White adipose tissue primarily stores energy while brown adipose tissue dissipates energy as heat, holding promise for therapeutic use. Brown adipose tissue in the anterior trunk is believed to derive from the somitic mesoderm, although some depots are of partially unknown origin. Here we show that the subscapular, lateral, cervical and peri-aortic brown adipose depots, but not the interscapular depot, are in part formed by a non-somitic source. Single-cell sequencing along with genetic lineage tracing indicates that at embryonic day 9.5 the dorsal aorta compartment harbors multipotent mesenchymal progenitors expressing the transcription factor Osr1. Spreading laterally from the dorsal aortic midline, these cells contribute to adipose, cartilage and myogenic lineages. This study uncovers an alternative source of brown adipose tissue and suggests that a fraction of dorsal aorta-associated mesenchymal Osr1+ cells may represent the in vivo correlate of a multipotent progenitor cell type so far only characterized in vitro, the mesoangioblast.

Secreted frizzled-related protein 1a regulates hematopoietic development in a dose-dependent manner.

Hematopoietic stem and progenitor cells (HSPCs) emerge from arterial endothelial cells (ECs) through a process termed endothelial-to-hematopoietic-transition (EHT), a process induced by paracrine signals and driven by a transcriptional cascade. Despite inductive signals being broadly received by ECs in the dorsal aorta (DA), only a subset of ECs undergoes EHT, while others maintain their vascular identity. The molecular mechanisms that determine this selective fate decision remain poorly understood. Here, we discover Apelin signaling as a critical regulator of cell fates in the DA, acting as a molecular switch to balance vascular and hematopoietic identities. We show that Apelin receptor (Aplnr)-expressing ECs retain their arterial identity, whileAplnr non-expressing ECs are primed to become hemogenic endothelial cells (HECs) and transition into HSPCs. Loss of Apelin signaling leads to excessive EC-to-HEC conversion and increased HSPC numbers. Conversely, forced Aplnr expression abolishes HSPC formation by maintaining EC identity. These findings reveal that Apelin signaling regulates HSPC formation by preserving endothelial identity. In summary, our findings establish Apelin signaling as a critical regulator for balancing endothelial and hematopoietic fates.

During vertebrate embryogenesis, hematopoietic stem and progenitor cells (HSPCs) originate from hemogenic endothelium (HE) in the dorsal aorta through endothelial-to-hematopoietic transition (EHT). While basal inflammation is essential for this process, excessive immune activation disrupts HSPC emergence. Here, we identify angiotensin-converting enzyme 2 (Ace2), a key component of renin-angiotensin system, as a crucial anti-inflammatory regulator of embryonic hematopoiesis in zebrafish and mice. Loss of Ace2 impairs HE specification and reduces nascent HSPC production. Mechanistically, transcriptomic profiling reveals that ace2 deficiency leads to aberrant activation of NLR family pyrin domain containing 3 (Nlrp3) signaling and pyroptosis in vascular endothelial cells. Importantly, pharmacological inhibition of Nlrp3 or Caspase-1 restores HSPC emergence upon ace2 deficiency, consistent with treatment with exogenous angiotensin-(1-7) [Ang-(1-7)], a downstream product of Ace2 enzymatic activity. Moreover, Ace2 knockdown in mouse embryos phenocopies the defects in zebrafish, demonstrating evolutionary conservation of ACE2 in developmental hematopoiesis in mammals. Together, our findings uncover an essential role for ACE2 in maintaining a permissive inflammatory environment for HSPC development and suggest therapeutic potential for targeting the ACE2/Ang-(1-7)/Nlrp3-pyroptosis axis in inflammatory hematopoietic disorders.

