Intermediate Mesoderm Research Articles

Kidney tissue regeneration using bioactive scaffolds incorporated with differentiating extracellular vesicles and intermediate mesoderm cells

BackgroundTo overcome the limitations of current alternative therapies for chronic kidney disease (CKD), tissue engineering-mediated regeneration strategies have demonstrated the possibilities for complete kidney tissue regeneration. Given the challenges associated with the reproducibility of renal basal cells, the incorporation of intermediate mesoderm (IM) cells and bioactive materials to control bioactivities of cells with supported scaffolds should be considered as a viable approach to enable the regeneration of the complex kidney structure via renal differentiation.MethodsWe developed PMEZ scaffolds by combining crucial bioactive components, such as ricinoleic acid-grafted Mg(OH)2 (M), extracellular matrix (E), and alpha lipoic acid-conjugated ZnO (Z) integrated into biodegradable porous PLGA (P) platform. Additionally, we utilized differentiating extracellular vesicles (dEV) isolated during intermediate mesoderm differentiation into kidney progenitor cells, and IM cells were serially incorporated to facilitate kidney tissue regeneration through their differentiation into kidney progenitor cells in the 3/4 nephrectomy mouse model.ResultsThe use of differentiating extracellular vesicles facilitated IM differentiation into kidney progenitor cells without additional differentiation factors. This led to improvements in various regeneration-related bioactivities including tubule and podocyte regeneration, anti-fibrosis, angiogenesis, and anti-inflammation. Finally, implanting PMEZ/dEV/IM scaffolds in mouse injury model resulted in the restoration of kidney function.ConclusionsOur study has demonstrated that utilizing biodegradable PLGA-based scaffolds, which include multipotent cells capable of differentiating into various kidney progenitor cells along with supporting components, can facilitate kidney tissue regeneration in the mouse model that simulates CKD through 3/4 nephrectomy.Graphical

Gonadal Germ Cell Migration and Proliferation after Transfer in Developing Chicken Embryos.

A germline chimera is a useful model for developing and differentiating germ cells in vivo. Gonadal germ cells (GGCs) collected from chicken embryonic gonads may be used to produce germline chimeras as donor cells. However, the migratory and proliferative abilities of GGCs after transfer into recipient embryos are unclear. Here, the migratory and proliferative abilities of GGCs collected from 7-day-old White Leghorn embryos and fluorescently labeled were analyzed following transfer into the dorsal aorta of 2.5-day-old Rhode Island Red (RIR) embryos. Five days after transfer, the numbers of male and female GGCs were significantly higher in the RIR gonads than those in non-gonadal RIR organs when 50 GGCs were transferred per embryo. To analyze the temporal migration of GGCs in intermediate mesoderm, 50 GGCs were again transferred. The numbers of male and female GGCs in RIR gonads increased significantly from days 3 to 6 after transfer. To analyze GGC migration and proliferation in the gonads, a single GGC was transferred into 100 male and 100 female embryos. Five days after transfer, the frequencies of settled and proliferated GGCs were 37% (37/100) and 24% (24/100) in males, and 23% (23/100) and 8% (8/100) in females, respectively. Thus, GGCs are a heterogeneous cell population that may or may not have migratory and proliferative abilities. The heterogeneity of GGCs may be greater in females than that in males. When 50 GGCs were transplanted, almost all those present in embryos had settled and proliferated in the gonads and mesonephros. The migratory and proliferative abilities of GGCs in recipient gonads were considerably diverse in individual GGCs or between donor sexes.

Blf and the drl cluster synergistically regulate cell fate commitment during zebrafish primitive hematopoiesis.

