pathway, and might be used for triglyceride biosynthesis to store excess energy in the form of fat. Furthermore, the tilapia with superior growth performance had lower ability to convert cholesterol into bile acids, but higher ability to convert it into sterols. We discuss the molecular mechanisms of the three key metabolic pathways, map the pathways, and note key factors that may impact the growth of Nile tilapia. The results provide an important guide for the artificial selection and quality enhancement of superior growth performance in tilapia.

Digestion characteristics of quinoa, barley and mungbean proteins and the effects of their simulated gastrointestinal digests on CCK secretion in enteroendocrine STC-1 cells.

The demand for plant-based proteins has been rapidly increasing due to sustainability, ethical and health reasons. The present study aimed to investigate the digestion characteristics of three plant proteins (quinoa, barley and mungbean) based on an in vitro digestion model and the effect of their simulated gastrointestinal digests on satiety hormone cholecystokinin (CCK) secretion in enteroendocrine STC-1 cells. The nitrogen distribution in the digestion process, the relative molecular weight (MW) of peptides and the amino acid composition in simulated gastrointestinal digests were characterized. Quinoa protein had the highest proportion of soluble nitrogen after gastrointestinal digestion (85.79%), followed by barley protein (74.98%) and mungbean protein (64.14%), suggesting that quinoa protein was more easily digested than barley and mungbean proteins. The peptides but not free amino acids were the main components in the gastrointestinal digests of quinoa, barley, and mungbean proteins. The gastrointestinal digest of quinoa protein had a well balanced amino acid pattern, whereas that of barley protein was lacking Lys, and that of the mungbean protein was short of sulfur amino acids (Phe + Tyr) but rich in Lys. In terms of the ability to stimulate CCK secretion, the gastrointestinal digest of barley protein had a strong stimulatory effect on CCK secretion, while that of quinoa and mungbean proteins had only a weak stimulatory effect. After pretreatment with a specific calcium-sensing receptor (CaSR) antagonist NPS 2143, CCK secretion induced by the barley protein digest was greatly suppressed, indicating that CaSR was involved in barley protein digest-induced CCK secretion. These results show that quinoa protein has good nutritional quality, while barley protein is an excellent plant protein source to stimulate CCK secretion and has a potential application as a dietary supplement for obesity management.

Integration of mRNA and miRNA Profiling Reveals Heterosis in Oreochromis niloticus × O. aureus Hybrid Tilapia.

Heterosis is a widespread biological phenomenon in fishes, in which hybrids have superior traits to parents. However, the underlying molecular basis for heterosis remains uncertain. Heterosis in growth and survival rates is apparent in hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂). Comparisons of growth and hematological biochemical characteristics and mRNA and miRNA transcriptional analyses were performed in hybrid and parents tilapia stocks to investigate the underlying molecular basis for heterosis. Growth characteristics and hematological glucose and cholesterol parameters were significantly improved in hybrids. Of 3097 differentially expressed genes (DEGs) and 120 differentially expressed miRNAs (DEMs) identified among three stocks (O. niloticus, O. aureus, and hybrids), 1598 DEGs and 62 DEMs were non-additively expressed in hybrids. Both expression level dominance and overdominance patterns occurred for DEGs and DEMs, indicating that dominance and overdominance models are widespread in the transcriptional and post-transcriptional regulation of genes involved in growth, metabolism, immunity, and antioxidant capacity in hybrid tilapia. Moreover, potential negative regulation networks between DEMs and predicted target DEGs revealed that most DEGs from miRNA-mRNA pairs are up-regulated. Dominance and overdominance models in levels of transcriptome and miRNAome facilitate the integration of advantageous parental alleles into hybrids, contributing to heterosis of growth and improved survival. The present study provides new insights into molecular heterosis in hybrid tilapia, advancing our understanding of the complex mechanisms involved in this phenomenon in aquatic animals.

Evaluation of pediatric hydronephrosis using deep learning quantification of fluid-to-kidney-area ratio by ultrasonography.

PURPOSE: Hydronephrosis is the dilation of the pelvicalyceal system due to the urine flow obstruction in one or both kidneys. Conventionally, renal pelvis anterior-posterior diameter (APD) was used for quantifying hydronephrosis in medical images (e.g., ultrasound, CT, and functional MRI). Our study aimed to automatically detect and quantify the fluid and kidney areas on ultrasonography, using a deep learning approach. METHODS: An attention-Unet was used to segment the kidney and the dilated pelvicalyceal system with fluid. The gold standard for diagnosing hydronephrosis was the APD > 1.0 cm. For semi-quantification, we proposed a fluid-to-kidney-area ratio measurement, i.e., [Formula: see text], as a deep learning-derived biomarker. Dice coefficient, confusion matrix, ROC curve, and Z-test were used to evaluate the model performance. Linear regression was applied to obtain the fluid-to-kidney-area ratio cutoff for detecting hydronephrosis. RESULTS: For regional kidney segmentation, the Dice coefficients were 0.92 and 0.83 for the kidney and dilated pelvicalyceal system, respectively. The sensitivity and specificity of detecting dilated pelvicalyceal system were 0.99 and 0.83, respectively. The linear equation was fluid-to-kidney-area ratio = (0.213 ± 0.004) × APD (in cm) for 95% confidence interval on the slope with R2 = 0.87. The fluid-to-kidney-area ratio cutoff for detecting hydronephrosis was 0.213. The sensitivity and specificity for detecting hydronephrosis were 0.90 and 0.80, respectively. CONCLUSION: Our study confirmed the feasibility of deep learning characterization of the kidney and fluid, showing an automatic pediatric hydronephrosis detection.