The generation of germline chimeras in chickens via transplantation of primordial germ cells (PGCs) provides a robust and reproducible platform for avian transgenesis, genome editing, and species conservation. Traditional approaches using blastodermal cells from EGK stage X embryos are limited by low germline transmission efficiency due to early cell lineage segregation. In contrast, PGC-mediated strategies exploit the intrinsic germline competency of these unipotent cells, enabling reliable incorporation into recipient gonads. Here, we present a stepwise protocol for isolating PGCs from HH stage 26-28 embryos, maintaining and expanding them in vitro, and transplanting them into the dorsal aorta of HH stage 14-17 recipient embryos. Short-term engraftment is monitored using fluorescent markers such as PKH26, while long-term germline contribution is confirmed by PCR analysis of recipient gonads. This approach ensures consistent generation of germline chimeras while preserving the viability and functional competence of donor PGCs. Additionally, the method is compatible with genome editing tools such as CRISPR/Cas9 and transposon-based vectors, enabling the production of transgenic and gene-edited avian models. Beyond basic research, this strategy supports conservation efforts through interspecies germline transmission and genetic resource preservation. Overall, this protocol provides a comprehensive and reliable framework for manipulating the avian germline, offering a versatile platform for both fundamental and applied studies in poultry biotechnology.

In vertebrate embryonic development, hematopoietic stem and progenitor cells (HSPCs) originate from a subset of arterial endothelial cells in the ventral wall of the dorsal aorta through endothelial-to-hematopoietic transition (EHT). Despite extensive research efforts, gaps persist in understanding the establishment of HSPC development. In this study, we demonstrate that DNA methyltransferase 3ba (Dnmt3ba), highly expressed in the hemogenic endothelial cells (HECs), plays a crucial role in regulating HEC survival in zebrafish. Dnmt3ba deficiency leads to hypomethylation at the itgα3b and itgα7 loci, diminishing the expression of these Integrins and downstream Akt signaling and Mdm2 phosphorylation, while concurrently triggering HEC apoptosis by upregulation of P53 activity. Manipulation of DNMT3B in an iPSC-derived human hematopoietic differentiation system indicates functional conservation. Collectively, our findings unveil an epigenetic mechanism governed by Dnmt3ba, orchestrating HEC survival through epigenetic modulation of Integrin signaling.

Spatial transcriptomics identified dynamic and spatial-resolved niche cell and signal architecture for hematopoietic stem cell specification

Introduction Variations in the vertebral artery (VA) origin and course, though uncommon, carry significant clinical implications, particularly in cerebrovascular surgery, stroke management, and endovascular interventions.Duplication of the VA is among the rarest anomalies, where one branch arises from the thyrocervical trunk (TCT)—a major arterial branch of the subclavian artery supplying the inferior thyroid artery, suprascapular artery, and transverse cervical artery. This anatomical variant can significantly affect cerebral hemodynamics, endovascular procedures, and surgical planning in the cervical region. Strub et al. reported a case of a left‐sided duplicate vertebral artery originating from the TCT during a neurosurgical evaluation. Material and method A 51‐year‐old male with no significant past medical history presented to the emergency ward with a sudden onset of vertigo and imbalance attacks. A magnetic resonance image (MRI) reveals multiple infarctions in the left cerebellar hemisphere and the left lateral medulla (Figure 1). Upon examination, the patient was alert and conscious but had impaired cerebellar function. The patient reported a minor trauma sustained while playing football the previous day. Cerebral angiography revealed a total occlusion of the left VA in the V2 segment. On the right side, a duplicated vertebral artery was observed. The first, hypoplastic VA originated from the subclavian artery and terminated in the mid‐cervical region. The second VA originated from the TCT and terminated at the level of the right posterior inferior cerebellar artery (PICA). Right and left carotid angiography demonstrated a bilateral fetal posterior communicating artery, supplying the basilar artery in a retrograde fashion (Figure 2). Dual antiplatelet therapy was initiated and continued for three months Discussion The VA develops from the longitudinal anastomosis of the first six intersegmental arteries from the dorsal aorta. Normally, these intersegmental arteries regress, resulting in a single VA with a subclavian origin. The most frequent variation observed bilaterally involved a variant origin proximal to the typical vertebral artery origin, with entry at the C5 level. Furthermore, multiple origins were more commonly observed on the right side.Variations in the vertebral artery (VA), particularly duplications with origins from the thyrocervical trunk (TCT), are clinically significant due to their impact on cerebrovascular interventions and surgical procedures. Conclusion A duplicated vertebral artery originating from the thyrocervical trunk is a rare yet clinically significant vascular anomaly. Advanced imaging techniques such as CTA and angiography are essential for accurately identifying such anomalies. Figure 1: Axial MRI of the cerebellar section showed multiple infarctions of the left cerebellar hemisphere and left side lateral medulla. Figure 2: Angiography of cerebral and cervical arteries.a and b: Right and left ICA angiography showing basilar supply from posterior communicating arteries retrogradely.C: An oblique view of the left vertebral artery that showed total occlusion in the cervical part.d and e; AP and f lateral view of the right subclavian artery showed a hypoplastic vertebral artery that terminates in the cervical region and an accessory or duplicate vertebral artery from the thyrocervical trunk that ends up in the level of the posterior inferior cerebellar artery.