Hematopoiesis is a highly coordinated process that generates all the body's blood cells, and perturbations in embryonic hematopoiesis may result in illnesses ranging from fetal anemia to various leukemias. Correct establishment of hematopoietic progenitor cell fate is essential for the development of adequate blood cell subpopulations, although regulators of cell fate commitment have not been fully defined. Here, we show that primary erythropoiesis and myelopoiesis in zebrafish embryos are synergistically regulated by blf and the drl cluster, as simultaneous depletion led to severe erythrocyte aplasia and excessive macrophage formation at the expense of neutrophil development. Integrative analysis of transcriptome- and genome-wide binding data revealed that blf and drl cluster genes are responsible for constraining the expression of vasculogenesis-promoting genes in the intermediate cell mass and monocytopoiesis-promoting genes in the rostral blood island. This indicates that blf and drl cluster genes act as determinants of the fate commitment of erythroid and myeloid progenitor cells. Furthermore, a rescue screen demonstrated that Zfp932 is a potential mammalian functional equivalent to zebrafish blf and drl cluster genes. Our data provide insight into conserved cell fate commitment mechanisms of primitive hematopoiesis.

Osr1 Maintains Renal Progenitors and Regulates Podocyte Development by Promoting wnt2ba via the Antagonism of hand2.

Knowledge about the genetic pathways that control nephron development is essential for better understanding the basis of congenital malformations of the kidney. The transcription factors Osr1 and Hand2 are known to exert antagonistic influences to balance kidney specification. Here, we performed a forward genetic screen to identify nephrogenesis regulators, where whole genome sequencing identified an osr1 lesion in the novel oceanside (ocn) mutant. The characterization of the mutant revealed that osr1 is needed to specify not renal progenitors but rather their maintenance. Additionally, osr1 promotes the expression of wnt2ba in the intermediate mesoderm (IM) and later the podocyte lineage. wnt2ba deficiency reduced podocytes, where overexpression of wnt2ba was sufficient to rescue podocytes and osr1 deficiency. Antagonism between osr1 and hand2 mediates podocyte development specifically by controlling wnt2ba expression. These studies reveal new insights about the roles of Osr1 in promoting renal progenitor survival and lineage choice.

PECULIARITIES OF SOURCES OF RUDIMENTS AND MORPHOGENESIS OF THE PELVICALYCEAL SYSTEM OF THE KIDNEY IN EARLY PERIOD OF THE HUMAN PRENATAL ONTOGENESIS

The aim of the study – to determine the peculiarities of the rudiments sources and thechronological sequence of topographical and anatomical transformations of the organsand structures of the human urinary system.Materials and methods. The material for the study was 14 series of consecutivehistological sections of specimens of human embryos and prefetuses (4.0-66.0 mm parietalcoccygeal length (PCL)) aged from 4th to 11th weeks of intrauterine development (IUD) A complex of modern methods of morphological research (anthropometry, morphometry,microscopy, 3D computer reconstruction, statistical analysis) were applied.The results. The first signs of mesonephric duct diverticula formation are determined inhuman embryos of the 5th week of the IUD (embryos 7.0-7.5 mm PCL). It is represented byan ampoule-like blind expansion of the diverticulum – the primary rudiment of the renalpelvis, which is surrounded by a condensed mesenchyme, that is the formation sourceof the renal parenchyma – a nephrogenic blastema. Starting from the embryos of themiddle of the 5th week of IUD, due to 3D computer reconstruction, the first topographicaland anatomical features of the primordia of the structures of the definitive kidney aredetermined. The rudiments of the renal pelvises – paired ampoule-like blind expansionsof the diverticulum of the mesonephric duct – are immersed in the metanephric blastema,which has the shape of a drop due to the upper narrowed end. Starting from the end of the6th week of IUD, there is a evagination of the wall of the blind end of the diverticulum (therudiment of the renal pelvis) in the cranial and caudal directions, i.e., the rudiments ofmajor calyces appears. The rudiments of the minor cups is formed by the evagination ofthe wall of the major calices and appears in prefetuses at the beginning of the 7th week ofIUD. Age-related topographical and anatomical changes of the kidneys were also traced.Conclusions. 1. On the specimens of embryos 4.7-5.5 mm PCL (4th week of IUD),mesodermal sources of rudiments of structures and organs of the genitourinary system,which originate from the paired genitourinary crest, are determined. At the border of thedorsal and ventral parts of the mesoderm, in the intermediate mesoderm, the source of theurinary tubules is carried out, which form the nephrogenic cord – the only source of layingof all three generations of the kidney. At the end of the 4th week of IUD, an evaginationappears on both sides from the dorsal wall of the excretory duct of the mesonephros in itscaudal part, which is the rudiment of the epithelial lining of the ureter and renal pelvis.2. The rudiment of the renal pelvis is observed for the first time in the 5th week of IUD(embryos 7.0-7.5 mm PCL), major cups – at the end of the 6th week of VUR (embryos12.0-13.5 mm TKD), small calices – at the beginning of the 7th week of IUD (prefetuses14.0-15.0 mm PCL). 3. In the process of intrauterine development, the kidneys move fromtheir original location in the pelvis, where the source of their blood supply is the commoniliac artery, cranial to the primordia of the adrenal glands, giving their own vessels to theaorta, which become renal arteries.

Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium

BackgroundThe kidneys require vast amounts of mitochondria to provide ample energy to reabsorb nutrients and regulate electrolyte, fluid, and blood pressure homeostasis. The lack of the human model hinders the investigation of mitochondria homeostasis related to kidney physiology and disease.ResultsHere, we report the generation of mitochondria-rich kidney organoids via partial reprogramming of human urine cells (hUCs) under the defined medium. First, we reprogrammed mitochondria-rich hUCs into expandable intermediate mesoderm progenitor like cells (U-iIMPLCs), which in turn generated nephron progenitors and formed kidney organoids in both 2D and 3D cultures. Cell fate transitions were confirmed at each stage by marker expressions at the RNA and protein levels, along with chromatin accessibility dynamics. Single cell RNA-seq revealed hUCs-induced kidney organoids (U-iKOs) consist of podocytes, tubules, and mesenchyme cells with 2D dominated with mesenchyme and 3D with tubule and enriched specific mitochondria function associated genes. Specific cell types, such as podocytes and proximal tubules, loop of Henle, and distal tubules, were readily identified. Consistent with these cell types, 3D organoids exhibited the functional and structural features of the kidney, as indicated by dextran uptake and transmission electron microscopy. These organoids can be further matured in the chick chorioallantoic membrane. Finally, cisplatin, gentamicin, and forskolin treatment led to anatomical abnormalities typical of kidney injury and altered mitochondria homeostasis respectively.ConclusionsOur study demonstrates that U-iKOs recapitulate the structural and functional characteristics of the kidneys, providing a promising model to study mitochondria-related kidney physiology and disease in a personalized manner.

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of <i>CEP83</i> in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous <i>CEP83</i> inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of <i>CEP83</i>-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (<i>PAX8</i>, <i>EYA1</i>, <i>HOXB7</i>), and an aberrant induction of LPM markers (e.g. <i>FOXF1</i>, <i>FOXF2</i>, <i>FENDRR</i>, <i>HAND1</i>, <i>HAND2</i>). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas <i>CEP83</i>-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that <i>CEP83</i> regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

Podocytes derived from human induced pluripotent stem cells: characterization, comparison, and modeling of diabetic kidney disease

BackgroundIn diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared.MethodsWe characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose.ResultsPodocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability.ConclusionsWe found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.

Abstract 3755: DNA methylation patterns, but not genetic sequence alterations, are concordant across multiply sampled sites within individuals with unilateral and bilateral Wilms tumors