Genetic variation among four bred populations of two tilapia strains, based on mitochondrial D-loop sequences.

The hybrid between Nile tilapia (Oreochromis niloticus,♀) and blue tilapia (O. aureus,♂) is an important strain in Chinese aquaculture industry. Two populations named AF (O. aureus, 29 samples) and NF (O. niloticus, 22 samples) were gathered from Freshwater Fisheries Research Center (FFRC). The other two named AG (O. aureus, 29 samples) and NG (O. niloticus, 28 samples) from Guangxi Fisheries Research Institute (GFRI). GFRI introduced AG and NG from AF and NF. The mitochondrial DNA D-loop was sequenced to assess the genetic diversity among four populations. A 580 bp fragment was sequenced. The 93 variable sites defined 39 haplotypes and three were shared. As a result, the genetic diversity of O. aureus AF and AG was much lower (H=0.497-0.532, K=0.69-0.714, π=0.0012-0.00124) than that of O. niloticus (H=0.849-0.866, K=24.286-24.807, π=0.04246-0.04337). Furthermore, the indices (H, K, π and D) was a slight increase between AF and AG, so did NF and NG. These results indicated that as the male parent, the AF and AG population was purebred and sustainable. And as the female parent, NF and NG had high genetic diversity. The conclusions might give reference to keep the germplasm diversity of tilapia and other introduced fishes.

Lymphatic function is required prenatally for lung inflation at birth.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

The secreted lymphangiogenic factor CCBE1 is essential for fetal liver erythropoiesis.

The secreted protein CCBE1 is required for lymphatic vessel growth in fish and mice, and mutations in the CCBE1 gene cause Hennekam syndrome, a primary human lymphedema. Here we show that loss of CCBE1 also confers severe anemia in midgestation mouse embryos due to defective definitive erythropoiesis. Fetal liver erythroid precursors of Ccbe1 null mice exhibit reduced proliferation and increased apoptosis. Colony-forming assays and hematopoietic reconstitution studies suggest that CCBE1 promotes fetal liver erythropoiesis cell nonautonomously. Consistent with these findings, Ccbe1(lacZ) reporter expression is not detected in hematopoietic cells and conditional deletion of Ccbe1 in hematopoietic cells does not confer anemia. The expression of the erythropoietic factors erythropoietin and stem cell factor is preserved in CCBE1 null embryos, but erythroblastic island (EBI) formation is reduced due to abnormal macrophage function. In contrast to the profound effects on fetal liver erythropoiesis, postnatal deletion of Ccbe1 does not confer anemia, even under conditions of erythropoietic stress, and EBI formation is normal in the bone marrow of adult CCBE1 knockout mice. Our findings reveal that CCBE1 plays an essential role in regulating the fetal liver erythropoietic environment and suggest that EBI formation is regulated differently in the fetal liver and bone marrow.

Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth.

Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.

Blood flow reprograms lymphatic vessels to blood vessels.

Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic forces. Although the genetic mutations that underlie the formation of many human vascular malformations are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program and natural history is not. Loss of the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) resulted in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice. Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic identity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter vessel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly lost expression of PROX1, a lymphatic fate-specifying transcription factor. These findings reveal that blood flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.

[Genetic potential analysis of six tilapia populations by microsatellite DNA markers].

Twenty five microsatellite loci were used to analyze two blue tilapia populations ["Xia'ao 1" (ZA), Guangxi population] and four nile tilapia populations [Egypt strain (ZN), 88 strain (XN), Guangxi population (GN), American strain (MN)]. A total of 7775 fragments ranging from 100 bp to 400 bp in length were obtained. Three to eight alleles were amplified in 25 loci and 143 alleles in all the six populations. The average number of alleles in each locus was 5.72. The average values of observed heterozygosity (Ho) ranged from 0.7253 to 0.8160, the average expected heterozygosity (He) ranged from 0.5146 to 0.6834, the average polymorphic information content (PIC) ranged from 0.4212 to 0.6105, and the number of average effective alleles (Ae) ranged from 2.20 to 3.23. The highest genetic similarity index was 0.9130 (between ZA and GA); and the lowest was 0.4352 (between ZA and ZN). The results showed that the four nile tilapia populations contained a high level of genetic potential, and the two blue tilapia populations were moderate.

CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish cardiovascular development and cerebral cavernous malformations.

Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Delta) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Delta proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20-like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role.

The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates.

Negative regulation of activated alpha-2 integrins during thrombopoiesis.

Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, alpha2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface alpha2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, alpha2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated alpha2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated alpha2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.