The aorta-gonad-mesonephros (AGM) is a region of embryonic mesoderm that develops during embryonic development from the para-aortic splanchnopleura in chick, mouse, and human embryos. The AGM contains the dorsal aorta, genital ridges, and mesonephros, and lies between the notochord and the somatic mesoderm. The aim of this historical note was to underline the fundamental role of AGM in the development of the hematopoietic system at the crossroads between endothelial and hematopoietic cells.

Unravelling early hematoendothelial development through the chick model: Insights and future perspectives.

Vascular scaling: A careful balancing act between proliferation and extrusion.

Hematopoietic stem cells and more committed progenitors (collectively referred to as HSPCs) emerge from vessels during development, via endothelial-to-hematopoietic transition (EHT). Recently, using the zebrafish embryo, we showed that two EHT cell types emerge from the dorsal aorta, raising the question of their subsequent fate. To address this issue, we established a complex pipeline based on single-cell photoconversion and transgenic lines to characterize the abilities of EHT cell progenies to conquer hematopoietic organs and to obtain their transcriptomic profiles. We show that the two EHT cell types lead to partly differentially fated cells, with significant differences in thymus colonization and T-lymphoid lineage commitment. In addition, we investigated implantation of HSPCs in niches, with the support of HSPC signatures (gata2b and cd34/podocalyxin), retrieved from our single-cell datasets. This revealed, at unprecedented resolution, the homing of HSPCs in niches of entire early larvae, including the pronephros, the sub-aortic and caudal regions, as well as the area contacting the supra-intestinal artery. Our work provides new insights into fundamental aspects of HSPC fate acquisition, from their emergence to their homing in specific niches.

The development of engraftable, long-term reconstituting hematopoietic stem cells (LT-HSC) from human pluripotent stem cells (hPSC) has been a long-sought goal. Since HSCs are formed by a subset of endothelial cells in the ventral part of the dorsal aorta, we analyzed heartbeat-mediated pulsatile displacement experienced by the walls of the dorsal aorta in zebrafish embryos. We found that pulsation-mediated circumferential stretch was restricted to the ventral part of the dorsal aorta and activated Piezo1 to stimulate LT-HSC formation. Stimulation of pulsation or Yoda1-mediated Piezo1 activation promoted the formation of de novo LT-HSCs from hemogenic endothelial cells derived from murine embryos or human pluripotent stem cells. These HSCs gave long-term multilineage reconstitution of hematopoietic cells upon transplantation into immunocompromised mice. The formation of transgene-free human LT-HSCs that can engraft and reconstitute the hematopoietic system will facilitate the generation of off-the-shelf HSCs from hPSCs for use in cellular therapies.