Abstract Wilms tumor, the most common renal malignancy of childhood, is a disease marked by heterogeneity. One tumor can harbor multiple histologic subtypes and multifocal or bilateral disease is common occurring in up to 10% of patients. Several molecular features have been associated with Wilms tumors and some are used for risk stratification. However, it is unclear which, if any, features are consistent throughout a single tumor or shared between tumors in cases of bilateral disease. To answer this question, we generated genomic and epigenomic data from multiply sampled Wilms tumors from 10 patients - 3 with unilateral disease and 7 with bilateral disease. Multiple samples from each tumor as well as matched normal tissues were included when available. All tumors and matched normal tissues had DNA methylation array data available. Segmental chromosomal aberrations (SCAs) were inferred from methylation array data. Sequencing data were available for all bilateral patients but not for unilateral patients. We found that SCAs were discordant both between pairs of bilateral tumors and within multiply sampled unilateral tumors. This discordance included SCAs used for clinical risk stratification (chromosome 1q gain, 1p loss). Sequence variants were also rarely shared between pairs of bilateral tumors. On the other hand, DNA methylation patterns were concordant across all samples for each individual. We previously described 3 distinct DNA methylation patterns in Wilms tumors and found that all samples from each patient classified into the same subgroup. Furthermore, DNA methylation at Wilms tumor-specific and subgroup-specific differentially methylated regions had high (&amp;gt;0.8) correlation in all samples from all cases. Our data demonstrate that, in contrast to genomic changes such as SCAs and sequence alterations, DNA methylation patterns in Wilms tumors are frequently shared between paired tumors in bilateral disease and in multiple regions within individual tumors. This is consistent with earlier studies demonstrating that gain of methylation at the H19 imprinted region is often the earliest event in Wilms tumors and in some cases likely occurs in the embryonic intermediate mesoderm prior to the development of individual kidneys. Our findings demonstrate the utility of Wilms tumor DNA methylation patterns in overcoming tumor heterogeneity for risk stratification in cases of bilateral or multifocal Wilms tumors. Citation Format: Jack Brzezinski, Sanaa Choufani, Cheryl Shuman, Rodrigo Romao, Armando Lorenzo, Rosanna Weksberg. DNA methylation patterns, but not genetic sequence alterations, are concordant across multiply sampled sites within individuals with unilateral and bilateral Wilms tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3755.

Large-scale engineering of hiPSC-derived nephron sheets and cryopreservation of their progenitors

BackgroundThe generation of human induced pluripotent stem cells (hiPSCs) has opened a world of opportunities for stem cell-based therapies in regenerative medicine. Currently, several human kidney organoid protocols are available that generate organoids containing kidney structures. However, these kidney organoids are relatively small ranging up to 0.13 cm2 and therefore contain a small number of nephrons compared to an adult kidney, thus defying the exploration of future use for therapy.MethodWe have developed a scalable, easily accessible, and reproducible protocol to increase the size of the organoid up to a nephron sheet of 2.5 cm2 up to a maximum of 12.6 cm2 containing a magnitude of nephrons.ResultsConfocal microscopy showed that the subunits of the nephrons remain evenly distributed throughout the entire sheet and that these tissue sheets can attain ~ 30,000–40,000 glomerular structures. Upon transplantation in immunodeficient mice, such nephron sheets became vascularized and matured. They also show reuptake of injected low-molecular mass dextran molecules in the tubular structures, indicative of glomerular filtration. Furthermore, we developed a protocol for the cryopreservation of intermediate mesoderm cells during the differentiation and demonstrate that these cells can be successfully thawed and recovered to create such tissue sheets.ConclusionThe scalability of the procedures, and the ability to cryopreserve the cells during differentiation are important steps forward in the translation of these differentiation protocols to future clinical applications such as transplantable auxiliary kidney tissue.

Leukemia-associated transcription factor mllt3 is important for primitive erythroid development in zebrafish embryogenesis.

MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains uncertain. Here, we examine the role of mllt3 in developmental hematopoiesis during embryogenesis using zebrafish. Cloning, sequencing, phylogenetic, and synteny analyses showed high evolutionary conservation between important functional domains of the zebrafish orthologue of mllt3 and MLLT3 in humans. Quantitative reverse transcription-PCR and in situ hybridization analyses revealed that mllt3 is maternally supplied and zygotically expressed throughout embryonic development, and that expression is highest between 10 and 24 hours post-fertilization (hpf) coincident with enrichment in the intermediate cell mass (ICM) and posterior blood island, which are the sites of the primitive and transient definitive hematopoiesis in zebrafish, respectively. Further, we found co-expression of mllt3 with the early hematopoietic progenitor markers tal1, gata2, and gata1a in the posterior ICM. By investigating zebrafish hematopoietic mutants, we discovered that mllt3 is involved in erythroid precursor formation. By 48-72 hpf, mllt3 expression proved to be restricted to non-hematopoietic tissues including head structures, pronephric tubules, and liver primordium. These findings establish a link between mllt3 and primitive erythropoiesis and provide the basis for future functional investigations.