Introduction: Bile acids, synthesized by hepatocytes, are metabolized by microbiota within the gut-liver axis. Recent reports indicate that both bile acids and gut microbiota significantly contribute to hypertension. However, the role of bile acid receptors, specifically the farnesoid X receptor (FXR), in blood pressure (BP) regulation is completely unknown. Therefore, we focused on FXR, which is the key bile acid nuclear receptor. Since FXR is a negative regulator of bile acid synthesis, which can also reshape the gut microbiome, we hypothesized that the genetic ablation of FXR lowers BP by increasing bile acid levels and remodeling gut microbiota composition. Methods: To test the hypothesis, we generated the first global FXR knockout ( Fxr KO) rat model on the genetic background of hypertensive Dahl Salt-Sensitive (S) rat using the CRISPR/Cas9 technology. Adult 10-week-old male Fxr KO (n=9) and control S (n=8) rats were studied, and BP was measured by radiotelemery for five weeks. Gut microbiota composition was analyzed using fecal 16S rRNA gene sequencing and Oxford Nanopore whole-genome sequencing. Microbial bile salt hydrolase activity, an enzyme involved in bile acid deconjugation, was quantified by ninhydrin colorimetry. Cardiac function was examined by echocardiography, and vascular function was studied ex vivo by wire myography. Serum total bile acids were measured via colorimetric assay. Kidney function was characterized by urinary protein excretion (UPE) and histopathological analysis. Results: The loss of FXR resulted in elevated serum total bile acids (45.6 μM vs 15.06 μM, p=0.0003). This was accompanied by a substantial reduction in systolic BP (171.1 mm Hg vs 199.6 mm Hg, p<0.0001), diastolic BP (130 mm Hg vs 151.4 mm Hg, p<0.0001), and mean arterial pressure (149.7 mm Hg vs 174.7 mm Hg, p<0.0001). Frx KO rats showed improved cardiac function, as evidenced by increased ejection fraction (87.24 % vs 81.48 %, p=0.0003) and fractional shortening (58.39 % vs 51.43%, p=0.0005). Vascular function analysis in the dorsal aorta of Fxr KO rats further revealed increased sensitivity to both endothelium-dependent, (acetylcholine, Ach) and endothelium-independent vasodilators (sodium nitroprusside, SNP), with both exhibiting lower EC 50 values (-log EC 50 , Ach: -7.5±0.3 vs -7±0.2, and SNP: -8.8±0.2 vs -8.1±0.1). UPE was significantly decreased in Fxr KO rats compared to S rats (28.2 mg/24-hour vs 69.6 mg/24-hour, p=0.0064), with reduced hyaline casts and fibrosis in renal tissues. Moreover, Microbiota analysis revealed significant rearrangement of gut microbiota composition in Fxr KO rats compared to S rats (α-diversity, p<0.01; β-diversity, p<0.001). Notably, the abundance of Akkermansia muciniphila, a gut commensal with known probiotic properties, was significantly higher in the Fxr KO rats (p<0.0001). Remodeling of gut microbiome in Fxr KO rats resulted in significantly higher bile salt hydrolase activity (1.24 mM/min vs 0.65 mM/min, p<0.0001), which showed a significant negative correlation with systolic BP (r= -0.7, p=0.0024). Conclusions: Our study is the first Fxr KO rat model that demonstrates FXR deletion in hypertensive S rats is protective against hypertension, which could be attributed to a combination of improved cardiovascular and renal function. Importantly, the remodeled gut microbiome and increased microbial bile salt hydrolase activity suggest a novel link between bile acid deconjugation and the observed BP reduction. While further mechanistic studies are warranted, these findings establish FXR as a critical regulator of BP through bile acid-gut microbiota interactions and highlight bile acid modulation as a promising therapeutic strategy for hypertension. NIH R01HL171401-01 to Dr. Bina Joe and AHA Predoc 24PRE1186688 to Ishan Manandhar This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Compartment boundaries divide the embryo into segments with distinct fates and functions. In the vascular system, compartment boundaries organize endothelial cells into arteries, capillaries, and veins that are the fundamental units of a circulatory network. For vascular smooth muscle cells (SMCs), such boundaries produce mosaic patterns of investment based on embryonic origins with important implications for the non-uniform distribution of vascular disease later in life. The morphogenesis of blood vessels requires vascular cell movements within compartments as highly-sensitive responses to changes in fluid flow shear stress and wall strain. These movements underline the remodeling of primitive plexuses, expansion of lumen diameters, regression of unused vessels, and building of multilayered artery walls. Although the loss of endothelial compartment boundaries can produce arterial-venous malformations, little is known about the consequences of mislocalization or the failure to form SMC-origin-specific boundaries during vascular development. We propose that the failure to establish a normal compartment boundary between cardiac neural-crest-derived SMCs of the 6th pharyngeal arch artery (future ductus arteriosus) and paraxial-mesoderm-derived SMCs of the dorsal aorta in mid-gestation embryos leads to aortic coarctation observed at birth. This model raises new questions about the effects of fluid flow dynamics on SMC investment and the formation of SMC compartment borders during pharyngeal arch artery remodeling and vascular development.

A double aortic arch is a rare vascular anomaly. However, this is the most common cause of a symptomatic vascular ring malformation due to the absence of involution of the caudal dorsal aorta. It involves the complete encirclement and compression of the trachea and esophagus. The disease usually begins to show itself in very early clinical signs, already detectable in the neonatal period. It may lead to significant morbidity for the patient, a wide range of clinical symptoms ranging from life-threatening symptoms to no symptoms can be resulting; regardless, their detection is extremely important before undertaking procedures or making surgical decisions. If associated symptoms are present, surgical correction of the vascular ring should be performed. For accurate diagnosis and evaluation of aortic arch anomalies, cross-sectional imaging modalities such as CT or MRI play an important role in providing three-dimensional reconstructed images. We here report one case of double aortic arch to highlight the contribution of imaging in the difficult diagnosis of this anomaly.

The contribution of the gut to the ingestion, production, absorption and excretion of the extra ammonia and urea nitrogen (urea-N) associated with feeding ('exogenous' fraction) has received limited attention. Analysis of commercial pellet food revealed appreciable concentrations of ammonia and urea-N. Long-term satiation feeding increased whole-trout ammonia and urea-N excretion rates by 2.5-fold above fasting levels. Blood was sampled from the dorsal aorta, posterior, mid- and anterior sub-intestinal veins, as well as the hepatic portal vein in situ. Ammonia, urea-N and fluid flux rates were measured in vitro using novel gut sac preparations filled with native chyme. The sacs maintained the extreme physico-chemical conditions of the lumen seen in vivo. Overall, these results confirmed our hypothesis that the stomach, and anterior intestine and pyloric caecae regions play important roles in ammonia and urea-N production and/or absorption. There was a very high rate of urea-N production in the anterior intestine and pyloric caecae, whereas the posterior intestine dominated for ammonia synthesis. The stomach was the major site of ammonia absorption, and the anterior intestine and pyloric caecae region dominated for urea-N absorption. Model calculations indicated that over 50% of the exogenous ammonia and urea-N excretion associated with satiation feeding was produced in the anaerobic gut. This challenges standard metabolic theory used in fuel-use calculations. The novel gut sac preparations gained fluid during incubation, especially in the anterior intestine and pyloric caecae, owing to marked hyperosmolality in the chyme. Thus, satiation feeding with commercial pellets is beneficial to the water balance of freshwater trout.

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Cell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, endothelial cells (ECs) can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs express aqp1a.1 and aqp8a.1 in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migration in vivo. Our study thus highlights an important role of hydrostatic pressure in tissue morphogenesis.

Cell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, endothelial cells (ECs) can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs express aqp1a.1 and aqp8a.1 in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migration in vivo. Our study thus highlights an important role of hydrostatic pressure in tissue morphogenesis.