25, 50 &amp; 75 years ago

25, 50 &amp; 75 years ago

Evolutionary Transition in the Regulation of Vertebrate Pronephros Development: A New Role for Retinoic Acid.

The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including Hox genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers (Pax2 and Lim1), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of Hox4 genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek’s nephridium. This suggests that despite the conservation of Pax2 and Lim1 expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and Hox-dependent regulation is a gnathostome novelty.

Fetal accessory renal arteries and other renal anomalies

Background: Human kidneys develop from intermediate mesoderm, in three successive stages; pronephros, mesonephros and metanephros. The definitive kidney, which is derived from metanephros occupy sacral region initially, which ascent later, reaching the adult position by 9th week. It receives blood supply from different sources during its development and ascent. Accessory renal arteries are widely studied in adults while the same in fetuses is an understudied topic. Aims and Objectives: The present study was carried out to find out the percentage of accessory renal arteries in fetuses and associated positional congenital anomalies with it. Materials and Methods: Fifty fetuses of gestational ages ranged from 14 to 40 weeks were collected from the department of Obstetrics and Gynecology, with prior permission from Head of the department and Dean. Results: The study showed a higher percentage of accessory renal arteries in fetuses (30%) and in 2 out of 50 fetuses dissected (4%), accessory renal arteries were found to be associated with positional anomalies. One was a case of bilateral ectopic kidney whereas the other was a horseshoe kidney (HSK). Lobulation were seen in most of the kidneys and 3 cases of bilateral accessory renal arteries without any obvious anomalies were noted. Conclusion: As the advancement in prenatal diagnostic techniques, screening for accessory renal arteries in fetuses can provide a clue for associated congenital renal anomalies. Knowledge on vascular variation in fetuses will help pediatric surgeons in planning abdominal surgeries in new born and children of early ages, where vascular anatomy may be different than in adults.

Horseshoe Kidney - A Case Report

Horseshoe kidney is a fairly common genetic renal anomaly. The caudal ends of the kidney fuse together to create a parenchymatous isthmus which cannot ascend out of pelvic cavity as the inferior mesenteric artery prevents its further migration. Urogenital systems are closely associated in their development topography and function and are developed from two important structures the intermediate mesoderm and cloaca.During dissection of male cadaver in the dissection hall of the Shareera Rachana Department a horseshoe kidney variation was observed and the same is emphasised in this article. It is important to be aware of this renal anomaly for clinical practice especially during renal surgical procedures transplants or other procedures.nbsp

Kidney development to kidney organoids and back again

Kidney organoid technology has led to a renaissance in kidney developmental biology. The complex underpinnings of mammalian kidney development have provided a framework for the generation of kidney cells and tissues from human pluripotent stem cells. Termed kidney organoids, these 3-dimensional structures contain kidney-specific cell types distributed similarly to in vivo architecture. The adult human kidney forms from the reciprocal induction of two disparate tissues, the metanephric mesenchyme (MM) and ureteric bud (UB), to form nephrons and collecting ducts, respectively. Although nephrons and collecting ducts are derived from the intermediate mesoderm (IM), their development deviates in time and space to impart distinctive inductive signaling for which separate differentiation protocols are required. Here we summarize the directed differentiation protocols which generate nephron kidney organoids and collecting duct kidney organoids, making note of similarities as much as differences. We discuss limitations of these present approaches and discuss future directions to improve kidney organoid technology, including a greater understanding of anterior IM and its derivatives to enable an improved differentiation protocol to collecting duct organoids for which historic and future developmental biology studies will be instrumental.

OSR1 Couples Intermediate Mesoderm Cell Fate with Temporal Dynamics of Vessel Progenitor Cell Differentiation

OSR1 Couples Intermediate Mesoderm Cell Fate with Temporal Dynamics of Vessel Progenitor Cell Differentiation

BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo.

ABSTRACTHuman embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos ( Xiang et al., 2020). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.This article has an associated First Person interview with the first author of the paper.

Meis1, Hi1α, and GATA1 are integrated into a hierarchical regulatory network to mediate primitive erythropoiesis.

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.

Osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.